Basic Biotechnology 2 ed

BaSK Biotechnology Biotechnology impinges on everyone-'s lives. It is one oftlle maJor rechnologies oftbe twenty..first...

Author: Colin Ratledge | Bjorn Kristiansen

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BaSK Biotechnology Biotechnology impinges on everyone-'s lives. It is one oftlle maJor rechnologies oftbe twenty..first century. les huge, wide-r.mging, multhlisdplinaTY activities ¡nelude recombiIlanr ONA: techniques, doning 3ud geoetics, and the application ofmicrobiology to the production ofgoods as prosak as bread, beer, cheese and antibiotics. Jt continue$ tO revolutionise 1:realnlents ofmany diseases, and is used lo provide clea.n tedlnologie5 and todeal with environmental probJems. Basic Blo:t'chtwlogyis a textbook. thal gives aJuU accouDtofthecu.r rentstate of biotechnology, providing!he ~ader with iruight, inspiratlon and instruction. The fundamental aspects lhat Uflderpin biotechnoiogy are explained through examples fiom me pbarmaceutical, food and environmental industries. Olapters on the public pen::eption ofbiot.echnology and the business aud economics of the subject are -82372 Penzberg Germany

DK 2800Lyngby

Denmark HENK] . NOORMAN

Gist-brocó1c\es NV

8J 0RN KRIHIANSEN

POBoxl

European Biotf.'Ch.nologist5 Gluppeveien 15 N-t6H. Fredrlkstad

2600 MA Delft lbe Netberlilllds

Norway CHRISTIAN P. ICUBICEK

Depanment ofMicrobial Biochemistry lnstitute ofBiochem.ical Technology Get reidemarkt9/l72 \Tienna a-1060 Austria

W. PFEfFERLE

Biotedmologie Deguna-Hüls AG PFllt2 0-33788 Halle

Germany COLlN R.ATlEDGE

Department ofBiological Sciences UruVl!nity ofHull

DAVID A. LOWE

HuU

Brlstol·Myen Co. Industrial Division

HU67RX, UK

PO Bax4755

Syracuse NY 13221, USA A . LÜBBERT

lnstiWt ffir Bioverfahreostechnik und Reaktionstechnik Martin Luther UniV{:rsital Halle Wirtenberg o.06099lialle{Saalc Gl!Tmany

H. SAHM

Insrirut für Biotechnologie Forscbung5zentrumJütich GmbH PO Bollt913 1).52-425 jülich GcrOlany R. SIMUTlS

Control Departrnent Kaunas Technical UniVl'fiity Kaunas lilhuania

DONALD A. MA CKENZ IE

Institute ofFood Research Norwich Research Park Colney. Norwich NR47UA,UK

JOHN E. SM ITH

Applle
1.2 I Public awareness 01 genetic engineering However, genetic engineering is sw-prisingly being subjected to a massivc leve! of criticism frem a deeply suspicious publico While [he American public seem to have agreater acceptance ofme potentials of genetic engineering. in Emope the technology appears to amuse deep unease among many consumers. Consumers demonstrate coneern abour 'unknowo' health risks, possible deleterious effects on the envirooment and lhe ' ullllaturalness' oftransfening genes between uorelated spedes. Also for many people there is an inereasingconcern about me ever.growing influence of technology in their lives ando in sorne instances. an unjustified mistrustofscientists. While genetic engineering is aD irnmensely compLicated subject, not easily explained in lay terms, that does not mean that it must remain. in decision·making terms. only in tbe control of the scientist. indusrrialist or politician. A Royal Society report in 1985 on 'Public Understanding ofSdence·. finished with the following stateme.nt: 'Our moS[ direct and useful message must be to the scientists themselves -

PUSUC PERCEPTlON OF BIOTECHNOlOGY

learn to ~ommunicate with the publi ~. be willing to do so and consider ic your duty to do so!' Thel'e is no doubt tbat many ofthe public 01' consumers are interest.ed in the science of genetic engineering but are uoable ro understand me complexity of this subject. Furthermore. genetic engineering and its myriad ofimplic.ations musrnot be beyond debate. Public attitudes to genetic engineering will infiuence i15 evolu· tion and marketplact': applicatioDs.ltis importantfor public confidence for everyone to recognise (induding scientisu) thar a1l sd eoce is fallible - esperially complex biological sdences. All too often press aod TV repom on genetíc engineering present tbe dlscoverie!i as absolute ce..· tainties when this is rarely me case. Whatthen mustbedone toadvance public understandingofgenetic engineering in the cont.ext of biotechnology? What does the public oeed ro know aod how can (his be achieved to emure that rhe many undoubted benefits thar trus technology can bring to manlcind do not suffer the same fate as (he food irradiatíon debacle in the OK in rhe early 1990s? While garnma·irradiation offoods was demonstrated to be a safc and effldent method [O kill patbogenic bacteria, itwas not accepted by tbe lay publjc following the Chernobyl disaster, sincemostwere unable to differentiate: het'Neen me pTOCess of irradlatíon and radioactivity. Effcctive communication about tbe benefits and risks ofgenetic engineering will depend on understandjng the underJying concems of (he public together with any foreseeable technical risks. Over recentyeaI"S tbe..e bave beco many efforts made ro gauge the public awareness ofbiotecbnology by questionna.ires. Eurobarometers and Consensus Conferences. Early studies rughlighted public artitudes to!he application ofgenetic manipuladon to a wide range ofscenarlos (rabie 1.1). While medical applications were more generally a.cceprable others sucb as the manipulation of animal aod hmnan genomes were: highly unacceptable. Eurobarometer surveys revealed a broad spectrum of opinions t bat were influenced by na.t ionality, religion, knowledgeofthe subjec( aod how the technology will be applied (Box 1.1). A major contriburory factor is the plurality ofbeliefs and viewpoints thar are held explicitly or implicitly abau!. the moral and religious status of Natu ..e and what out relationshjp with it sbould be. Do we view Nature. in the. context of man's dependency on plants and animals, as perfect and complete derived by natural means ofreproduction and rherefore not to be taropered with by 'unnatura l' methods. or do we set': it as a source of raw material fu .. tbe benefit of mankind? For centuñes now man has beeo indirectly mampulating tbe genomes of plants and animals by guided matings primarily to enhance desired characteristics. In this way, food plants and animals bear little resemblance ro their predecessors. In essence, such cbanges have been driven by the needs a od demands of tbepublic or consumer, and have beeo readily accepted bythem. 1n the tradiriooal methods that have been used. tbe changes are made at tbe level of tbt': whole organism, selection is made for a desired phenotype and tbe genetic cbanges are often poorly characterised and QCcur together witb othe[: possibly undesired gent':tic changes. The oew

S

6

SMITH

Microbial production af bio-plastics Cell fusian ta improve crops Culing diseases such as cancer Extensron of shelf life oftomatoes Oeaning up ait slicks Oetoxifying Irtdu$trial waste Anti-b!ood-dotting enzymes produced by rats Medical research Making medicines Making crops to grow in the Third World Mastitis-resistant Ct::/INS by genetic modification of CO'HS Producing disease-resistant CIUps Chymosin production by micro-organisms Improving crop yields Using viruses to attack crop pests Improving milI< yields Cloning prize cattJe O1anging human physical appearance Producing hybrid animals Biological warfare

Comfortable

Neutral

Uncomfortable

91 81 71 71 65 65 65 59 57 54 52 46 43 39 23 22 72 45 4.5 1.9

6 10 17 11 20 10 14 13 26 25 16 29 30 31 26 30 18 9.5 12 2.7

3 10 9.5 19 13 13 11 15 13 19 31 23 17 29 49 47 72 84

82 95

PUBlIC PERCEPTION OF BIOTECHNOLOGY

m ethods. in contrasto enable genetic material (O be modified ilt the celo lular and m olecular level, are more precise and accurntc. and consequently produce beUer charactetistics and more predicrable results while still rctaining (be aims ofthe cJassical breeder. A great many sucb. cbanges can and will be done within species giving better and faster results tbanby traditional breedingmethods.

1.3

Regulatory requirements - safety of genetically engineered foods

Much debate is now taking place on rIle safety aod emital aspects of geneticalIy moditied organisms (GMOs) and their products destined for public consumption. Can such products with 'unnatural' gene changes lead to unforescen problems roc present and future gener.ations? The safetyofthehuman foorl su pply is ofcritical importance to most nations and all foods mould be fit forconsumption Le. not injurious to health or contaminated. When foods oc food ingredients are derived from GMOs tbey mustbe seen to be as safe as. orsafer tban, their tradi· rional counterparts. The concept oC subrtantial equivalence is widely applied in the detennination ofsafety by comparison with analogous CODVl!ntional roed products rogetherwitb intended use and exposure. \Vhen substantial equivalence can be shown then norrnaJly no further safety consideratiOIlS are necessary. \-Vhen substantial equivalence is DOl dearly established tbe points of differcnce murt be subjected lo fu.n:her scrutiny. When sucb novel produc{s aTe moving ioto the marl-..-etplace tbe con· sumer must be assul'ed oftheir qllality aod safety. Thus tbere must be tnxicological alld Jlutritiona l guidance in the cvolution ofnovel food s and ingredients to bighlight any potential risks whic:h can tben be dea lt: with appropriately. Safery assessmcnt of novel foods and food ingredients must satisf}r!he producer, the manufucturel'. Che legislator ana Che consumeroThe approach should b e in line witb accepted scientiJk considerations, the r:esuJts of the safet)' assessment muse be reprodu· cible a nd acceptable to the responsíble health authorities and tbc outcome mllstsarisfy una convince thc consuffier! A comprehensive regulatory framework is now in p lace within tbe EU with the aim to pr'otet:t human health and the environrnent from adverse activities involving GMOs. 'l'here a re two Directives providing horizontal controls i.e. (1) contained use and (2) delibera te release of GMOs. Thecontained use ofGMOs isregulated under theHealtb and Safety al Work Acr through the Genetically Modified Organisms (Contained Use) Regulations which a re administered by the Hea lth and Safety Executive (HSE) in the UK. The HSE receives advice from the Advisory Com.mittee 011 Genetic Modification. These Regulations, wbich implemen t Dh'eCtive 90/219/EEC. covet tbe use ofaU GMOs in contai llment and will incorporate GMOs used to produce food addirives oc processing aids. AH programmes must carry out detailed lisk assessmenrs witb

7

8

SM1TH

spedal emphasis 00 the organism thar is being modified aud the effect ofthe modificatioo. Anydeliberate release otGMOs into theenvironment is regulated io the UK by the Genetically Modified Organisms (Deliberate Release) Regulations, which are made under the Environmental Profection Aa and implement EC Directive 90{220IEC. Such regulations will cover the release into me environment ofGMOs foc experimental purposes (Le. field trials) and the marketing of GMOs. Current examples could inelude the growingofGM food crop planes orthe marketingofGM soya beans for food processing. AU ex:perimentall'elease trials must havegovernment apPfO\r.lI and the applicant must províde detailed assessment of the risk of harro ro human he.alth andjor the environrnent. AH applications and tbe risk assessmentsare scrotinised by the AdvisoryCommiU~ on Releases ioto the Environment which is largcly m .. de up otindependent experts-who tbeo advise tbe Ministers . The Ee Novel Foods Regulation (258/97) carne inro effect in May ]997 and reprcsents a mandatory EC-wide pre-market approval process forall novel foods. TIIe reguJatioll defines a nowl foad as one that has llot previously been coruumed ro a signific:mt degree within the EU. A partof their regulations will include foad containing or consistingofGMOs as defined in Directive 90/220and food produced byGMOs but notcontaining GMOs in the final producto In tbeUK the safetyof all novel foods including genetically modified foods is assessed by t be independent Advisory Committee on Novel Foods and Processes (ACNFP) wbkh has largely followed the approach developed by the WHO and OEDe in assessing the safety ofnovel food s. The ACNFP has encouraged openness in 311 ofits dcalings. pubUshing agendas. reports oi assessments and annual reports. a Newsle.tter and saoo a ComnlÍtree Website. By such means it llopes ro dispe1 any misgivings mat may be harboured by memben of the publk. The ultimare decisions are nor influenced by industrial pressurc and are based entirely on safety factors. Thereis undoubtedly going ro be a steady increase in the range ofGM foods coming to tbe market in the US and in Europe (rabIe 1.2). A comprehensive HU regulatory framework covering GMOs is now firrnly established and the specific legislarion now in force will ensure the safety ofCM foods. In aU ofthe foregoing. the risk assessments ofGMO products ere. have beeo made by experts and judged on the basis of safety to the consumer. However, ir must be recogni5ed thar subject experts define risk in a narrow technical way. whereas the publicor consumerwitboutsufficientknowledgegeneraUy displays a wider, more complex view ofrisk tbatincorporates vaJue-ladeo considerntions 5uch as unfamiliarity, cato astrophic patential and controllability. Furtbermore, the public, in general. will almost atways ove.restimate risks associated with technologkal hazards such as genetic engineering and underestimate risks associated with 'lifestyle' hazards such as driving cars. smoking. drinking, fatty foods etc. Ir is puz:.r:ling ro note that food-related technoJogies

PUBlIC PERCEPTION OF BIOTECHNOLOGY

C"'p

Trait

Company

Tomato Soya beans Tomato Potato Tomato

Modirted ripening Glyphosate tolerance Modified ripening Insect resistance Modifled ripening

Zeneca Plant Science Monsanto Ca, Monsanto·Co. Monsanto Ca. DNA Plant Technology

Cotton Tomato Squash Corton Oilseed rape

Bromoxynil tolerance Delayed ripening Vir1Js resistance Insect resistance Glyphosate tolerance

Calgene Calgene

Asgrow Seed Co.

Cotton Maize Oilseed rape Maize Oilseed rape

Glyphosphate tolerance Insect I"esistance High laurate - oil Glufosinate tolerance Glufosinate tolerance

Monsanto Co. Ciba Geigy Corp. Calgene Agrfvo Ine. Agrf vo Ine.

Maize Oílseed rape Maize Petato Maize

Male sterile Male sterileJfertility restorer Insed re5istance Insed resistance Insect resistance

Plant Genetic Systems Plarrt Genetic Systems Northrup King Monsanto Ca. Monsanto Ca.

Maize Cotton Maize Tomato Soy,

Insed resistance. glyphosate tolerance Sulpnonyiurea tolerance Glufosinate tolerance Modified ripening High aleic acid content of oH

Monsanto Ca. Du Pont Dekalb Genetic; Corp. Agritope Ine. Du Pont

Maize Maize

Herbicide tolerance Herbicidetolerance and ¡nsed resistance Herbicide tolerance and ¡nsed resistance Male sterile

Monsanta Ca. Monsanto Ca. Calgene BejoZaden

Cenon Chicory

rend to be perceived as high i.n risk relative to benefitwhen compare
Monsarlto Co. Monsanto Co.

9

10

SMfTH

1.4 I Labelling - how far should it gol Perbaps the most maten tiou! issue related to foods derived fro m genetic engineering is to what extent should chey be labelled. nlC' purpose oflabellinga food product is lO providesufficientinfonnation and advice. accurately and clearly, to allow consumers to select praducrs according ro their needs, to store and prepare them carreed)' and la consume tbeOl wiUl safety. With respeet to the principie oflabclling. infonnation should be accurate. t ruthful. suffidentlydctaiJed. not m isleading and above all understandable. Labelling ofa productwill only be relevane iftbe con sumer is ablt' to understand (he information printed 011 the labels. The Food a nd Drug Administration of tbe USA considers thar labelling should n othe based on the way a particular pl'oduct is obtained. Thisis, orshould be, a part ofno rmal approval for agricultural practice orindustrial processes. and j f approved. then JabeUing should be unnecessary. whkh i5 the COmIDon pTactice for rnost food products. lt c.lDbe argued th
D

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.'

'""

,:%! )

16

SMITH

ehain as theywould be anathema tovegetacians and especially vegans.

(4) Use oforganisms containing human genes as animal feed (e.g. micro-organismsmodifled ro produce pharmaceutical human proteins ruch as insulin). Clase eonsultation wjth a wide range ofre1igious fuims strongly suggested lhat there were no overwheJming objections to necessitate an absolute ban on tbe use of food prodUClS containing copy genes oC human origino(Ibis was particularly notlceable when the conce pt of the copy gene was understood.)The report, however. strongly advocared tha( the.insertion ofethicallysensitiwgenes into food organis ms bediscouraged espedally when alternative approaches were available. lf transgenic organisms conraining copy genes that are unacceptable to specific groups of che papulatioo rubject (O religious dierary restrictions were lO be presenr in sorne foods, ir should be rompulsory (hat rum foods were c1early labelled .

1.7 I Conclusions The safety and impact ofgenetically modified organisms continues ro be addressed by scientific research. Baste research into the natwe of genes, how Chey work and how they can be ttansferred between organisms has served to underpin the developm ent of rhe rechnology of genetic modificabon, [n tbis way. basic. informarion about the behaviOUT of genes and of GMOs will be builr up and used to address th.e concr:rns about the overall safety of GMOs and thciT impact on the environment.

1.8 I Further reading Frewer, L1. & ShqJherd, R. (1995). Ethical concems and risk perceptions associated wi t h different appllcationJ orgenetic engineering: intcTrelationship with the. pe~ved need for regulalion orlhe technology.Agric. Hu m. V
me eell. We can also distinguish between organisms which ca.rry out their metabolism aerobkally. using O] from the airo and those tbatare able to dothis anaerobically. thatis. witbourO r Theoverall reaction ofreduced carbon compounds witb 2 , to givewater and COl' is a highlyexothermic process: an aerobic organism can therefore balance a relatively smaller useofits substrates for catabolism ro sustain a gjven leve.! ofanaboLismo that ¡s, ofgrowth (see Fig. 2.1 a). Substrate tlOmsformatio ns for anaerobic organisms are essentiaUy disproport:i.onations, with a relatively low 'energy yield'. so tbat a larger proportion ofthe substrate has to be used eatabolicaJly to sustain a given level ofanabolism (Fig. 2.1b). The difference can be illustrated with an organism 5uch as yeast. Saccharomyces cereviSiae , wbicb is a facultative anaerobe - that is, ir can exist either aerobicaUy or anaerobically. Transforming glucase at the same rate, aerobic yeast gives COl' watfi and a relatively high yield of new yeast. whercas the yeaS! grown anaerobically has lawer yield of energy and reducing powet. Consequently. fewercells caDbe made tban under aerobic conditions. A1so it is nat possible (or the eeUs. in the abseneeof0l' (O ox.idise aIl the reducing power that is generated during catabolism oConsequentIy, sutplus earbon intermediares (in the case of . yeast it is pyruvic acid) are redueed in arder to recyde the reductants '(see Fig. 2.2) and, in thecast oryeast. cthanol is the product. Overall. this process can be described by the simple reaction:

°

X+ NAOH -+Xl4 + NAD't'

Reduced carbon metabolite o r H20

I C.. '~n liubstrntc ICmHnOpNQ) Nitroge" source

INH3)

K'!PO:-IMgl'I

SOl·tetc. 020r surplus carbon metabolites

HU

Thermodyl'wnk IMbnce

19

20

RATLEDGE

wb~re X ¡S3

metaboliteand NADH is tbereductant, and NAD·· is its oxid¡sed form (see Fig. 2.3a, bJ. NAO stands for nicotillamide ade.nine di· nucleotide; NADH is therefore reduced NAD. There is abo the phosphorylated form of NAD+, NAD phos:phate designated as NADP+. TltiJ; can aIso be reduced lO NADPH and it can also functiou as a reduc· tant but u sually in anabolic reactions of me cell, wbereas NADH is usually involved in the degradative reactLoos. All four forros (NAD+, NAOP". NADH and NAOPH) occur in both aerobic as well as anaerobic ceUs: in tbe former (ells re-oxidation of NADH can ocror with 0 2' but mis cannor take place in the a naerobe, hence the need for the altemative re-oxidation scrategy (see Section 2.6). A ce:U that grows obviouslyuses carbon but manyother elemen ts are needed to make up the final composition ofthe cell. These will inelude nitrogen, oxygen - wbich may come from the air if the organism is growing aerobically (otberwise the 0 2 musí come from a rearrangement oflhe molecules in which the organism is growing, or e.ven water ilSclf) - togetherwitb other elemellls sucb as r. Mg H , S {as SOr). Pías PO! - ) and an array of minor ions S"uch as Fe H , Zn H , Mn1-t , etc. The: dynamics ofthe sysrem are set out in Fig. 2.2.

2.2.2 Catabolism and energy

(a) NAD '· /NADP+ (olddlsed): (b) NADH / NADPH (rrduc:e d). ln NAD ~ ~nd NADH, R = H: In NADf>'" and NAOPH,

R=POI-·

The necessary linkage between catabolism and anabolism depends upon making t he catabolic processes 'drive' the syn.t hesis of reactive reagents, few in numbC!r, whicb in tum are used to 'drive' the full range of anabolic reactions. These key intermediates, of which the most important is adenosine triphospbare, ATP (Fig. 2.4), have what biologists t/!'TIU a 'high-energy bond': inATP itis the anhydride linkage in tbe pyrophospbate residue. Directly or indirectly the potential energy released by splitting chis bond is used fur the bond.forming steps in ana.bolic 5yn· t heses. Molecules such as ATP then provide me 'energycurrency' ofme cell . Wben ATP is used in a biosynthetic reaction ¡tgenerates ADP{adenosine diphosphare) or occasionally AMP (adenosine monophosphate) as the hydro1ysis product: A+B +ATP~AB +AD P + Pt

or A+B+ATP -+ AB+AMP+PP¡

(where A and B are botll carbon metabolites of the ceH and Pi'" morganic pnosphate, and pPJ "" inorganic P}'rophosphate). ADP, whlch still possesses a 'hjgh-energy bond', can aIso be used to produce ATP by the adenylate kinase reaction: ADP + ADP-+ ATP + AMP Phosphorylation reactions. which are very (ommon in living ceUs, usually occur through the mediation of ATP:

o 11

- e -OH + Al'P -+-C - O-P - OH + ADP

I

I

I OH

GROWTH ANO METABOUSM

The phosphorylated product is usual1y more reactive (in arre of sevual ways) than the original compourrd.

2.3 I Catabolic pathways 2.3.1 General considerations of glucose degradation

,l

, ,~

f ¡ t

The purpose of breaking down a substr.lte is to provide the microOIganiSDlS with: • building units fur the synthesis of new cells: • energy. prinopaUy in the forro ofATE, by which to syntbesise new bon4,s,and newcompounds: • reducingpower, which is main1y as reduced NAD (i.e. NADH) or reduce
DNA)

RNA -

ATP ) ____

Nucleotides.

daoxynudootides,

¡

con .... .,/"'otc.

""''''''0''.''- Polysac:charide$ ¿"

---p",to,,~'P'

histldine

Phenylalanlno. Iyrosine,

lipids

~-- S";nÓ- --'G"do,, , :-Cysteine. mothionine

NAD. etc. -tryptophan, p-hydroxyoonroate

quinooes

.

Purines,

pyrimldinE

Porphyrim tnc.

p"amlnobenzoale,

Aespiratory .........

slora~

Stori\ge lipida

Glyeerol - ~Membrane

ADP

Folle acld _

..............

/

= =

= =::AJanlne

Va line, ¡eucine

= =: : Fatty &Cids. lipids, PHB, polyketldes

Mey¡¡lonato, !taroid!, carotenoids

Glut8mate, glutamine- Argini prolin,

l

Folie acid

Anabalic P'othway, (synmesls) and lhe centnl atJ.bo~c ~th-rs. Only me main DIo5)'flrhu15 rouUt~, and tlIelr maln COOlllCDOf\t with o.aboVc

Pilthways:are $hown, aY 11'1 h\gJIly slmplifled ~ions. ConnKtJgns through 'energy' (ATP) ilnd 'redox' (NAO·, NADP ') metaboVsm a!"ld throu¡h die metabo~.\Ill cA nitrogen, etc., are al omltted. (PHB

=pcty.,8-r.yd,.~j)(ybutyra~; p= phos.pooVOUp). The nIM principal pr«ursors ~ in the lihade
and tetrose (Col) phosphates. This is the pentase pbosphate patb sometirnes referred te as a 'shunf or as the hexost' monophosp pathway (see fig. 2.7). Thepurpose ofthis pathway is rwo-fold: to PI"( C~ and C4 unirs for biosynthesis (see Hg. 2.5) a nd .1.lso to provide NA furbiosyntheris. Although the EMP pathway and the pentose phosphare (PP) patl botb use glucose 6-phosphate. the extent to which each mute ope. depends Lugelyon what the cell is doing. During tlle most active ! ofceD growth. botb patbways operate in the approximare ratio 2: tbe EMP pathway over tbe PP pathway. Howev&, as growth sJows d the biosynthetic capacity of the cel1 also slQV.Is down and less N,Il and C5 and C4 sugarphospbat"es are needed so tbat tbe ratio betwee pathways nowmoves ro 10:1 or even to 20:1. Ir is therefore apparent mat metabolic pathways are control systems capable ofconsiderable refinement to meet tbe eh anging 1 ofthe eell. '!ltis is discussed lacu (see Section 2.8). Although lhe EMP and PP pathways are found in mon n organisms, a few bacteria have an alternative pathway to the fe patbway. This is the Enrner-Doudoroff pathway (see Fig. 2.8) ... occurs in pseudomonads and re1ated bacteria. Tbe pentose phos· pathway though soll operares in these bacteria as the En Doudoroffpathway does notgenerate C, and C4 pbosphat.es.


2.3.2 The tricarboxylic acid cycle lbe. degradation of glucose. by whatever route or routes. invariably leads to the formation ofpyruvic acid: CH3 .CO.COOH. The fate ofpyruvate is diffe:rent in aerobic organisms aud anaerobic ones. ln aerobic sys~ms. pyruvate is decarboxylated (i.e. loses C0 2J and i5 simultaneously activated in r.he chemical sense. to acetylcocnzymeA (abbreviated as acetyl-CoA) in a complex reaction also involving NAD+: pyruvate+ CoA + NAD+ ---t acetyl-CoA +C0 2 + NADH This reaction is cataIysed bYPYf1lvate ddtydrogtttast'. (lbe fate ofpyruvate

in anaerobic cells is described latero) Acel:yl-CoA. by virrue ofit being a thioester. is highIy reactive. It is capable oC generating a large number of intermediates but iu principal though Dot sale tate is to be progressively oxidised through a cyelic series ofrcactions knOWD as the citrJc acid cyde. TIris is also known as the tricarboxyUc acid cyde or me Krebs cycle after its discoverer. The reacrions ofthe amc acid cyde are shown in Fig. 2.9. This cyele fulfils two essentiaJ functions: • it provides key intennediates fur biosynthetic reactions (sec Fig. 2.5), principal ofwhich are 2-oxoglutarate (to make glutamate and r.henceglutamine, arginine and Qroline), succinate (to make porphyrins) and QXaloacetl'l.te (to make aspartate and the aspartate famUy of amino acids - see Chapter 13): • to produce energy from me complete oxidationofaceryl-CoA to COz and Hp. (1bis process is desmbed in detall in Scction 2.5.) However tbe citric acid cycle canDOt fulfil either funcD oll exdusively: ifintermediates are removed fur biosynthesis. then sorne energy production must be sacrificed: if all the acelyl-COA is oxidised to CO 2 and RzO there wUl be no intermediates left for biosynr.hesis. Consequently. the cycle runs as a balance between the two objectives. Pyruvate, commg from glucose, provides the input and the cyele p~ vides [he output in fue way of energy and biosynthetic precursors (see Fig. 2.10). In meeting its twin objectives. the cyc1e cannot entirely replenish the ¡uitial oxaloacetate [hat is needed as a priming reactant tO makecitrate as sorne ofthe inrermediates must inevitably be used for biosynthetic purposes.. (lftheywerenotsoused. there would be no poillt in the cydejust producingenergy as this could nor then be used in any

Glucose

ATP

(i)

f= AOP

Glucosa &·phosph8te

®! Fructo68 6·phosphate

®

ATP f= AOP

Fructose 1.6-bisphosphate

~

1

1

®

OHA-P...- G3-P

P'yNAD ®f--NADH l,3-dlphospho-glycerate

F=

ADP

(!)

' - -ATP

3-pllosphog Ivcerate

® .

The

Embden--MlI)'erhof-Pa~ p;ithw~y

of IIy~ysb. O~nll:

2·phosphoglycerate

Glueose + 2 NAD + + 2 ADP + 2P, ..... 2 p)'T1JVlte + 2 NADH + lATP TIlo reacrions In! catalysed by:

(1) hexokinase, (2) gIucosM~bate iromerze, (3) pho$phofructokln:ue. (-4) aldolale. (5) ulose phosphate l5omerue, (6) gtrceraldehyde--J-phOspllate dehyd~ •. (7) 3-phc~glycerate kio
®! PhO'PhO'"f=o IPY'O:::: @ Pyruvate

ATP

2

24

RATlfOGE

Al?

NADP~

AOP

GIllCOse-"""-)~,

01'00"

NAOPH

5-P U6-P

NAOP' NAOPH

.,",on.,,~ RIb"o," '-P

(1)

EPi:'~'1 \ om."w Ribose 5-P

Xylulose 5-P

T'' ' ' '/{

Glycor¡"ldehyde 3-P

Sedoheptulose 7-P

T"n"/dO/'~ Fructose6-P

'--------c,

e,

Erythrose 4-P

TrlJnsketofaso

fructose 6-P

Glycereldehyde 3-P

Ttw. pentoH pho~te cyde (toexose monophosphue slwnt). Th~ number.d emymes are; (1) &lucose.fo-pho5phate dehyG'osenas., (lJ pl'losphacluconaa dehyclrogenue.lr'I$ft; summar)' ~.nx stoldliometry wnen fruCWs. 6-pho~le b recyded to IkicO$e 6-phosphale by:llll,~; glyeeraldehyde l-pnosphale an Ño be reqclcd by revur'Se glycolysis (Fl¡. 16). WIth MI r~fin(the pathway function5 as a llenM'UOf"of NAOPH. bUt!he ,~nsaJdolaH and tnnIb!olue roctions abo permlt sugat Illten:onvel'$im'15 whlch are us~ in o!her ways.

Ne' reodkwl: glucose+ATP+ lo NAQP+ .... gtycenldehyde 3-P+ADP+ 6 NAOPH

(Afrt ",maYal of~ and C. ,usarE lar bIosyntheril wllI dimJnhh me nqdiflg?fOCHJ and thus!he yield oif NAOPH wiJI 6ecntase..) sensible manner as tbere can be no biosynthesis witbout precursors.) It is therefore essenti ... 1for t here to be a seoond pathway by which oxaloacetate can be formed a nd this arises principally by fue carboxylation of pyruvate: pyruvate+ COl + ATP -+ oxaloacetate + ADP+ PI 'Ibis reaction is carne
NADH ATP

ADP + PI

~HsOH~ CI\CHO~CH3COO-~ aceryl-CoA ethanol

acetaldehyde (ethanal)

acetic acid

CoA

1he manner in which acetate units are converted to C4 compounds 5!i::nawn as the glyoxylate by-pass (see Fig. 2.12) fur which tw'O enzymes

aiditional ro tbose ofthe tricarboxylic acid cycle are needed: isocitrate: f!est and ma!ate synthast. The former enzyme cleaves isocitrate mto succli:!are and glyoxylate. The latter enzyme rhen uses a second acetyl-COA if)zdd te the gIyoxylate to give malate. 80th theseenzymesare ' induced' (chal is they are synthesised ooly wben the spedfic signal is given - see Sel:rion 2.8.4) when micro-organisms are grown on el compounds . The a::rivity ofborb e nzymesincreases by sorne 20 to 50 times under such growth conditions. The glyoxylate by-pass docs nor supplant the operatil:moftbe tricarboxyLic acid cyde; fer example 2-oxoglutarate will stUI iEre ro be produced (from isocitrate) in arder to supply glutamate for ~ syntbesis etc. Succin;¡¡te. the oeber producr from isocitl'ate Iyase. __:JI be metabolised as before toyield maJa te, and tbence oxaJoacetate. l31!3 tbrough the Eeactions oftbe glyoxaJate cyde, the C4 compounds Gil now be produced frem C units and are tben available fur synthesis l fIÉ an ceU metabolites (see Fig. 2.5). Their convenion into sugars is *'t:;¡¡jJed in Section 2.4 .

2..3.-4 Carbon sources other than glucose ~compound

that is used by a micro-organism and can feed ioto any afme intermediates of glycolysis. 01' even the citric acid cyde. can be Simdled by the orgaaism witb its existing complement of enzymes. 6I:M~ a great many other substrares can be handled by micro~ms . 1n other words. aU natural compounds are capable ofdegra.eation and me majority of this degradative capacity is found in iiii!f::robia1 systems. The application ofmicro-org.misms as 'waste dispos;¡! units' is therefore paramounr aDd this activity forms aD intrlnsic

Glucose

f==m ADP

Glucose 6-phos phale

f==NAD P

NADPH 6-phosphogluconate

G)f'-H~ 2-tet0-3-deo)fy-6-phDSl)l1ogluconale

n

Gr

l____...... Pyluvate Entner--Doudoroff

pathwly. Thls rometimes repbcl$ ttH! Embd~n--Meyerhof--Paml$

pathway (see Rg. 2.6) in sorne psaudomonads an d re!¡ued baaeri.l. Numbered enzyrna 11,..: (1) pllruphoglucon:lte ~dntDlR. (2) 11 speclflC .!doIase. Gtycenldehyde J-pho:\'~ (G3p) b c.onverted to pyruvate by ÜMI re:levant enzymes- gfven In Al. 2.6.

o(

Z

O

..:

O-

()

Ue, (6) weclnatl! thiolmase, (7) succinate dehydn),ena$(!, (8) fumara se. (9) rmbte dehyd~nue.

3AOP + 3P,

3ATP

(oKphos)

Oiagr;mmaric preunutioll 01 1M du.al role ~ el IIIfI U"h:.lrt>o~ytic add cyde: to prod!.Jtl! Intl!rmedllte~ lnd Me!X)' (ATP).

Citrie aeid cyele

GROWTH ANO METABOclSH

pan of cnvironmeo ral biotedtnology which is {'xpJaineu in detall in OJ.apter24 . To illustrate this diversity, the exa.mple of microbial degradation of fanyacids will be collsidered. The ability ofmicro.arg;misms to growon oils and fats is widespread. The difference betweeo 3D Di! and a fue is wherher one is liquid or salid at ambient temperatures: mey are both chemically the same, that is chey are fatty acyl triesters of glyceroL:

CH,OH I CHOH I CH,OH

CH,O.OC{CH,I.-CH, I

giycerol

triacylglycerol

wbere o. m and p are rypically 14 or 16: me long alkyl chain maybe saturated as indicated ormay b.aw ooeor more double bonds givingunsat· unted. ur polyun satttrated . fauy aeyl groups. TIle oils, when added to microbiaJ cull'ures, are initially hydrolysed by aUpase enzyme into ils constituent f.J.tty acids and glycerol. The latter is then metaboLised byconversioo lO gIycel'3Jde hyde 3·phosphate (ree Fig_ 2.6). The fany acids are raken iota the ceU aod immediately converted into their coenzyme A thioesters. The fatty acyl-CoA esters are degraded in a cyclic sequcnce of reactious (sce Fig. 2.13) in which the

Acetyl-CoA

($Ce A¡. 2. 10), are ( 1) isocltr.ate

¡

trasO! and (2) malatB synthue. ~

Isocitral6

~S""i"," 1

Glyoxylate

'umm"

Malate ~

¡

Oxaloacelate _ _ ATP

--J.

0

Hcw ceH ensures equal wpplies of onIoacetaU (OAA) and acetyl-CoA (AcCoA) for citric acld biosynt~esls . Tht activily of pyruvate urboxytUt (2) Is nimubttd by acetyl-CoA fOl'med by ~1"UY2[e ¿dYydrogen:ue ( 1)

The ¡Iyoxylate by-pm.. Th e acklitlonal reactloos. beyond tho~ 01 me trlarboxrl;( acl¿ cyde

Citrato

~j®

y

• Acetyl-CoA

tne

I Cf!,O.OCíCH,I,-CH,

I

~

Oxaloacetatll

Citrato

CHO.OC-{ CH2)m-C~

1Acotyl·CoA

Pyru vate

ca,

Aop .-/l - Phospnonnolpyruvate

~

_ _ ...J

~cheme ¡ llO shows oow che bypass functions lO permit rugar formatlon from acetyl-CoA. wim lhe ad~ ruetlo n (1) pOO~hoeno lpyruvat.e

arboxykl nne. folowed by nlVemd IIyc~s (d. Fi,. 2. 1'1).

27

28- 1_ RATlfOGE " ._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

IRCH,.CH¡.CODH)

,8-0xldaticll\ cyde of

f8try acid

"., K"~:oA "~r\.. En:r:yom.s are:

wn ZJ crotona¡e): (1) 3.hydroxyac:y\-

ATP= t m H-S-COA

G)

AOP

+

HR NADPH + 3ADP + 3P¡ + ~z -. NADP~' + 3ATP + H;eQ

NADH + 3ADP + 3Pi + ~Ol -. NAO I + 3ATP + H20 FADH+2ADP+2P¡+ YzO~-t FAD + 2ATP + HP

w

phosphoenolpyruY:ite by

phosphoenl>lpyruva« car-boxykinase (2). nls i$ ~rted by che rtvened glycolytic sequence ef erlZ)'me< (l)

¡ntofructD~ 1.6-blsphos phate (see

abo F"1g. 2.6) vffljdl is therJ hydroly,ed -...fth me n!leas. of

lno rganic ph05pN te (P¡) by fructes",-I .6-bisphospha~1 e

number of ro-factors (NAO+, NADP-¡'. FAD) produdng the correspon ing reduced forms (NADH. NADPH and PADHz). The reducing power I these products is released by a complex reaction seque.nce which, i aerobic systems, is linleed eventually to reduction of aonospheric e This pro
Emyme

Reaction cataJysed

OCCUITeOCe

l . Phosphoglycerol kinase

I ,3-bisphosphoglycerate + ADP ~ 3.phosphogly<erate + ATP

Wldespread, 5ee Fig. 26

2 Pyruvate kioase

phosphoenolpyruvate + AOP -Jo pyruvate + ATP

Widespread. see Flg. 2.6

3. Acetate kjnase

acetyl phosphate + ADP ~ acetate + ATP

Widespread

4. Butyrate kinase

but)ryt phosphate + ADP ~ butyrate + ATP

E.g. ef1terobacteria on allantoin

5. Carbamate kinase

carbarnoyl phosphate + ADP -Jo carbamate + ATP

E.g. dostridia en arginine

6. Formyl-tetr.Jhydrofolate synthetase

N,o.rormyl-H 4 folate + ADP + p¡ -Jo formate + H4 rolate + ATP

E.g. clostridia on xanthine

RATLEDGE '.._34_.L...... _ _ __ __ __

_

_ __

_

_

_ __

_

the accumul ation of.lactic acid i n musde fusue during byper-activity of an athlete. In micro-organisms we can see a whole range of redueed carOOn compounds being accumulated. by organisms growing allacrot>. ically. Examples may abo inelude lactie acid itself(produced by laetic bacteria), but would ¡nelude short ehain fatty acids such as butyric or propioruc acids, and aleohoIs such as butano!, propano! 3nd ethano! (see Fig. 2.17).Some orga.rnsms, such as me methanogenic.bacteria, may go even further and produce, as the comple:tely reduced end-product. methane (notshown in Fig. 2.17). It is important ro poinr out that pyruvate. produced by che glycolytíc pathway (Fig. 2.6). will still enter the tricarboxylic acid cyclewhich muse continue. aC lean in part ro provideessential precursors furbiosynthesiso pnnopally 2-oxoglutarate. and oxaloacerate. but not to p roduce energy. The NADH produced in the cyde cannor be converted inro ATP as the cells have no supply ofO~ to drive oxidative phosphorylarion; however in certain bacteria there are terminal electron acceptors other than 0 2 which are capable of being eou pled m to the E1l' system (Fig. 2.15) and which will allow the fOl'mat'ion of ATP ro take place. This indudes microbes tbat can use nitrate (which is reduced to nirrite). nitrite (rccluced to NH~ or in sorne cases N 2 in a proeessJcnown as denitriftcation), COl (reduced to merhane by metbanogens) or su1phate (reduced to HlS by rulphare reducing bacteria) as alternatives [O 01. ln all cases. although theyield of A'IPis less than occurs in aerobic systems. ir is muc.h greater than obtained by substrate-Ievel phosphorylation alonc.

2.6.2 Products of anaerobic metabolism Figure 2_17 summarises sorne ofthe maln reactioos leading to the for-

mation ofreduccd end-products in anaerobic micro-organisms. The major products are: • glycerol. produced byyeasts when the conversion ofpyruvate to ethanol is bloeked; • lactic add, forrned by lactic acid bacteria; • formic acid, formelnd sometimes purines. Metbane(notshown in Fig. 2.17) is pcrhaps me ultimare redueed ca.l·bon compound and is produeed by highIy specialised An:hatMcteria by eleavage ofacelate to ca} and CH~ or in sorne cases by red.uction ofCO, .

GRDWTH AND METABOLlSM

2Py'+"'HE-_L_ _""''''''''''''' .-'!-

OKaloacetate _ _ _

IR

~1t~r-Su CcinYI-COA

¡x, Acetoacetyl-CoA

P(O PjOL,-CoA

- - --1 R

" '- - - CoA, CO~ Butyryl·COA

/

~";~""a:.""'i!1il""j,, " ""~·

jR

1!íiU~1

memanal (CH30H). etbana l ( C~CH 10H) 01" fonnic acid {HCOOH) a1l in me presence of~ gas.

2.7

I Biosynthesis

The provision of energy (AIP). re ducing power (NADH and NADPH) and ;a variety of monomeric precursors (see Fig. 2.5) from the degradation of .2 substrate provides the cell with lhe neeessary means oC regenerating iiself. The cel! undertakes roe biosynthesis oCtbe macromolerules of me cell~ nudeic acids (DNA and RNA). proteins (for enzymes and other functions).lípids for membr.mes and polysaccbarides as eomponents of me ce11 envclope. from fuese simple buiJding blocks. As rnanyofthe bio-syntbetic patbways are eOYere
"' -

MicrubAlgrowth: prlm;¡ryand 5K01ldary mt'tJboIic

I

tropoPhase,~·-tI~·~--i dloPha se ----;o

...... , ..

I

,,

Iomass

I

I

, ~

I I

limit ing'\ nutrient ...

"

B~ .ot

,

E

...-

I \

I

\

I

."

.../--

,

......

............

phases. ln the initii!1 pha~e (babnced growili=~) all nutrMnU are kl e~ When one nutrMAt (not arlJon) G consumed (- - ) cell gvwth ( - -) s~ down ~d J«ondilry mr.aboite(5) (- - - - _o) are fol'm4td in the idiophue.

./ secondar y ./ metabolite

.../ "

\ I !' '.1 /" '. ./

t ime

Pyruvate

Formadon of !.eConebry metabolil$! from ¡cet}i-

~co,

CoA. The .$eCOIIcDry mnabclites are showo tri lhe slladed ttxts.

There can be considl!rab!. onl"latlon In s uu~ of sorne of mese mllltabollte5.

x3

Mevalonate (Cd

~co, Isopren e units (es)

l~

----1ij~. Mi~ ií·!i~

38

RAnEDGE

2.8 I Control of metabolic processes 2,8.1 Metabolic flux

NewDNA

Rapl~tlVI1

DNA

Tmnscnpt"on ma g ene Mosse oger RNA (mA NA)

Atlach ea 10 11

ric.osome mRNA .. rlbo.ome

Tra"s/a¡IOtI 01 mfl.NA

(ccde. for ,m¡!'Io acids 10 be .clded logetlteo- In conlKl

sequ~)

PnMein I IduHsed db.gn.m .$howing how DNA C\n elther be

repliuted to "", nI!'" DNA (for new ce l! synthesls) or be ¡ran¡tribed Into meUflnxer P.NA (mRNA) chal is decoelld (or tTatlw.ted) by It becomllll 3.ttached ro a ribotome whlch moo ITlilkes ¡¡ proteln rnolecule by 1.qoentiat .addition or amino aelc:fs. Thu$ lbe original sequen« oí b,u as ¡ long

the ONA tlhe gene)" flrst conven:ed to a correspcndlng sequence ofbau~, (mRNA) mat ¡¡:/YQS r!se f.O

a I'\(!W proteln; see also

Ch;¡pter 4, Flg. 4.1.

The concept of metabolic flux (or flow) has been developed th¡ attempts to describe iIunatbematical (ertnS the1
2.8,2 Nucrienc uptake

Control of cell metabolism begins by th e cel! regulating its uptake ( nutrients. MeS! nutrients, apart" from oxygen ¡¡nd a very few carho compounds, are {ak.eo up by specific:transport mechanisms so that tht maybeeoneen tra ted within the cell from dUute sol utionsoutside. Suc 'active' transport systems require an input ofenergy. The processes al controllable so that o nce the amount of nutnent tak.en into the cel] he reached a given concentratioD. further unnecessary (or even demmel tal) uptake can be stopped. (This is abo discussed in Sectioo 2.8.5 o catabalite repression.) In sorne cases the rate atwhich a earoon sourel


such as glucose.is takenup inta the cell may be the limiting process for growth oftbewhole cell and themore should receive particular attenrion when evaluating potential bottle-necks to increased productivity ofa bioprocess.

2.8.3 Compartmentalism A simple form of metabolic control is the use of compartments, or

organelles, within the ccll wherein separate pools ofmetabolites can be roaintained. An obvious example is the mitochondrion of tbe eukaryodc ce.U which separates (amongstothers) the tricarbaxyl:ic acid cycle reactions from reactians in the cytoplasm. Anothe.r would be the biosynthesis of tatty acids which occurs in tbe cyroplasm of eukaryotic cells whereas the degradanon offatty acids (see Fig. 2. l3) ocrurs in the peroxisome organeUe. Separating [be two sets ofenzymes prevents any (aroman interDlediate being recyded in a futile manner. Qther organeUes (vacuoles, the nudeus, peroxisomes, etc.) are simHarly used to control other reacrions ofthe cen. Bacteria, bowever. do Dot have such compartments witbin tbeir celIs and therefore must rely on other means of metabolk control.

2_8A Control of enzyme synthesis Many enzymes within 3 ceU are present constitutively; that is. they are there under al1 growth conditioI1.'l. Other enzymes only 'appear' when nl'eded : e.g. isocitrate lyase aftbe glyoxylate by-pass (see Fig. 2.12) wben the cell grows on a C; substrate. This is termoo induction of enzyme synthesis. Conversely enzymes can 'disappear' w hen they are no looger requieed: for example, enzymes for histidine biosyntltesis stop being produced ifthere is sufflciem externa] histidine avaiJable lO satisfy the needs ofthe ccl1. 'Ihis is (emted reprt'ssion: when thegraruüous supplyofthe compound has gone, the enzymes for syntbesis of the material 'reappear' ; their synlhesis is de-repressed. The key to both induction and repressian is that the genes codingfor the synthesis ofthe proteíns bythe processes of transcription (see Fig. 2.20) are either switched on (indu etion) or off(repl'ession) according to the metabolite~ present (or absent) in the cell. These processes are shown diagrammatically in Fig. 2.21.

2.8.5 Catabolic repression This type ofmetabolic control is an extensioo ofthe ideas already set out with respect to enzyme ¡nduchon and repression, being brought ahaut by external nutnenlS added (Q tbe microbial culture. 'Ole term catabolite repression refers 10 seveJ.;:¡ ] general phenomena seen, for example. wben a micro-organism is able to select, from MO or more different carbon sources simulraneously presented to it, tbat substrate which it (Irefers lO utilise. For example, a micro-organism presented with both glucose and lactose may ignore the lactase until it has con· sumed all the glucose. This scquential utilisation oftwo substrates is referred ro as diauxic growtb. Simila r selection may cernr ror the choice ofa nitrogen source ifmorc than one is availablc. The advantage ro tbe cell is tbat it can use the compound which provides it with

)9

RATLEDGE

10

¡a)

DNA

~

~

2.

Transcriptlon

o· 00 • •

DNA

lb)

e

2. • e

J\JV' mRNA

~

Transcription

o· ¡¡D

JV\I" mRNA

!

~

Translatton

¡TmM"t'"" ~

$

2inding

•

~

O

z

O ~ w ~ O

~

• O

Proteio

~

••

•, o

¡;

"2 lE

1ha attached protei"

~tiv e

No binding 10 ONA. possible

StrucUlrlll genes IlOt exprossad as signal from the operator geno Is not given dUlI 10 the presence of

Oparator

'Switch 00' signal

Z

O ~ w ~ O

••e •

~

" , ~

. ~j

J\f\J' m"NA, J\f\J' mRNA ~ JV\F" mANAl

1

Proteína produced

lE

Con1rol of enzylT\ol! s)'TlI:htik thl'Ol.lgh re,gul;atlon of DNA I!Kpression. (a> Represslon: In the absence of any Indudng molecuhl the mes$t1'lser RNA (mRNA) from the reJUlatory gelMl produces a pl'O'te lo tN.t binds te an 'oper.Itor' ¡ _ funher down dl4t

ONA mo!ecllle. As a resultofthis binding. tM

openuor gene ¡~

Inact/vated ~nd no signal is given te allow the strUcwl'lll genes (tlut

WCluld makllcdve enzymes) te be eXprased. (b) InductiO!1: in me presellCe

ot ~n Induclrlg molecule.

tlw. protein arlslllffrom me "'g"blo')' , _ 1, now no 'on&er able ce bllld te> d'M>~r g~. Consequently, me openlotor 'switche,' Qfl the 1171.JCt:UraJ cenes and active prou:ins (enI)'mes) are

now made.

the most useful substrate ror production of energy aod provision of metabolites. The mechanisms by which catabolite repression is acl1ieved varies from organism to organismoA simple caseis with E. colt where control is exerted via 3n effector moLecule, cyclic AMP (cAMP). (lo cAMP (he single phospho groupofAMP-see Fig. 2,4 - bridges across IToro the 3' -hydroxy group ofnoose to tbe 5'·hydroxy group, tbereby forming acydicdiphosphoester.) cAMP interacts with a specific protein. catabolite activator protein(CAP;! (also known as the CRP "" catabolite receptorprotcin), and the cAMP-CAP comple>: binds to DNA causing the genes that follow afier (ordownstream of) the binding site to be tl'anscribed (see Ag. 2.22). Tbese genes may tben be used to syntbesise new proteins for uptake and metabolism ofthe next substrate (e.g. lactose if [he cells aregrowing on a glucoseflactose mixture).1his positive system ofgenetic conl:l'ol is the reverseofthe negativecoDl:I'ol systern described in Fig. 2.21. The key molerule is Lherefore cAMP. As long as gluoose or its catabolites are present, cAMP is not formed as ilS syntbesising enzyrne (adeny· 1a1'e cydase) is inhibited by these catabolites and thtlS Jactase uptake and metabolism cannotoccur. The catabolites therefore repress the syn· tbesis ofnewenzymes. Thc repression is removed when the catabolites disappear- i.e. a11 the gtucose has been consumcd.

2.8.6 Modification o( enzyme activiey Once an enzyme has been syntbesised, its activity can be modulated by a variety ofmeans.

GROWTH AND HETABOlISM

ONA

Catabo!lI:. rep!'Msion.

•e • D

CAP mRNA

j

~

Inhlblted by gJucose m81aboJites

CAP

e

~ cAMP --- ATP

AM:M~ ~

~

_~" _ v_V_V

"" mRNA') Proteins

~ mRNA, -~mRNAa

Th. stn.I(tur;a1 gen6anl therefore 'swI~

off' (I.e .• ~mud);u Ion¡ as ~a o r lu c:ar:aboliles anI pruefll. Sevenl operoru. may respond 10 the cAt1P-CAP slf:naL

) .

Th. mech;r,nism lhown ís medj¡ted by cyOlC AMP (cAMP). An operon Is 3.2.2 Measuring yields It: is stressed that stoicbiometric yield coeffidents ;¡re ratios of conver·

sionrates (rx is given as e·mol X m- 3 reactor per h. ti in mol i m - 3 peth).

"

y . {)( ri

(3.1)

These tates are ca1culated fram measurements in experirnents which may be either batch, cantinuous or fed batch cultures. using correct mass balances. TIte mose frequentIy rncasured gtowth stoichiometric coeffident is the bíomass yield on substrate (or e1ectron donor ) Ysx (or Ypx)' In a constant volurne batch culture (O indicating tUne O), y ~ will be: (3.2a)

In a chemostat. where inputand o utflow cates are equa!, a similar equa· tion holds . wher-e ~ = Oand ~ is r-eplaced by the concentratioo ~ of the incoming substrilte. If volume variations Decur. more complex relations can be derived from rhe mass balances.Forbatch reactorswith variable volume V. WE':get:

(3.lb)

3.2.3 Maintenance effect lnitiaUy Ysr was inteoouced by Monod as a constant. However aftee the introduction of the chemostat, cultivanon of micro-organisrns undE':t diffe.rent growtb cates showed thar Ysx was dependent 00 the specific growth rate Jl.. The.proposed explanatían is basE':d on the maineenance concept (Herbert-Pict). ln this concept it is assumed that maintenaace of cellular functioIlS requires thE': expenditure of Gibbs E': nergy (ror restoring leaky gradienrs, protein degradation. erc). 1his Gibbs energy is produced by the catabolism ofa certain amountofelectron donor (= substrate). lfthis maintenance tate is ms or Irry e·mol substrate per C-mol X'h, the following equation holds 1 1 m,

- =~ +-

Ysx

Gene ... 1stoich lornetrlc

nlpresenQtion of t he fonn3 don of

ma.:

JI.

(3.3)

unknown stoichlometrlt compounds.

48

HEIJNEN

Dependtrn:e of biomass yleId Yu on sptelfic ¡rowth r.J.te (maintenance efl'ect), m,ls me

Y"

t

substnte maWltenill10Ct coefficlent.

r;-1s the mu.ima.1 biomus ylt!ld

0.5 Ys:;"

on sulmnote.

ExperimeoralJy y sx is rueasured in a chemostat under different speciflc growth mIes 1-/-. From the obtained Ysx and 1-/- values, one calrulates. llsingEqn(3.3). thf" modeJ parameters y~a:< and m,. Figure 3.3 shows how Y5X depends on Ik. Fot high valUe5 of JL. ysx approaches the value ofthe model paramf"tf"[ For low p. values Ysx drops signiticantly. becoming Y.! Y for p.=m.Y~. For mOSl conventiooal processes, itcan be shown that at nonual growth temperatures theeffectofmaintenanceonyield can be neglected ror 1-/- > O.OSh- ' . This means that in batch cultures ducing exponencial growth (where p.= JLmll strate (or eJectron donar). me electron acceptor and biomass occur. TIlis gives d = -1.857. being identical lo the full solution ofeonservatiOD eODstraims obtained before_ The other coefficients follow from appücation of (be regular conservauon constraints. i.c. the N-soutce coefficient fl"Om the N-balance. me HCO; from the C-balance etc. From me example severa! po¡nts mu!íI: be stressed: • the balance ofdegree of reduction specifies atways a linear relation between the stoichiometric coeffi.cients ofelectron donor, electron acceptorand biornass, making this relation extremclyuseful in prarnce; • the bala nce ofdegree ofreduction is not a newconstraint. it bjusta l>uirable combinatio," oftheC. H. N charge conservation constraints.

Degree of reduction

+6 +8 +8 O +8 +8

SI

Other useful applications of me conservatiotl constraims are outlined in tbe references and indude: • selection ofthe yield measuremenlS wbicb provide the least errors i.n tbe calculated otber yields (dlle ro e rror propagation in the meas' uremcuts); • improvementoftbe elTon in all yie1ds by measuring more than the minimal required yields (measurementredundnncy allowing data reconciliation); • use ofredundant measurements to investigate l he occurrence of systematicmeasure.ment errors 01" errors i.n the system definidon (e.g. a product has been forgatteo).

3.3

Stoichiometry predictions based on Gibbs energy dissipation

A number cf methods have previously been proposed to estimate biomass yields fl'ox) from corre.1atioDS. Aparticularly sitnple but useful and recent method has been the thermodynamically based approach using Gibbs energy dissipation per unit biomass (l(YGX) in kJ perC-mol X. This is a stoichiometric quantity which can ¡similar to che. biomass yie.ld YDX on e.lectron donor as in Eqn j3.3)1be written as

_1_ = _'_ + me Yex

y~x

(3.4)

¡J.

mG is the biomass specific rate of Gibbs energy dissipation fur maincenance purposes in kJ per C-mol X h and y~ is me maximal bioOlasS yield on Gibbs energy in Cmol X kJ- l. Eqn (3.4) show$ that the Gibbs eneTg)' dissipation contams a growth and a mainte.nance re.la ted termo Simple correlations have becn propased for 1{yGX' and for me (see Further reading. Section 3.5). Thcse correlations cover a wide IdIlge of microbiat growth systems and temperatures (heterotrophic. autotrophic. aerobic, anaerobic, den.itrifying growth systems on a wide rangeorC·sources. gTOwth systems with and withoutre.versed electron

transport - RE'I).

3.3. 1 Correlation for maintenance Gibbs energy The following corre1ati on has becn fouud to be valid for maintenance .

Gibbs cnergy Illc=4.sexp [

(! -.)_)]

690.00 8.314 T

(3.5) 298 This correlation holds for a temperature range of5 to 7S OC. for aerobic and anaerobic conditions. It does not depend an tbe C-source or electron donor OT acceptOr being applied and only shows a significaD[ ~m perature effec[. This see.ms IOglcal beca use maintenance only involves Gibbs enerID'. irrespective ofthe electron dOllorfacceptor combination which provides this Gibbs energy.

STOICHIOMETRY AND KINETICS OF GROWTH

3.3.2 Carrelatian far Gibbs energy needed far grawth For the growth-rela ted Gibbs energy requiremenr lfYGf. the following correlations can be used: Por her.erotrophic Ol" autotrophic growth witbour RET: 1 Yg';" = 200 + 18(6 - c)U + exp[({3.8 - 'Y.)2r l ~{3.6 + OAc)[

(3.6a)

Por autotropbic growth requiring reversed dectron transporl : 1 """iiii=3500 Ye
and (he number of C-atoms {parameter el per mole ofe·saurce. Eqn (3.6a) shows that lfYr.x ranges between about 200 and 1000 kJ af Gibbs energy requirement per Cmol biomass dependenr on the e source use
+aHCO; +bNH; + cHp+d02 +eFe H + 1 CH,.lPo.,No.z+JF¡:l-t- +gH'"

+ lfYal( Gibbs energy.

We can specifY six consewation constramts and one Gibbs energy balance ro calculare the seven (a to g) unknown stoichiometric coeffi· cients. Using Eqn(3.4) lfYcxfoUows from the correlarions (knowingthat RET is ¡nvolved, tbat p.=0.01 h- ¡ :l.nd thal r=323 K) as: . 38.84

1fYcx = 3500 + - - = 7384. kJ/C- molX

0.01

The- six conservanon constraints and the Gibbs energy balance (using 6G~"-va.lues froro Table 3.1) are as follows: C
6CCAT) I')'¡) ~ 4.5J f'X [ 1 fY~;l P

-

(!- ~~)l

69000 R T

298

(3.9)

Eqn (3.9) can be shown toprovide rea.~onab l e estimates of JLm ....·values for a wide varieeyofmicro-organisms (e.g. nitrifiers, methallogeos, heterotrophie aerobes). A final aspect to be discussed is the occurrenee ofsoll. K. Ch. A. M. (1994).linearconstraintTelatiamin biochemkalreartian systems: 1. Classif'iration ofthe calCU1.1bility and the balanceabilityof conver· sioll rateo .lI!o r~ch7l ol. Bloe-ng. 43. 3~ 10. van der Heijde n, R. T.J. M.. Romein, B.. Heijnen,J.J .. Remnga. C. and Luyben. K. CtL A. M. (199-4). Linearconstraint relations in biochemical reartion systems: Ji. Diagnosis and estimation of g~ errars. B!orechnoL Biorng. 43. 11-20. van der Hcijden, R. T. J. M.. Romein, B" Heijnen,j.J.. Rellinga. C. and Luyben. K. Ch. A. M. (1994). Linearconstraint reJatiOTU in biochemicall?lct:ion systems: 10. Sequen tial application of data reconciliation for sensitive defection ofsystematic enoTS. BiotertlTlol. Bioeng. 44. 7Bl-791. von Storkar, U. a nd MatisOD, 1, W. (19B9), The use of cal onmeny in biotechnol· ogy.t\d.... Blocru-m. E"8 . .lIlotechnol. 40, 93- 136. Westerboff. H. V. and va n Dam , K. (1987). MlJSaiC NOTl~'1uilibrium Thermodynam ics and rhc Control of8ioJoglcaJ Fm CTlergyTrUTlSdlóC!:wTI. El$evier. Amsterdam.

Chapter4

Genome management and analysis: prokaryotes Colin R. Harwood and Anil Wipat lntroduction

Bacterial c.hromosomes and natural gene transfer What is genetic engineering and what is ¡tused for? The bas ic OOols oC gene tic engineeriog Ooning vecto.rs and tibraries Analysis o f genomes/proteomes AnaJysis ofgene e..''{pression Engineering genes and optlmising products Proouction of hetl':rolagolls produC'(s In sl1ico analysis ofbacterial genomes Further rt'ading

4.1 I Introduction Gene manipu latioo is nowadays a eore technology u sed for a wide variety ofresearch and industrial applkations. In addition to representing an exttemely powerful analytical too1, it cm be used ro: (i) ¡ncrease the yield (ami quality) of existing products (proteins. me tabolites or even whole ceDs): (ii) improve che characteristics of exining products (e.g. proteio e ng ineering); (iü ) produce existing prodUCES by new routes (e.g. patbway en ginecring): and (iv) deve10p novel produces Dor previously found in Natuce. ln this chapte.r we assume a knowlcdge in the

reader of the basic structure and properties ofnudeic acids , m e organisatiOll of the genetic information into genes and operons amJ the mechanisms by which bacteria transcribe and translate this encoded infurmation f Ol" the synthesis ofproteim (see abo Chapter 2).

Bacterial chromosomes and natural gene transfer 4.2. 1 Bacterial chromosomes Chromosomes are tbe principal repositories of the ceU's genetic information, m e sire of gen e clq'ression and the vehide of inherita nce. The

60

HARWOOD ANO W!PAT

Genetic. element

Size range

Transposons Plasmids Prophages

800 bp te 30 kbp I kbp to ISOkbp

3 kbp

Bacteriophages

Bacterial c.hromosomes

''''' bp.

nlJcleolid~_ lx!sc

'o

300 I
Organism

Type

Number Size

MS2 <j> X 174

bacteriophage bacteriophage bacteriophage bacteriophage eubacterium eubacteril.m eubacterium eubacterium eubacterium euoocterium eubactenum archaea

1 1 1 1 1 1 1

lambda T4 M ycoplosmo geniwlium Borre/io burgdOt(eri

Campylobocter lelunJ Rhodobaclff sphaerOldes Bacillus sub61is Esch erichio wli

2

Myxococcus xonthus Methanococrus jonnaschii An:haeog/obus fUlgidus arcnaea Schizosoccharomyces pombe eukaryote

3

Socchoromyces cerevisioe

16

eul
Con;ugatlve plasmid

conjvgatlon. (a) The
by othcrorganisrns. This technology, illusU"ated in Fig.4.4. has nor only facilita te
4.4 I The basic tools of genetic engineering lbe techniques for isolating. I.:utting and joining molecules of DNA, developed in the cady 19705. bave provided (he foundations of OUT current technology foT e ngineeling and analysing nucleic acids. Tbese. allow fragments ofONA from virtually any org;:¡nism to be doned..in a bacterium by inserting rhem into a vector (carl)'ing) molecule lhat is stably maintained in tbe bacteria] hos!.

4.4. 1 Isolation and purification of nudeic acids Biochemical techniq ues for preparing Iarge qua n tities of re lati\'ely pure nucleic acids from mkrobia] ce.lls are an essentiaJ prerequisite (or in vifmgene technology. The firsrstepin the ¡solarion of nucleic acids is!he mechanical Or e nzymatic disruptioD of the eeU ro release the intracellular components {bar indude the Ducleic acids. Once re1eased from the eell, the nudeic acids mustbe purified from o thercellular componen.r:s such as pl'oteins and polysaeeharides [O provide a substrate of appropriate purüy for nudeie aeid modifYing em:ymcs. '!be released nudeie adds are recovered using a combina non oftec.hniques induding centrifugation . electrophoresis. adsorption to inert insoluble substrare Ol" by precipitation with non-aqueous solvents.

GENETIC ENGINEERING: PROKARYOTES

4.4.2 Cutting DNA molecules Thc abiJity ca cut molecules oto NA. ei lher randomly or atspedfic target sites. is a requirement for Ulany reco mbinant DNA temnlques. DNA may be deaved using mechanical or enzymatic methods. Mechanical shearingresulr.s in thegene.rationofrandom DNAfl'agmenn which are onen lLsed for tbegeneration ofgenornic libraries (Section 4.5.5). When DNA molccules are mechanically sbeared ie is not possible to ¡solat,.. a specific fragment containing, for exalllpLe. a particular gene or operon. ll contrast, when che DNA is cutuslng restriction endonucleases whicb recognise and cleave sped.fic target base sequences in double-stlOmded (ds)DNA, specmcfragmems can be isolatcd. Restnction endonucleases cut the pbosphodiester bac:kbone ofboth strands ofthe DNA to generate 3'-OH and 5'-P0 4 [crrniDi. Several hUlldred restrictiOll endonudt.'ases have been isoLatcd frOID a wide vatiety of microbial spedes, and thf'ir numbf'rs continuc to grow. Different dasses of restriction codonucleases witb distinct biocbemical properties have beeo identifled. witb the type n enzyme:s being themain dass used for genetic engineering purposes. Resu'iction endonucJeases are named according to the species from which they were originaliy isolated; enzymes isolated from liaemopllilus f!lflucnzae are designated Hin, those fi:orn 1!aci1!us f!!!!)'IDlfq ue[adens, &1m etc. Whenmore tban one type of enzyme is isolated from a particular strain 01' species, tbe strain and isolation number(in roIDan nurncrals) are added to the name. Thus the three restrictioo endonudeases isolated from H. infiuenz.ae strain Rd are designated Hi1!dl, Hind ll and HindIII. The targetrecognition sequences (restriction.sites) oftype II restriction enzymes are usuallysbort. typically four lo six b phosphodil!!"Ster badlbone d tile ONAmd 1lAk.es a covaIent bond ~!he exposed ] 'OH VId S·PO.groUjK olll!!illm sideofthe

b...k

69

70

HARWOOD ANO WIPAT

DNA. Akeyfeat1l.ré ofthe PCR is thatthe entire DNAampli1kation reaction is carried out in n single tube containing enzyme, template, primers alld ~ubstrates. Each cycle of amplificaDon therefure involves annealiug. exteruion and denaturation reactions, eat"h brougbt about atdifferent temperarures (Hg. 4.7). Since the di.ssociation reaction may occur at temperatUTeS as bigb as 95·C. and mere may be as many as 35 cydes in a single PCR. a bigb1y thcl'mostable DNA po]ymerase is a basic requirement for PCR. Taq polymerase. isoLate

AA a\llOndlo~ of pare of l ONA nquentinggel ~rated by me Saoger cNm ttrmhmlon meehod. The I~ne$ l~ la"lIed acuordill¡ lO nud.otIdu al whlch mey l~ urmlnaeed. namely: A. aden!ne; C.

T G

A G A

me

e T

A

G G A

Directlon of elec1rophoresis

G

A

T

e e

T

A G

e

T

g~ 4.4.9 Site-directed mutagenesis Site-direcred muragenesis. the specific replacement ofnudeotides in a sequeoce afONA. is use
75

76

HAR-WOQO AND WIPAT

gene.lt is transformed iDtoE._~repair ofthe mismarches by the bost's mismatch repairsystemsis~byuseofa mutant(e.g. mutS) tha r is defective in this function. Aft:i5' ~ eac.h strand will forro a double-stranded molecu1e without mimla.lI:bes and mese will segl'egate into separate daughter cells. One molecuJewill oontain the mutations introduced by tbe oligonudeotides. whilst the other wm be ideDtieal to the original plasmid_Cells harbouring plasmids with the desired mUlation are selected byvirtue oftheir newly aequired 3mpidlIin resistance phenotype.

4.5 I Cloning vectors and libraries A c:loning vector is a molecule ofDNA into which passenger DNA can be cloned to allow it ro be replicated inside a bacteriaLhost cell. The vector and passenger ONA are covalently joined by ligation (Sel.-tion 4.4 .3). CJoning vectors have four basic characteristics: (i) they must be easily introduced into the hon bactenum by O'ansformation 01', after in vilro paekaging, by phage infection; (ii) chey must be able ro replicate in me host bacterium. prefel'd,bly so thar the number of copies oftbe vector \CQpy-number) exceeds that ofme host chromosome by between SO and 200; (jii) they shouId contain unique sites for the action of a varietyof .restriction endonucleases; and (iv) tbey should encode a means for selecting 01' screening host celis that contain a copy of the vector, Cloning vectors are derived from narurally oa:urring DNA moleculcs, such as plasmids and phages, which are capable ofrepLicating indepeIl"" dently of me host chromosome. A wide variety of cloning vectors llas been deveJoped for specific applicatloru and these are briefly descnbed below.

4.5.1 General purpose plasmid vectors General purpose plasmid vectors are designe
GENETlC ENGINEERING: PROKARYOTES

Polylinlter multiple --:::::::~¡¡;;_ ~/aC~/ Clonlng sita

pUC19 2686 bp

Multiple cloning site:

..,,,

Aval

Ec/13611

"".,

,~ ,

~"

~qt\lunc.G.\GL'1'CCG'l'.I.('Ct" GO" G ·,~~~~ ~",,~ ... t =t>¡~(C

EcaRI

Knpl

Apal

,01",,651

B~mHI

S~II

$phl

/11",,11

C-Sg,Ams..rs.. rP, aV~lArgPraA."
Ta il

genes

genes

DNA synthesis genes Non·essential genes B.g.lntegration Regulatory l ysi s genes genes alld immunity

Le'

cohesive end

"'" '''''

Right cohesive Righ1 a"" e nd

Thc prese:nceoftbecos siteel1ables [hese plasmids (O be: used in conjune= tion witb a lambda iJl \/itro pack SO kbp) sequences ofDNA. BACvectors are usu allybased on che F plasm.id ofE. mil and are able to accept DNA inserrs as largeas 300 kbp. pBACs are maintained as single copy plasmids in E. eoli, excluding the reptication of more dlan one pBAC in tbesame hostcell. Ord~ pBAClibrariesofbactcrialgenomes maybeconstrtlcted in wruch theentiregenome sequence is represented by a series of dones with overlapping ¡nserl!.

4.5.4 Spedal purpose vectors In add itioll to the vectors described aboYe. a range ofspedal purpose vcctors have been developed and are described briefly below. E:c:press Loh vettOTS Expression vectors are designed to achieve high level , controlled expreso sion ofa targetgene with resultingproductionofa protein productat concentl'ations as high as 40% total cellularprotein. Expressioll vectors often incorporate a system that .adds an. affinity tag to the protein to facilitate ies purification by affinity chromatograpby. .Expression vectors are mostly plasmid-based and ofien use the tight1y controlled and highly efficientphage 1'7 RNA po1yrnerase gene expression sysrelll. The targetgene is doned downstream ofthe transcriptional (promoter) a nd rranslational (ribasome binding site) control signili derived from T7. The ve C..:IomHln

Hydrophoblc amioo acid! forming lI·he/leal strul:"l.ure (>8 residuul

LeS!! hydrophobie. wilh . 19nal peptid ase re.::ogni,ion site ( ~ 8 rlsl tloosJ

lb) Socretion vector: lnéuc ibh'l promoter

che tilrgetproteio1 fu~ed ....fram. dowNlream el ,lgnal

---Ciimm;,';-i_ _llIIlHe.-

me

/

l equence.

(c)

\

Signal

Clerning

Anllbiatic

$&Quunce

!lite

."lst' nc8gl'lnll

-

Rtlpllcatiern o rigin

Secretion lIac1or wi\h insert: -Yi¡i IlIl1 WVUZUII

e

largal gene MC¡uence fus ed in·frame with thlt s ign ai sequence

Secretion Veclors Currently most systems for the production of TeCombinant protcins lead to the in tracdluLar acrumulation ofthe productoHoweyec, mm· cellular accumulation can ¡ead to lower production levels, protein aggregation, proteoJy5is and perm¡¡nent 105s or biologicaJ activity (Section 4.9.41. T.his can sometimes be oven:ome by 5ecrcting the target protein diIeclly into the cuJture medium since secrete
GENmC ENGINEERING: PROKARYOTES

Single-str.mded phage and phagemid vectors It is sometimes necessary to generate single-stranded DNA, partirularly for DNA sequencing and oligonucleotide-directed mutagenesis. Mcssingdeveloped a series ofvectors based on bacteriophage M13 ,
4.7

1

Analysis of ge ne expression

Promoten influence the frequenc:y of transcriptioD iniba.tion rathe.r than me rue oftranscription. and strongpromoters bave a high affinity for RNA polymerase binding. A comparison of a number of E. coH promoters has loo ro the recognJtion of a consensus promoter seqll~ce: S' -TATAAT-J ' .cenrred around 10nudeotides up.streamof(prior to) the transcriptiol1 ¡niriarlon site (- lO regian) aDd 5 '-TI'GACA-3', located about 35 nudeotides upstream (- 35 region). The strongcstpromoters are mese which show tite closest identities ro [his sequence. Additionally. the spaang between tbe - 10 and -35 regions is impor· tant the optima! being 17 nucleotides. The ability te analyse gene expression is an important prerequisite for optimising the biotechno· logieal potcntial ofbacreria and many highly sensitive and precise techo niques have been developed ror this purposc.

4.7. 1 Analysis o( messenger (m)RNA transcripts 1hree methods are used for the analysis of mRNA u-aflScripts: Nortbem

blotting. S1-nudease mapping and p.ri.mer extension analysis. The lauer two techniques have tbe potential to identif}r the transcription ¡nidaDon sitcs. Northern blotting involves lhe separation ofrnRNA species by cleclrOpboresis ttlrough agarose Ol" polyacrylamide. Formam.ide. urea or other dcnatur.mts are indudOO (O avoid the sjngle-stranded molecuIes fonning secoDdary strucrures (e.g. duplexes. loops) that might affect Lheirmobility. The separated mRNA species are transferred to activated

83

84

I

HARWOOD ANO WIPAT

Promoter

l'1ipf>lng 01 tl"al'lstrlptlon Inltlarlnn poinl$ u51ng

SI

noclUSI.

mRNA Í!5 hybrid~d lO

a dflMWred ONA rr'alrnent th;¡.t has been bbelled at iu 5'...end. The ONA fra¡rrterlt is cho~en 50 mat la S' ·.,.d b Intel'nal to the t:lrxet mRNA. whUfI th,l' -eod extenm beyond da putativa mRNA St3rt

DNAII mRNA

~....

TIE

Gene 2

Gene 1

9

.....

'i

I!

Hybridlse extracted mRNA

transcripts 10 a deneluroo, 5'-laba lled, ONA Irll9mont

5'·labelled

1

;;... _e:

'M

polnf. Thll RNAJDNA hybrid moleal~

__o

hu sift&le-stnnded

UQnsiOlU Úlatal"O!. d~raded by

1M sln¡le-smond-:;pecific anivity of

S I nuc!e¡¡s-e. TheJ'-endofthe DNA l~lIlnt is ootennlned by r unning II o n .. denaturing gel ¡pinst a DNA sequencll8 of me ongml fngmem geoer.J.ted by me Mu;¡¡m;aml Gilbert chernial d~'Bge

method.

,, , ,,, ,,

,,, ,,,

I

ElectrophoreS8

,. product on a denaturing

,

T polyaerylamlde gel

GG

ee

1

-j

nylon membranes by blotting and theu covalentlycross-linked. Specific mRNA species are detected by hybridisation (Section 4.4.7), using labelled oligonucleotide. DNA or RNA pro bes. The use ofmarkers wilh different mo!eLwar sizes allows the sizes af specific transcripts to be estimated whkh provides ciLles as ro the organisation ofthe transcripriona! unit from which rhe transcriprwas syntbesisro. Sl-nudease and primer extension analyses facilitate the identificarion ofthe 5' -prime eods ofmRNA transcripts or the processed products of primary transcripts, In rhe case of Sl-mapping (Fig. 4.14), mRNA is hybridised [O a speciflc spedes of ssDNA that overlaps tbe start afthe targct !r.lnscl"Ípt. Tbe resu!ting RNAfDNA hybrid malecule has an overll'lpofDNAat theJ'-end thatisdigested bythe singl~strand specific Sl-nuc1ease. The size ofilie processed ssDNA molecule, which is labelled al ¡u unmodified 5'-end, is determined by denaturing polyacrylamide gel elecrropboresis using a ONA sequence ladder as molecular size markcr. ID the case of primer extension analysis (Fig. 4.15). a 5' -end la belled oligonudeotide, hybridising about 60-100 nucleatides downstream ol' the predicted tr.:mscription initiation rite. is used to prime the synth esis of a ONA copy of the mRNA tr.mscript, using the enzyme reve rse tl"atlscriprase. Syntbesis ofthe complementary DNA strand terminales at me 5'-end of the transcriPl, ro genera te a prodUL1: of defined length. Again t.b.is can be sized using a ONA sequence ladder. geneI
4.8.1 Protein and pathway engineering A well studied example of enzyme o ptimisaLion is that ofsubtilisin. an albline protease ¡solatOO from Rsubnlis and c10serelatives fe.g . Rlichenifonnis, B. stearotherlflopl.i1l1s) and used asa stain remover in the detergent industry, Subtili5in i5 used ln95% ofwashing detergent fonnulatiolls a.nd allows prorein stains to be re moved mote effective1y and a tlower temperatures dlan are usually needed for laundry processing. The ideal requirements far such an cnzymc are stability up to 70 ~ C and wi thin the pH range 8- 11. resistance te non-ioruc detergenrs and oxidising reagents such as hydrogcll peroxide. and the absence o( metal ion require ments. Based on an exte.ns ive knowledge of its catalytic activity a nd Lhree-di01ensional structure, subtilisin was engineereerby

Si

:!8

J.lARWOOD ANO WIPAT

site-directcd mu tagenesis (Section 4.4.9) to produce variant enzymeS with combmations ofthese improved charactenstics. An alternative approach bas beco used to improve tbe enzymatic characteristics ofBaciUu5 a-amylases. In this case natural recombina tion was used to generate fun ctionaI hybrid amylases from genes encodlng c10sely related enzymes from B. arnylolfquejacic!lts, B. licheniformis and B. stearothermophilus. Tbe genes ror tbese enzymeswece doned in pair-wise combinations and then aUowed toundergoroundsofreciproca.l recom· bination. TIlis gene.ca.ted a population of cells witb a. large numbcr of hybrid a-amyJases, which were then screened for ceUs produdng amyJases witb improved catalytic oc structucal characteristics. More recently, k.nowledge: ofthe tJu-ee.dimenslonal structure of tbese amylases. togetherwith information on (he relationship between structure and fundional cha.racteristics such as thermostabiliry, has enabJed more directed approoches to be used. These have induded [he use oC PCR gene splidng techniques ror the construction of spcci.tic hybrid a-amyJases. Comparative DNA and proteinsequence studies have demonstra ted the importance of recombination of blocks of sequence rather than point mutation alone in m e evolueon of protein structure. These studies have loo to the development ofmolecular techniques such as DNA shuffiing or sexual PCR 10 facilitate the rapid evolution of proteins_ The principIe involves mixing randomly fragmented DNA encoding dosely reJated genes and then using PCR (Section 4.4.4) to reassembJe tbem into fulllength fragments, witb the individual fragments acting as primen. The PCR produces are used to generate a tibrary (Section 4.5) of chimaeric. genes foc lhe se.lection of proteins with modified or improved characterisr-ics.Ihis system hasbeen used to generate a variant of an E (oli ,B-galactosidase with a 6()"fold increase in specitic activity for sugar that is normally a poor substrate fo. this enzyme and a variant of the green fluorescent protein (Section 4.7.2) with a 45-foId increase in f1uore scent signa!. Bacteria produce a number of compounds mat, if syntherised at suitabl e concentrations, represent commercially viable products. Traditional1y. bacteria tbat make a significant amount ofa potentially commercial product can bedirected to synthesise larger amounts. This may be acrueved by randomly mutageuising a population of the target organismo and screening fo. mut:mts producing higbe.r concentrations ol the product, or by doning the synthetíc genes together and placing them under the l'Ontrol of efficient transcription and transIation signals. While there are examples chat testifY to the success of such approaches (e.g. antibiotic production. syuthesis of amino acids), recentJy more ratioual approaches have becn made by engineering merabolic pathways_ either to increase. productivicy or to direct syntbesis towards specjfic products.


4.9 I Production of heterologous products Traditionally, genetic techniques have been applied by indllstry to increase the.production ofnaturaJ products such as enzy:mes, antibiotics and vitamins. Dnly a limited numberofprotein prodllcts wereproduced commercia1ly. and these were produced using existing technologies frorn their natural hosts (e.g. proteases frorn Badnus species j. Specific genes can now be ¡sola red from.virtua Uy any biological material and c10ned lnto a bacteri um or other host system. However, cloning a gene does not, pe.r. se, eru;ure its expression, nor does expression ensure the biological activity ofits producto Many factors need to be considered to ensure commercially viable leve1s of prodllction and biological activity. FoI example, the choice ofhosr/vector systems determines both the strategy used fur thedoning and expression, and these in tUIn can affect the quantity and fidelity ofthe product.ln sorne cases itis nor possible lo use a bacterial system to produce a biologicallyactive product or one that is acceptable for phannaceuticaJ purposes.lnstead hostfvector systerru based on higher organisms. fuI example mammalian or insect cell culture systems. may be used.

4.9.1 Host systems and their relative advantages Prior tú tbe 1970s, the only methods fur obtaining proteiru or polypeptides for analysis or fm: therapeutic purposes were to ¡solate them frOID natural sources or, in a limited number of cases (e.g. bioactive peptides). to synthesise the-m chemically. Recombinant DNA technology opened up the possibility rodone the gene responsible fur a particular product and to produce it in unlimited amounts in a bacterium sllch as E. eolio Initially, the only eukaryotic genes that couId be doned were those encoding products that were already available in re1atively large amounts and that hao very sensitive assays (e.g. insulin, human growth honnone. inteñeron). This was because it was necessary to have extensive information about theiI amino acid seqllence. and proteinsequenc· ing techniques available al that time required relatively large amounts oftbe purified protein. Current teehnical improvements permitalmost any characterised protein to be doned, either directlyvia copy DNA syn' tbesis from mRNA extracted from biological material, or indirectly by gene synthesis. Although recombinant rechnology was developed in bacteria! systems. it has been increasingly expanded into a range of eukaryotic organisms, using a wide variety ofinteresting and novel technologies (see Chapter S). However, from an ~onomic point ofview. bacteria are still the organisms of choice because ofthe case with which theycan be genetically manipulated, their rapid growth rate and relatively simple nutritional requirements. Proteins derived from recombinant technology are expected to mee! the same exacting standards as conventionally produced drugs; particularly with respect ro product potency, purity and identity. Sensitive analytical techniques are used ID cbaIacterise the products,

8~

90

HARWOOD AND WIPAT

with particular attentiOll being paid to undesirable biologiC'al activities such as adverse immunogenic and allergenic react.ions. It is therefore me exacting requirements of regulatory authorities, sucb as tbe US Food and Drug Administr.ition (FDA), for increasingly .mthenti c phormacological produc.:ts that has led producers to switch from ba
GENETlC ENGINEERING: PROKARYOm

4.9.3 Transl.tion The effldent translation ofmRNA n.mscripts requires the incorporaoon of an efficient ribosome binding sire (RBS), located about 5 bp upstream of (prior to) the translational start codon. The structure of Che RBS tend5 to vary frorn bacterium to bacterium acccrding to the sequ ences at che 3'OE{ end cfthe 16S ribosomal (r)RNA that interact wito the mRNA The genetic code is degenerate. that is many amino acids are specified by more than one codon (triplet ef nucleotide bases). Codeus t hat sped1y the same amino acid are said to be synenyroous but are not nocessarily used with similar ftequencies. ln facr. mon bacterial spedes exhibit preferences in their use ef codons, partic ularly for highJy expressed genes. Variations in CodOD usage are, in partoa refJection of the %GC oontent of the organism's DNA. with favoured codous correspondiog to che organism's most abundant transfer (t)RNA spec.ies. Since most codous a re recognised. by specific aminoacyl tRNA molerules. the use of non-favoured codons results in a reduction in me rate of translaoon and an increase in the mis-incorporation of amino acids above that of me normal error rate oí aboue 1 in 3000 amino acids. Codon bias can be determined by calculating the Relative Synonymous Codon Usage (RSCU) ofincj.ividual codens: RSCU "" ohserved number orrimes a particular codon is used expccted number ifall codons are used with equa J freqllency therefore: ifRSCU equals 1. fue codon is used without bias ifRSCU is Jess [han 1, the CodOD is 'non-favoured ' ifRSCU is lno/l: chan 1 thecodon is ' favoured '. Gene syn tbesis technology alIows genes to be conrtructed so as te optimise the codon usage ofilie host producer strain. The signiflcance of codon usage was dernQnstrated in srudies on the production of interleukin-2 (lL-2) by E. coH. When (he native IL·2 gene (399 hp) was analysed for 115 coden u sage only 43%ofthe codons were .favoured' byE. mil. When an altern.ative copy ofme [L·2 gene was generated by gene synthesis , it was possible to adjust the codon usage such that 85% ofthecodons coro responded to those faVOllred by E. col!. When the twOvel'5iOllS were d oned and ex:pressed in E. coli on identical vectOTS. despite tbeir producing identical amounts ofmRNA, e.ight times more biologicaUy active 11-2 was produced.from tbe syn thetic gene as compa.red wi th thenative gene.

4.9.4 Formation of indusion bodies Many .recombinam proteins. particuJarly when produced al high conccn(rations, are unable te fold properly witrun lhe producingcell and instead associate with each other te fel'm large protein aggregates referred to as indusion bodies. lnc1usion bodies are particu1arJy oomm on in bacteria expressing mamrnalian proteins. The proteins in inclusion bodies can vary frOID a native-like state mat are easilydissociatoo. to completeJy misfolded molecules t bat are dissociated onIyunder

9I

n

HARWOOO ANO WIPAT

highly denaturing conditions. 111e size, stat.e and aggregation deDsity of indus ions are affected by the characteristics of the recombirunt protein itself and factors lhat affect ccll physiology (e.g. growth rateo temperature. culture medium etc.). In sorne cases aggregation can be prevente
4. 10

I In si/ico analysis of bacterial genomes

The availability of e.mire genome sequences fuI' a significant num ber of micro-organisms apeos up new approaches Cal the anaJysis oCbacreria. and the rapidly expanding field ofbioinformatics has the potential to reveal relevant and novel insights on bacterial evolution and gene tunetion, lt has the potential te provide a nswers ro long-standing questions celari ng toevolutionary mecbanisms and (O the relatiollships bel:Ween gene order a nd function . One of the mast significant advances in metbods for studying and analysingmicro-org;rnisms has come about through the availability of powcrful personal computers which, togetherwith the developmentof the inrernet, provides researchers with access to powerful bioinformatkal mols, Bioinformatics, often retened to as jn silfco analysis, nicely complements In Vivo and in vitro methodologi es. An ultimare goal ís to model rhe bebaviour ofwhole organisms, including asptttsof theirevo-lutíon. Although nor currently a subsotute for in vivo and in \litro experimenration, bioinformatics has already demonstrated jts potential [O direct rhe focu s of more tradicional approaches. Severa l rypes ofcomputerprogram are avaUable for analysing bacterial genome sequences. These ¡nelude prognms thata ttempt to identify protein-encoding genes. sequence signals slIch as ribosomc binding sites, promoters and protein. binding sites, a nd rclatioruhips ro previously sequeneed DNA oC whatever somec. Programs thar attempt ro identifY protein-e.ncoding genes translate t he consensus DNA sequence in a1l sixreadingframes and then anaJyse tbe resultingdata forthe presE'nce oflongstretches ofamina acid-e.ncodingcodons, uninterrupted by tcnnination codons. These so-called open t'eading fuunes (ORF) are usuaUy at least 60 aminoacids long, burmay be several thousand amina adds in lengtb. The more advanced prOgr.'lms for predicting protein roding genes are ab!e te search for the presence oC ribosome binding


sites locared immediately upstl'e3m ofa purative srarr codon and even to idenrify potential DNA sequencing errors that generare frame-shift mutations. Once purative prateins have been identified. orher bioinformarical t001s can be used to derermine their relationships to previousJy identified proteins OT putative proteins. A prerequisire for this type of analysis is rhe availability of data libraries which act as repositories of l."11tJ'eDtly available DNA and prorein sequem;es. The databases can be routinely accessed via the internet. using programs such as FASTA and BLASf that provide a list of DNA sequence.s and proteins. respective.ly. sbowing bomology to alt oc part oftlte query sequence. The rnternetis a1so a souree of molecular biological tools tltat facilitate a wide r.mge of analyses including the identificatioll of putative transmembrane domains. secondarystructures and the signa! peptides of secretory pl'Oteins.

4.1 1

I

Further reading

Davies.J. E. and ~ain. A. L (1999). Manual o/Industrial Mirrobi%gy amI

Hlotichnology. 2nd Editíon. American Society foe Microbiology, Washington OC. Glazer. A N. and Nikaldo. H. {1995). MiCTObill! Hiotechnology: r'llndamentuls oJ Jl.l'p!itd Mlcrobl%gy. W. H. Freeman and Company. NcwYQrk. Lewin. B. (2000). Gt'It~ VJ1. Oxford University Press. Ox:ford. Old. R. W. and Prlmrose. S.a. (1994 ). Pr1nc1pl~ ofGcnt Manipuladon: AA lruroductian toGcnetic E"gincmng. 5th I:dition. 8lackwcl1 Scientific

Publicatiolls. Oxford .

Snyder. L. and Champoess. w'(1997). Mo!rcularCeuma oJj'Bacwria. American Society fur Microbiology. Washi ngton Oc.

93

Chapter 5

Genetic engineering: yeasts and filamentous fungi David B. Archer, Donald A. MacKenzie and David J. Jeenes Glossary IntroductiOll

Introduong ONAimu fungi (fungal transform:uiun) Gcnedoni ng Gene SI1'UCIUrt", organisatioll alld e.xpres.s ion Spccial llle[hOdolugies Biotechnologit:al apptications offungi rurther rt"ading

1

Glossary

Auxotrophic lllufauon A mutation in a gene t.hat confkr.; the requiremcnt fur a g rowth factor lO bt! ru pplied ratber than syntheslsed by the organism./\ gene that complemears this auxotr0IJltic mutation is o ne that can ¡'etu ro lbe o rg
GENETIC ENGINEERING: FUNGI

Sonthern blotting DNA fragments ilre separated according ro size bye1ectrophoresis. tr.msrerred to a membrane and probed with a labelle
GENEl'IC ENGINEER1NG: FUNGl

Organism Soccharomyces cerevisiae Schaosoccharomyces pombe Condida orbiccms

Numberof Genome chromosomes size (MW web sit~ 16

12. 1

3

14

8

16

Neurosporo crossa

7

43

Aspergillus nidulans

8

JI

http://genome-www.stanford.edulsaccharomyces http://www.sanger.ac.uk/ProjectslS-pombel http://aJces.med,umn.edulCandida.html http://sequence-vvww.stanford.edu/gtoup/ candida/lndex.html http://w#w.unm.edu/-n?PI http://gene.genetJcs.ugaedu

Notn

• Ilapl id &Cll r¡m~ oompkm"nl ",
Inoculate wilh fungal spores

IncuIJate tor 16-24 h

minimal medium (lacking che

Remove cell walls with carbohydrase emyme for 1-2 h

requlred growth suppl\lment which 15 now supplied by me activlt)' of me Introduced gene) or it can contaln an antibiotic. For anclbiotiI'llwlamyd n (G 418)-resistance gene (Kan) under the control of a runla! prornotet (P) and short !link;"g sequences ofonly about 40 bp (O) whlch corrtipClnd 10 !he ends oftMl:ene lO be deleted. A doubl. o:roJ~r by homologou1retOlTlblnation between tht..SOl endsand me chrorTlOSOIl"OfI ~d:s 10 lhe

deletlon of d-..gene. At th e tron·OYer 11tU, cJvumo$OlNl ONA molKule$al""ll broken en¡ymlc.1l/y ¡nd DNA $tr;lnd, ei'!d\arJ&ed with mat ofthe ¡ncomin¡ cassette ONA viii DNA repair mechani$m whlch re-joins me DNA molecules_ BecaU5e many genes I I""Il essentiaJ ror surviYlll, gene deleooo is flrst arded OUt in dlploid ceh wheNl on/yone of lile [WO copies o/ th.gene is deleted. Y~diploid tr.ansformoonu COI"Iulni ng Ihe dele'".ed

gene ue seletttd on moolum contailllnll the ;votiblotic G418, which is more effective -

"gB

J'

•

:::::o

Spetlfit gen!! deletlon In f~~ment~ funll. Upstreml (5 ' ) and down5tr'Um (l') reglons of a~ le¡¡5t I kb In Jlze, Immedlalfllyadjacllnt te thII chromowmal ¡en. to be deleted (gene X). Incorponl\e
GENETrC ENGINEERrNG: FUNGr

geuebutwltich can be lost during the process ofcell division. Extractio n of tbe total DNA from su ch Ullstable A nidul,ms colonies, and transforrnationintof. eoli, pennj ts the isolation aftbe plasmicl s containüIg the rungal DNA fragment which complem ented che original A nidulans mutation. Advantages ofthc.method are that itjs rapid and it is nOl necessary to construct a library of individual genes from m e total DNA of tbe fungoso

5_3.3 Gene isoladon by the polymerase chain reaction Extensive use of tbe po lymerase chain rearnon (PCR. (see Fig. 4.7) is now mOlde to isolate many specific genes. This approach requires profetnS with rhe same function ro have been identified in other orgOlrnsms and for the gene sequent.:es ro be available in a DNA sequellCE' data.base (e.g. 0 0 the intemet)_ Alignment of tbe protein sequences eucodecl by these genes Olg III

112

ARCHER, MACKENZlE AND JEENES

'Nested primer'· PCR. Redundant primer mike~ (00 and
Fungal culture

' . ANA ~

2. cONAsyntl"l6!till

cONA

~

, . """' " !

4 . E. cailr.lnltormtlion

E. coJllibmry

11s.

1

P!asmld DNA ex!tllctlcn

6. Yeas! trans/otm¡¡11on

7. RepIca platng

Veast screenlng

1

8. P!ur 95%) S. cen:risiae genes appear not ro require CCAAT-boxes for function althoughthis motifisknown tofunction in A.1'IiduJolls. [tshould be stressed that in the majority of fungal promotet'S mat have been ¡solated, the funcrlonal significance DI sequences identified in rhem has uot been detennined and therefore remains uuclear. With a constitutive promoter, the basal level of transcription is determined by me binding to the 'core promoter' ofa proteio cornplex which eontains RNA polymerase and tbe so
lumcn

(b ) An'sted protein 'olding in t he ER lum en p< o-sequenclI

Cr-í~ COOH~

=

G ~

he~u5

~

prole;n

C.

(e) Endopeptld... el....eg. In tIMo I.t. PC•• tory pathll\lIlY

LLI O

(001,,0 .nd .'!aa.. o,

O

ca,rla. p.otl!Ó n and /"utterolollO'ls prou!n 10

...

tha c" l osteril>l'"

O

lQ

cK~T C?OOH~ •

FoIdlng and proCeJ$ln&

of ffcre10ry l'ullon pIO{eÍll$In flIunenlOU$ '~ (a)

Entry of

JUs(ent polypepdde Imo the tumen oIthe e~opWm!c reticulum lE"R).

n. sI¡nal s.qllel"lCt whkh d lreca erTll'y of!h. polypepdde is r.mo-v.d by slgnal pcpcld.ase so th:.Ic ma emar¡ln¡ poIypepudc wlthln malllmen IlW me slpl SllqUiIn". SIP Is m ~bI.r1d~l1t chaperone wlchil me IUll1en whtch 11 nroclated wich nrly prot.eln fQldtn, e-.n:nt$ , Other chaperones

an d foldues (lee tElxt) are also preient. (b) Fokl ing of!he fuI!· length fu$ion proce~, within the ER. (e) The rus Ion proceln is

clea~.d

withln til . Golgl body by a lpeclfl(

peptldan (KEX2I n S.cereviJioe) to release me hetw-otogous prote;n tO m e ceUextMlo r fOIIowl"g tnnsport of me prot eln by membra.r.e·boun d ycsJ.t:1e5 (el).

126

ARCHER. MACKENZIEANO JEENES

a1ready c1ear tb3t. as wirh ycast expression, severa} factors can conspire to present a bottle-neck and l llat their relative importance depends on the heterologous protein. Foreign genes which use CodOllS Ilotcommon in fungi , the presence of sec¡uences which destabilise mRNA, differences in the protein folding/secretOlY pathway and the abuodance of protcases aO contribttte ro me observed bottlenecks. lo addition, although hyperglycosylation of heterologous proteins Is not such a problem with1i.1amentous fungi as it is with S. cerevisiae, it can still be a difficulty. In addioon. [he patterns of gIycosylation diffcr from those seen in mammalian ceJls wh ich could be importa nt for therapeutic protein production. TIte glycan su-uctures in fungaJ glycoproteillS are being analysed aod the genes tbat enrode enzytDes respons ible fur gIyc3n assembly are being doncd, providing the possibilil)' in che fuorre of manipuladng glycan synthesis. The essential details ofthe secretory pathway in filamentous fun gi appearto be qualjtativelyverysimilar ro those in theyeastsystem whích has becn studied more exrensively. Sorne of the genes that encode chaperones aud foldases have -been d oncd, as have genes that encocle proteins lnvolved in vesicular traosport.Although successful manipulation o( the protein sec.retory pathwayusrng these genes has notyetbeen reported, the nccessary rools to do so are becoming available.

5.7 I Further reading Atuubel. F. M_ Brent. R., Kingston. R. E.. Mool'e, D. D.. Seidman.J. e .• Smith,J_A. and Sttuhl. K. (1995). CllrrmtE'roI(l(.l,¡'S in Molmslnr f:liology. John W iJey, New

York, Broda, P" Oliver, S, G. aud Sinu, P_ F. G. (1.993). The cuknl)'tlllC Cfflcttw:Organisution ¡HIt! Rlgulatioll. Cambridge University Press. Cambridge. Gel lissen, e, and HoUcubcrg, C. P. (1997). Application of ycasts in gene expresslcn studies: a comparison ofS[J[charomycts cercvisiac, HanuIJu la polymorpha and f(!uyveromym: lactis - a review. Gm~ 190. 87- 97. Gow, N.A. R. and Gadd. G.M. (eds.) (1995). The Growing Funsus. Olapman and Hall, London . Kinghorn,j.R. and Tumer, G. (eds.) (1992). J\pplied Molf'CU larCenalCJ 01 FilatlU'ntollsFungi. Blackie Academic!lr Profusional. GlilsgtJW. I.uban.J. 31ld GoEr, S.I'. (1995). The.ycasl lwo-bybrid s~te m for studying proteinprorein inter.1roolls. Cun: apill. Biotl'dlnol. 6, 59- 64. Oliver, R. P. and Scb weizer, M. (eds.). (1999). MoIcculur FlIngtll Diolug)'. Cambridge

Universiry Press. Cambridge. Wolf. 1
JI

Y,.

SubscripLS t growthcnhancingcompound f.th SUbStT3te o r produce e.ssential growth compound o initia] conditions substra lc x biomass p product Supe.rn:riprs

r

fero

Greek letlers a.f3 cOó!fficients in equalion 6.11 6.G,¡

free enerxY change spttifir gl'owlh mteofthe total biomass (g g- "lLorsimply b- 1) I'inu maximum specljir growth rote of[he rotal biomns (gg-l'b 01' simply b- I) p.

6.1

Introduction

Quantitative desc:ription of cellular processes is an indispensable tool in the design offennentation proc:esses . The cwo most importantquantitative design parameters. yield and productivity, are quantitative measures that specify how the cells convert the substrates to the producto The yield specmes the amount of product obtained from the substrate, and it is ofpartirularimportance when the rawmaterial costs make up a large fraction ofthe total costs, as exemplifled in the produc:· tion of solvents. antibiotics . alcohol. and other primary rnetabolites, The productivity specifies the rate af product formation, and is partic· ularlyimportant when the capital investments play an important role. such as in a growmg market where there is an increasing demand for producing the product by a given capadty(or factory). These two design parameters can easily be derived from experimental data but, what ts more difficult to predlct. is how theychange with che operating condi· dons. e.g. if the mediurn c:omposition c:hanges or the temperature changes. To do this it is necl!:Ssaryto set up a rnathematical model.

MICROBIAl PROCESS KJNETlCS

A modells a setofre1ationships between the variables in rhe system being studied. These relationships ate normally expressed in the form of mathematical equations. but they may also be specmed as logic expressioos lar c3use/eff'ect relationships) which are used in che opera· bon ofa process . The variables ¡nelude any property mat are ofimportance fol' the process, suc.h as me agitaban mte, [he feed rateo pH. temperature. concentrations of substrates. metabolic produces and biomass, and the stateorthe biomass - ofien represented by {he caneentration of a set ofkey intracellular compounds. To set up a malhematical mode! it is necessary to specüy a control volume wherein aJl the variables ofinterest are taken ro be uniformo For fermentadon processes the control votume is typic:ally the whoLe bioreactor, but fu, large bioreactors the medium may be nonhomogeneous due to mixing problems and hereitis necessary to divide fue bioreactor into several control volumes. When me control volume is tbe whole bioreactor ir may either be of constant volume or ir may changewith time depending on the operation ofthe bioprDCess. When fue controL volume has been defined . a set ofbalance equatioos can be spedfied for the valiables of interest. These balance equa· tions speci:fy how material is flowing in amI out of the control volume and.how materiaL is converted withjn the control volume. Rate equa· tions (or kinetic expressions) specify the convenion ofmarerialwithin the control volume. They may be anything from a simple empirical corretation mar specifies rhe product formatlon rate as a function of me medium composition to a complex model mat accounts for all the major cellular reactioos involved in the conversion ofthesubsuates to rhe producto Independentafthe model structure. the process ofdefining a quantirative description of a fennentation process involves a number of steps.assbown in Fig_6.1. A key aspect in setting up a model is to specify the model complex¡ty_ This depends on what rhe model is going to be used rOl' (see Secrion 6.2.1)_ Specificatioo of the model complexity invoLves defining the numhe,ofreactions tobeconsidered io che modelo and specification oí' tbesroichiomerry for these reactions . When the mode! complexity has been specified. mtes afrhe cellular reactions considered in the model are described with.mathematical expressions, Le. the rates are specified as functions af the variables: namely rhe concentrarion of me substrates (and in sorne cases the metabolic praducts). These functions are normally referred to as kinetic expressions. since rhey specify the kioetics ofthe reactions considered in che model. !bis is an important step in the overall modellingcyde ando in many cases. differentkinetic expressions have to be examined befare a satisfaclory model is obtained. The next step in tbe modelling process is ro combine the kinetics of the cellular reactions wim a model for the reactor in whicb the ceUular process occurs. Such a model specifies how the concentradons of substrates. biomass, and metabolic products ch.ange wirh time. and what fiows in and out ofme bioreactor. These bioreactor models are

Specify mooel complexily Sel uptkioellQ

I

~

&~'~IPlinces mass

Rtdenne model compkxity

llii

..timol'

~-"J

¡

Simu[a1.~ ferm~nlllliDn proces ~

Dlffenlllt ueps In fermematlon prcx:esse5.

12~

1]0

NIa5EN

nonnally represented in terms of simple mass balances over the whole reactor, bu! more detalle« reactor models may also be applied, if inbomogeneityofthe medium is likely toplay él role. Thecombination of the kinetic and the reactor model makes up a complete matbematical description ofthe fermentation process and this model can be used la simulate the profile ofthedifferentvanables ofthe process, e.g. the sul> strate and producr concentrations. However, before this can be done ir is necessary to assign values to the par.uneters of tbe mnde!' Sorne of these pararneters are operaring par.uneters. which are dependent on bow me process is operated. e.g. the volumetric flow in and out oftlle bioreactor. whereas otbers are kinetic parameters which are 3ssooated with !he cellular sysrem . To assign values tu these parametcu ir is necessary to compare model simulations witb experimental data and herebyestiman.' a parameter setthatgives the bestfltofthe model tothe experimental data. This is referred to as paramete.r estimation. The evaluation oftbe fitofthe modellO the experimental data can be done by simple visuaJ inspection ofthe fit. but gener.illy it is preferential to use a more rationaJ procedure, such as minimising the s um ofsquared eITOl'S between the fiodel and the experime ntal data. ln the fullowing wewill consider tbe two different elements o.eeded for setting up a bioprocess modeJ , na melykinet ic modelling and mass balances. This wilIlead to a description ofdifferenr types ofbioreactor operation. and bereby simple design probtems can be illustrated. Even thOllgh parameter estimation is an imporrantstep in theoverall modelling cycle. we will notconsider tbis, since the tools available for this are extensively described elsewhere.

6.2

I

Kinetic modelling 01 cell growth

All researcbets in life saences use madels when resuhs froro individual experiments are in lerpreted aod when results froro several different expe.riments are compared wü.,h the aim ofsetting up a rondel m a r may explain the differeot observations. During rhe last 10 years: rhere has been a revolution in experimental tecbniques applied in life sciences. and this has made possible (ar more detailed modellingofcellularproct'sses. Furthermore. theavailabilityofpowerful computers h as made it possible to salve complex: nume rical problems with a reasonable computational time; l'Ven complex mar.hcmaticalmodels for biological pro· cesses can be handled and experimentallyverified. However, often such detailed (or mechanistic) models are oflittle use in tbedesign ofa binprocess. whereas they mainly serve a purpose in fundamental research ofbiological phenomena.ln t bis presentation we wiII fucus on models which are usefuJ fordesign ofbiop rocesses, bur in order to givean ove.cview ofthe different mathematicaJ models applied to describe biologj· cal processes we start the presentation of kinetic models witb a discussion of model comptexity.

MICROBIAL PROCESS KINETICS

StructurillO al !he eell 'aval

Unstructured No n-segre9ll t ed

Slructured Non·segrngated

Unstructurad Segregated

Structured Seg¡egaled

6.2. 1 Model structure and model complexity BioJogical processes are per se ex:tremely complex. CeIL growth and metabolite formation are the remlt ofa very large- number of ce!lular reactiOllS ¡¡nd cvcnts like gene express ion, translation of m.RNA iuto functional protejns. further processing of proteins into functional enzyrnes 01" structural proteins. and !K'Cjuencesofbiochemical reactions leading to building blocks needed for synthesis ofcellularcomponenbi (see Chapre.r 2). lt is cJear that a t:Ompleu" descriptioll of 311 these reactions and events cannor possibly be incJudcdin a mathemalical model.ln fennen· tation processcs, wheTe there is a large populanoo of cells. nonhomogeneity of the cells with respect to activity and function may add funher to the complcxity. In setting up fe rmentation models lumping ofcelJular reactions and e...-ents is therefore always done but the detall leveI considered in che model. i.e. the degree of lumping, dcpends on the aim ofthc mode.lling. Fermentation mode1s ca n roughly be divided' iota fout groups depending on the detaillevel induded in the mode l. see Fig. 6.2. The simplest description is the so-
,~

,

(6.1)

The doubling time td is equal (O the generation time rar a cell . ¡.e. [he lengthofa cell cyde for u¡úcellular organisms. which is frequentlyused by life scientists to quantify the rate ofccll growt.h. The speciftcrates. ar the flow in and out ofthe cell. are very impor· tant design paramcters since they are related to the productivity of the celL Thus , the specific productivity of a given met.abolite directIy indicates the capacity of tile cells ro synthesise uds metabalite. FuI'thennore. iftbesped.fic raCe is multiplied by lhe biomass concentra· tion in tbe bioreactor one obtains the volu.meni.c productivity. or the capacity of tbe biomass papulation per reactor volume. In simple IUnetic models the speci1ic rates are specified as runctions oC the vari-

MICROBIAL PROCESS KINET1CS

ables in tbe system, e.g. the 5ubstrate coneentratioo!!. In more complex models where tbe rates ofthe intracellular reactiODs are spedfied as functions ofthe variables in the ~ys(em, me substrate uptake rates aad productformation rates are given as funcnons ofthe intraeeUular reaetion mtes. Another dass oC very important design parameren; are the yield coefficients, which qu:mtify the amount of substr.lte re
41

Thupe
Glucose Glucose Glucose Glycerol Glucose Arabinose Frudose Glucase Glucose

Name

Kinetic expres"¡on

5 4 9 9

10 50 10 4 180

Tessier Moser Contois

e,

Blaclrm3n

J.I.=

l' ~' ( s 2K rr,al
and forinhibition by a rnetabolic product:

(6.9)

MICROBIAL PROCESS KINETICS

-

~ ---'+ plKt

P - ~DWtc.+X. 1

(6.10)

Equilrions (6.9) and (6.10) may be a useful way ofinduding product or substrate inhibition in a simple model . Extension ofthe Monod rnodel with additional terms or factors should. howevcr. be done witb sorne resrr.aintsince the result may be a modeJ with a large number ofpararn· eters hut oflittle value outside the range in which rhe experiments were made.

6.2.4 Linear rate equations In the black box modelaUthe yield coefficients are taken to be constanL This implies that all the cellular reactions are lumped jnto a single, QVerall growth reaction where subsO"ate is conver~ ro biomass. A requiremencfor this assumption is thar the re is a constant distnouoon of (luxes through al! the differenr cellular pathW Oand FQU ( = O. Le. me volume ¡ncreases. The mass balances for the different bioreactor modes can all be c1erived from a set of general mass balances, and we therefore start to consider tbese general balances.

6.3.1 General mass balance equations The basis for derivation of the general dynamic mass balances is [he mass balanceequation: Accumulated=Nct fonnation rate + ln - Out

(6.20)

The term AccumuJated specifies the rate of change of a compound in the. bioreactor, such as the..rate ofincre.ase in me biomass ooncentratioll

141

1-42

NlELSEN

du ring J. batch fermenlaDon . For subsrrates. tbe tenn Net formatlon r.ate is given by a substrate uptake rate (tha.t js regarded as neg-dtive being tbe withdrnwal ofcarbon from tbe system), whereas for metabolic products and biomass tlús tenn is given by the fonnation rate ofthese variables , TIIe tenn In represents the flowofthe compoundinro t he bio· reactor and the te.rm Out tbe fl ow of tbe compound out from the bio· reactor. For the ith substráte. which is added to the bioreactorvia the feed and is consu med by the ceOs presen t in the bioreactor, the mass baJancc is: d{coJV ) - - - ""' -'r xV+Fcf-F

dr.

IJ

.J

In"r

_ 'J

(6.21)

The first term in Eqn (6.21) is the accumulation term, the second term accounts foc su bstrate cOll5umption (or net farm ation), the (hird tClm accounts foc the ¡nlet, and the last rerm aCCOlmts for rhe oudet. Rearrangement ofEqn (6.21) givcs; dc,j

F ,

(Fout 1~

Tt=-r"ix+V'·j- V .+Vdt¡'· j

(6.22)

Since for a fed-batch reaLtor: dV dr

F--

(623)

and PO Dl = O the term within tbe parentheses becomes equal to the socalled dilUtiO D rate given by: F

D~

V

(6 .24)

Fo!' both a continllOUS and a batch reactor, che volume is constant. i.e. dV¡dt=O. and F=F"u,' and also foI' mese bioreactor m odes tbe term witbin tbe parentheses becomes equal to che diLucion cate. Eqn (6.24) thetefore reduces to Lbe mass balance (6.25) foI' any type of operatioo . dco.l _ _ f d~ - T~x + Dfc'J - 'I~)

(6,25)

The first tenn on the righthand sideofEqn (6,25) is thevolumetric mte ofsubstrate collSumpdon, whicll is given as the product ofthe specmc tate of substrate consumptioll and the biomass concentration. The second term acrounts for the addition..and re moval of substr.1te from the bioreacror. Oynamic mass balanl'es for the metabolic products are derived in analogy with those fo! me substrate5 and ralces tbe form:

~ = -T .x +D(cf -e ) dt '.' p.! p.!

(6.26)

where the first term on the right hand side is thl! volumetric formation r.Ltf oftbe ¡th metabolic pl·oduct. Normally the meraboLic products are Dot present in thesterile feed (O the biorcactor and c:¡is therefore ofien zero.

MICROBIAL PROCESS KlNmcs

With sterile feed me man balance for the total biomass is:

dlxV)

-- =~V-F

dt

16.27)

:Ji

0111'"

which in analogy with thesubstrate balance can be rewritten as: dx - :(,,- D)x dI

(6.28)

6.3.2 The batch reactor This is the classical operation ofthe bioreactor thac is use
(6.29)

d~

-de =- rlt'c( r=o)=c•.0 r' •

(6.30)

According ro these mass balances the biomass concentranon will increase and the substrare concentration will decrease until its coneentradon reOlches 'lero and_growth stops_ Assuming Manod kinetics, tlle mass balances for biamass and rhe lirniting substrate can be rearrangcd into one first-ordel' differentiaJ equation in [he bio1ll3SS concentration a.nd an algebraic equation relating the substr.Jte concentratioD to tlle biomass concentl'arion. The algebraic equanon is given by: (6.31)

and the saludan to the dlffe.rential equationfor [he biomass concentration i5 given by: P. n=r

f;=

- (1 + c"o +K.Y.,.}Io) In (X) XII

(X,-X)

- - K,- -In 1 +- Cl,lI

+ Y~,X()

Y",

c"o

16.Ja)

Using these equations [he profLles oftbe biomass and the glucosc con· centrations dllring a typkal batch culture are easily derived. as shown in Fig, 6,8. Since the substraJe cOn
'.

DK, Pm",, -D

(6.38)

lbus, [he coucentration of [he limiting substrate ¡ncrea.ses wilh the dilution rateoWhen substrate concentration becomes equal ro che substra te concentration in tbe feed the dilution rate attaios in maximum value, which is often called [he critical dilution rate:

O

-

c:ri, -

~

Pm..~cr + ~

(6.39)

When the dilution tate becomes equa! to 01' larger thao this va tue the biolUass i5 washed out of the bioreaclor. Equation (6.38) c1early shows thar the steady slate c.hemostat is well suited to study the influence of che substrate concentranon on the cenuJar function , e.g. produce formation, si nce by changing tbe dilution tate it is possible to change the substrate concentration as the on1y variable. Furtbennore, it i5 possible to study the influence of differe.nt limiting substratts on lhe ceHular physiology, e.g. glucose and arnmonia.

1
(6.43)

and ifDis lo be keptconstant there needs tO bean exponencially inacasing ~ed fl ow ro the bioreactor. Iftbeyield coefficient is CODstant combination ofthe mass balances for the biomass and the substrate gives: (6.44)

orsincep.=Y.. r.

di' -

y.( (6.46)

where xo' eJ.o and Vodefine the biomass conce.ntration , the substrate con· centration and tbe reactorvolume at the startofthe fed-batch process. As mentioned aboYe the substrate conc~tration in tbe feed e~ is normally very high and much higher than both the iniria1 substrate

147

148

NIB..5EN

concentration and the substrate concentratíon during tbe process (C.), Furthermore, a very high c! means that Y is larger than [be biomass concentration, both initiaUy and during the proeess. Consequently the ¡nerease in volume can be kept low even when tbere is a very largc increase in the biomass concentration. If there is an exponential feed f10\\' to che bioreaetor there will be substantial biomass growth a nd, SLnce the biomass concentration ¡ncreases, this may lead to limitations in Che O}supply. The feed Oow is therefore t:ypically increased untillimitations in tbe O} supply set in and thereafter tbe feed flow 1s kept constant. lhis wiU give a decreasing specific growth rateo However, since the biomass concentration nor~ mally will.increase. the volumetric uptake rdtc ofsubstrates (induding oxygen) may be kept approxirnately constant. From the above it 15 dear {bat mere may be many different feeding strategies in a fed·batch process and optimisation ofthe operaDon is a complex problem tbat is difficult te solve empiricaUy; and, even when a very good process Olodel i5 available. calculation of the optimal feeding strategy i5 a complex optimisation problem, In an emprncal search fOT rhe oprimal feeding policy the two most obvious criteria are : (1) keep the concentration of {he liruitingsub5trateconstant. and (2) kcep the volumetric growth rate oftbe biomass (or uptakeof a given substrate) constant. A constant volumetric growth rate (or uprake of a given subsn-ate) is applied if there are limitanoos in the supply of oxygeD or in heat removal. A COD5tant concentration of the timiting substrate is often applied ifthe substrate inhibits producr formation, and tbe chosen concentration therefore depends on tbedegree ofinhibition and thedesire te maintain a certaingrowth ofthe cells. The required feeding profile to maintam a constan t 5ubstrate concentration c•.ocorrespondlng to a constant specific growth rate iJ.o is quite simple to derive:. From Eqn (6.27)

uc!'

withFOU/ = O,

d(xV)

(6.47)

Tt=J.I.oXV oc

(6.48)

Since me: substrate cont:t!Iltration is constant the 5ubstrate balance gives: (6,49) 0,

(6.50)

Finally, the biomass concentration I'(t) is obtained from Eqn (6046) with.

c. = (,.o:

:!lE "" Xo

e¡z[X

1

QX(I

+ axoei'tlI

(6.51)

MICI\OBtAl PROCESS KINEnCS

~

..

0.3

lOO

,

~.s 80 t BIOREACTOR DESIGN

sterility considerations, vessel design and surfuce finishes, clean-inplace issues, and aspects ofbioreactor performance are ofien coromon to bioreactoI'll_ The bioreactor types used extensively are the airlift, stirred tankand bubble column bioreactor_

7.2 I Bioreactor configurations 7.2. 1 Stirred tank reactors

D--CJ

Stirred tank bioreactors consist of a cyJindric-dl vessel with a motordriven central sbaft thatsupports one or more agitators. The shaftmay enter through the top oc the botlorn of the Teactor vessel. A typical stirred tankreactoris showninFig. 7.1. Microbial culturevessels are generally provided with four baffles projecting into the vessel from tbe walli to prevent swirling and vortexing afthe tluid. The baffle width is Ylo oc Yu of the tank diameter. The aspect ratio (Le. height-to-diameter ratio) of the vessel is 3-5, except in animal ceU culture applications where aspect ratios do not normally exceed 2. OCten. the animal cell culture vessels are unbaffled (especially small-scale reactocs) to reduce turbulen
A flvidbed bed

I biofe;JttOf".

SenUng ~"

Fluidised biocBllIlyst

Llquid

-

~,

• • • • •• • • • • • •

Rueyele

Pump

th;;m compensate for any additional resistance to flow due to the separator.

7.2.4 Fluidised beds F1uidised bed bioreactors are suited to reactions involving: a fluidsuspended particulate biocataLyst such as me hnmobilised enzyme and ceU partides or microbial fiocs.An up-Oowing stream oftiquid is used to suspend or 'f1uidise' che solids as in Fig. 7.5. Geometrically, che reactor is similar [o a bubble column except that the top section is expandec1 to reduce che superficial velocity of the Ouidising liquid ro a level below titat nceded [O keep tbe solids in suspe.nsioll. COllsequently, the solids sedimentin the expanded zone and drop back into (he narrower reactor column beJow; hence. the solids are retained in the reactor whereas the liquid flows out. A liquid fluidised hed may be spa:rged with air orsome O[her gas to produce a gas-liquid-solid Huid bed. lftbe solid partides are too light. tbey may have to be artificially weighted. for example by embedding stainless steel balls in aD otherwise Iig ht solid matrix. A high density of solids improves solid- liquid mass tt"ansfer by increasing the re[ative velocity belWCen the phases. Denser solids are also easier [O sediment

BlOREACTOR DESIGN

TI

but me densi(}' should not be too high relative to that ofme Iiquid . or fluidisation wilJ be difficu)t. Liquid Ouidised beds tend ro be fuirly quiescem but introduction of a gas subsrantially e nhances turbuJence and agitanan. Even withrclatively Iight partides. the superfidal liquid velocity needE'd to suspend mE' soJids may be so high that the liquid lE'avE's tbE' reactor much too quickly, i.E'. thesoLid- liquid contact time is insufficient for tbe reaction. ln mis case. me Iiquid may have to be recycLed to eruure a suffidently longcumularive contact time with the biocatalyst. The minimumfluid¡sacian velocity - t.e. the superficial liquid velocity needed to just suspend the solids from a settled state - depends on severa! factors. induding the density difference between (he phases. the dia.meter of the particles. and theviscosity ofthc liquido

pad matrix ofsolids tharrnay be porous or a homogeneous non-porcus gel. The solids may be particles of compressible polymeric Or more rigid materiaL A fluid conraining nutrienr.s flows continuously through the bed to provide the needs of the immobilised bioca.talyst. Metabolites and products are released into the fluid and removed in the outflow. The flow may be upward or rlownward . bur downflow under graviry is the norm. Ifthe fluid Oows up the bed. the maximum flowvelocity is limited because the velocity cannot exceed the minimum fiuidisation velocity orthe bed will fluidise. The depth of the bed is limited by several (actors. including the density and fue compressibility of the solids, rhe need {O mainrain a certaifl minimal leve! of a critical nutrient. such as O~, through the entire depth. and the fI.ow rate mal is needed Cor a given -pressuredrop. For a given void volume (Le. solids-fi·cc volume fraction of che bed) che gravity-driven.flow rate tbrough the bed dedmes as thedepth ofthe bed toereases. Nutrienrs and substrates are depLeted as lhe fluid moves down the bed. Conversely. cODcentraUODS of metabolites and products iucrease. Thus. tbe environmem of a packe
• "l,

t u

'N.,

t

Harvest

A pacbd bed

¡ biore.:lctor.

157

158

CHt5TI AND MOO-YOUNG

7.3 I Biore.ctor design fe.tures

'"

19 21

"

,

•

SteBm

." , ==,F~'

A typical b ioreaetC>r.

( 1) reactO r vessel; (2) jacket;

(J) insubtlon; (-4) ~roud: (S) itloa.olum COmfleOon; (6) polU for pH, temperawreand dlnc>lved oxygM senSOrl; (T} aglt>ltor; (8) gas sp~rger: (9) mer.hanlal ~a!s; (10) n1d ucitlg gurbole: (11 ) mexor; (12) harvest nozzle; (13) Jadoot connecdoM 5;; ( 14) u mple ,.., I~ wlth ltl!am

co nnecrion; (IS) 51g1lt glass: ( 16) connecliom ror a
60

I

CHISn ANO MOO-YOUNG

A biore:actor with alr

inlet and ewustgroups arranged

elean steam

_ ~" Fllter Exhaust

for In-place sterillsadon wlth

steam.

SI,h'

.. (

g1885 .......

(SG)

Filter

.~-"~

Air=:-

r--

'0

Bioreactor

Sparger

Thermody namfc

s-t ¡~

Hal'\lest

____----- valve Product

st!l3m trap

Clean

k, candBnsatf Waste

path through thevalve body. The valves maybt': operated manually, but pneumatic operation under automatic control is more efficient aud reproducible. '!be bioreactor is sterilised either filled with the mffiium orwitbout.Empty sterilisatiou is the norro in cell culture applications where the media are invariably heat sensiti~. In this case. filter sterilisation is osed to srerilise themedium. Proper closing aud opening sequence ofthe various valves is important for attaining sterility aud I?Teventing recontaminatiOIl from the adjacent non-sterile areas. Once the sterilising steam supply is shutoff, the bioreador is immediately pressurised with sterile ah' through tbe air inletfilter so that any leakage from tbe outside to the sterile vessel is prevented. In bioreactors with stirrer 01' foam breaker shaft penetrations. the shaft seals require. suitable. piping and valves for steam ste.rilisation and maintenance of a sterile barrier fluid betwee.n the·('ontents of the fennenter and the outside.

7.4.2 Clean-in-place considerations Industrialbioreactors and much ofthe other processing equipment are deaned in-p11ce using automated methods. Automation ensures consistency of c1eaning and reduces down-time (Le. unproductive time of a machine). Attaining an acceptable state of cleanliness is essential to prevent contamination and IToss-contamination of biophannaceuticals and tbod products. An effective and trouble-free cleaning capability requires atlention to design ofthe bioreactors and the dean-iD-place (ClP) systems.At any given time a plant may have. several bioreactors at differentstages ofprocessing and some emptyreactors which need ro be cleaned along with any associated transfer piping. The CIP devices and procedures must be matched to the speci.fic configuranon ofthe biareactor and to the ferme.ntation process to emure satisfactory cleaning. GeneraUy, a bioreactorwhich has processed hybridoma or othe1' animal ceU cu.lture broth is far easier to dean than one whic.h has processed broths oí Srreptomyces or rnycelial fungi such as PenidUium.

SIOREACTOR DESIGN

e,P ,.

,",

exhaust

I

liquid

~. ~",: ...Air ¡"1m

r

-

-

¡

CIP s pray 0011 - I rem ovable)

L

CIP IIquid Transferl1ne

Bioreactor

rI

el P return

Design aspects To ensute removal of solid particles and avoid sedimentation. the minimum fiowvelocity through piping sh ould be 1.5 rn 5- 1. but ahigh~ value of2.0 m 5 - 1 ¡s preferred. ln addition , the piping should be free of dead spaces as much as possible; ifunavoidable, the depth ofthe dead zone must be less than twopipe diam eters te ensure adequate deaning using CIP tcch.niqucs. OnJy valves with a metal·bel1ows-sealed mm . or diaphragm and pinch valves are recommended as all othervalves carry a significant risk of contaminating reactors with accumulated debris during the final rinse cycle. For adequatecleaning. the ClPsolutions are sprayed into the reactor through one or more removable. static or dynamic.spray baBs. ordynamic spray nozzles (see Fig. 7.9). In addition. the piping fo!" air exhaust. which 15 upstream oftbe exhal1st gas fiI{er, and the air ínlet piping. shouLd also receive the deaning 5OJutions. For deaning with jet spray. pressuI't$ of 308 ro 377 kPa (absolure) are optimal Permanently installed spray heads are no( reconunended for bioreactors because oi potential difficulties with steriJisation, These devices must be inserted into the reactor through one of [be ports on the head plate. Ofien. the spray heads are designed to spray the upper one-third ofthe cankand the falling liquid film inigates the remaining sw:face. For bioreactors for parente!"al (inject:able) products and othcr biopharmaceuticals. potable quality deionised water i8 recomlnended for all pre-rinsing and detergent fonnulations. Pre-rinse should be on a once-through basis witbout reciI"culation. A five minute pre-rinse is usually sufficient for bacterial, yeast and animal ceH culture reactors. Following pre-rinse. 1% (wfv) NaOH ar 75-80 ~C should be circulared through the equipmentso tharall productcontactsurfaccs are exposed ro this solution for 15-20 minutes. TIte alkali should be discaroed afterwaros. Dilution. contarnmation withsoil and microbial spores that can survive for long periods aud los5 of quality definition of che starting material far the Dcxt deaning. are sorne ofthe arguments against l'e-use of c1caning chemicals. A deionised or reverse osmosis water rinse al 25- 35 oC is used to remove all alkalifrom the system. Process equipment

DeI..... ry 01 me dean-In(ClP) Ilqulds. [O tN bioreactor. The fIow of CIP rolutions is '~nted throu¡h the tnnsfer in
16 1

161

CHISTIANO MOO-YOUNG

fur ptoducts mar are injected into the body mUS! nndergo a final wash with hot water-for-injection grade water. This erunres that 0111 residual water compl1es with rhe requisite quality standards. In mechanically agitated bioreactors. the spr.Jyofd eaning solutions may be unable to achieve proper deaning of m e agüators, magnetic couplings. mecha nical seals and the lower partions of baffLes. Therefore, filIing afthe vessel ro at (east above thelevel ofthe lowerrnost hnpeHer and agitation at impeller Reynolds numbers (see Se¡;tion 7.6) of lOL IQ8$ is recommended during pre-rinse. alkaJi recirculalion and the final rinse. Agitation for 2- 3 minutes is. sufficient ro dislodgc adhe.ring dirt or sollo These recommcndations assurne thar re'H.:tDrs are being deaned in·place sooo afte.r use and caking ofdirt b.ts nor occurrcd.

7.5 I Photobioreactors Cenain micro-a1gae and cyanobacteria provide imponant chemicals. such as as.talGlnthin and ,B
ofthe heatoutput typically comes from microbial activity. Heat production is especially large when the biomass is growing rapidly in highdensity fermentations and when reduced c.lrbon sources suro as hydrocarbons and rnethanol are oseo as 5ubstrate. Themetabolic heat generation rate in kJ l-l·h 15 typicalIy about 12% ofthe 01 consumption tate expressed in mmol 0ll- l·h. Heat removal in large vesscls bccomes difficult as the heat generation rate approaches 20 MJ m - J. corresponding to a n 0l consumption rate orabout S kgm-3.h.ln addition [O metabolic heat mecharncal agitanon ofLhe broth produces up to 50 MJ m-l. In ait driven ferment~. a1l e:nergy input due to gassing is eventuaUy dissipated as heat. Consequently. a fermemer must be cooled to prevent temperatute rue and damage te culture.As the scaleofopt'r.ltion increases, heat transfer and no[ 01 mass transfer hecomes (be limiting process in bioreactors because the available surface area rOl" cooling decreases as Lhe fermenter volume increases. Thmperature is controlled by heating or cooling through external j ackees and internal coils. Lcss frequently. additional double walled baffles, draft tubes or heatexchangecs locared ¡nside the fermentation vcsse! are needcd to provide suEfkient heat transfer su:rfuce aTea. TIle rate ofheat removaJ. Q.¡, i5 related to (he surface area,J\•. avail· able for heatexchange and (he mean temperature difference. tJ.T. thus, (7.1)

The overall heat transfer coe.fficient, UH' is the inverse of!be overall resistance. to heat transfel". During cooIing, heat flows from the broth side to the roolin gwat~ in a jacketOl"coolingcoil. TIletransferring heat

BIOREACTOR DESIGN

t - - - Jacket

Cutlure

broth

--1--- Cooting water

1'-----11--- Water

film (1'hl l

A+i-----t--- deposils Fouli ng (1/h¡)

Broth film l1ih.,)

...-Cooting

water Direction ----'Lb~-f1fL. of heat tran sfer

---11--- - - - - --

Fermenter wa lt (dwfk..,)

eocounters several resistances in senes as illustrated in Fíg. 7.11 : the thin sta'goant film of broth on tbe [mide wall of tbe fennenter ; the ml."tal wal l ofthe fermenterorcooling coil; scaleor 'fouLing' de posits on the cooling water side; and a thin S[agnant fibn of the cooling fluid 00 the jacket side of the fennenter waU. These individuaJ resistances are related to t he overall resistance as folJows: 1

1

U.

h,

OYer311 resislanct"

broth lilm

resistance

1

+

+ vessel waJl n!Slst.am:e

h, foul1ng fl.lm resistanct"

+

(7.2) water film resistance

'Ihe film heat tTansfer coefficient is infIuenced by llumerous fac..:tors, inc1uding (he density and the viscosity of the Huid, tbel'mal conduc tivity and heat capacity, the velodty offlow a l' sorne otber rneasure of tu 1'bulence (e.g. power input. gas flow rateoete.). and the geornetry of the bioreactor. 111e manyvariables that affectheat transfer can be grouped into a few 'dimens ionless numbers' to. greatly simplifY the study and description ofthose effects. The groups relevant to heat transrer a nd the corresponding fluid dynamics (e.g. Ulrbulence) are as fo110W5: totaJ beat tr.msfer Nu (Nusselt number)= condu~üve bea[ tra.nsfer

Pr(Prandtlnumber) -

momenrum diffusivity h iffu' . termal d SlVlty

h.,d

k,-

(7.3)

(7.4)

inertia I force PlVId Re fReynolds number) : . , VlSCOUS orce ¡.LL

(7.5)

' ~a~ti~o~n force Gr (Grashof number) - -~gr~'7_"~'r VlSCOUS force

(7.6)

165

166

CHISTI ANO MOO-YOUNG

In these equations. d is a charaC'teristic length (e.g. diamerer oftube or impeller). The above noted dimcü5ionless groups expl'CSS (he relative significance of the various factors infiuencing a given situatÍon. The value ofthe Nusselr number rells us about the relative magnitudes oí total bcat transfer and that transferred by conduction alone. Ihe Grashofnumber is importanr in situations where 60w is produced by density differences thar may tbcmselves ~generared by thermal gmdientS (hence me!ll'f3 in [he Grashof Dumber). The Reynolds number is employed indcscribing fluid motion in situations where fol'ccd con Ve(:· tion [$ predominant. Correlatioru fur estimating thc film heat rransfe!' coefficient, h", are often briven in rerlllS ofthese dimensionless graups. Equations for quantifying the heat transfer resistances of tbe fouling films and films of heating and cooling fluids are disclISsed in readily availablc process engineering handbooks. Suitable corrclations fuI" cstimating the heat tcansfcrcoeffidem. h D• for m e film ofLiquid OI culture broth in various (:onfigurations ofbioreactors are summarisoo in Table 7. 1, Note thar me couelatíons given for mrred ve.55e1s utilise a Rcynolds number thar has becn deflned in terms of the tip speect ofrhe impelJer. lo somecases, the correlations in Table 7.1 require the rhermal conductivity. "-r. and the specific heat capacity. Cp ' ofthc ferrnentalion broth for estimation of the heat tró\nsfer cocffici.ent. For most bl'oths. the va lu e~ ofthase paramerers are close ro those ofwater. The film coefficielltgencrally illcreases with increasing turbuJence. gas flow ratc, 3nd the agitation power input. lbe roeffident typically declines with increasingviscosity ofthe culture broth. The geome nyof tbe bioreactor affccts the film heat trnnsfer cocfficient mainIy by illflu· cncing the degree of turbulence 01' related parameters such as the induced liquid cirrulation rate in airlift vessc1s. lo bubble columns the film coeffil.'ient i~ independent ofthe column diameter so long as che diamerer excecds abour 0.1 m. SimUarly, in bubble columns the ho value is nor affcc:ted by the height ofthe gas-free fluid . 111e value ot'h" mereases with increasing superficial gas velocity, 01: power input. bu t onJy up ro a veladt)' of abour 0.1 m s l. Furthennore. for jdenticaJ specificpowerinputs. bubble columru and stirred vessels providequite similarvalues ofthe hcat transfer coefficient. Litcratu.re on heat [rarufer in airlift reactors is sparse. Equations developed fu r bubble columns fTable 7.1) may be used to provide a low estimateofh" in aidiftvessels when the induced liquid circulatiOll ratcs are small. Undel' other conditions, the coefficient in airlift reactors can be more than two-fold greater rhall in a bubble column. When liquid flow velocity does Dot exceed abaut 0.015 m S- l . [he film hcat transfer coefficient is largely independent ofliquid velocity: however, for higher liquid velodties hn increases with Iiquid velocity as follows: (7.7)

A large amoune ofpublished datais available on heat rrarufer in verti· cal two-phase Oows. Sorne oi' this information may be applicable to airlift reactors provided tha[. the fluid properties, gas hold·up and rclative velocities ofthe two phases are idenrical in rhe airlift and the

. ·iiiiik;~;¿i'~w.áiaiá~aihlmb~~;f#~OO~~~t:i~~tiji.i!ñ~~¡~iW~:.@iis!~~rm!i!~~ . ..H~,lt~H#¡¿¡tH , .m ·'! · t~· .. ¡;"'liffl "r.¡'·" ... • ...... ....... "::·![""t··¡" ·h .• •• " ....... . ....... 'P In~ \ ...... . U ..... :::¡: .• • _.. .. :, ' . ·,:m.,:fH~~i¡Hgñi;.·imi,~m, •• • ..... ........ ¡~mnml~¡H¡mn , .. .. ¡

Bioreactor configuration

t. Stirred tanks (coiI5)

Ranges

Correlatlon

(",w)"" = 0,87 (dl NP )"" (Ck",), r

-ho
3. Bubble columns

ho = 939 I U~(::r3S

4. Bubble columm.

S. Airlift vessels

For cooling coils; Ne'Ntonian tluids

:::1'-

/J.i..

Forjacketed vessels: Newtonian fluids

Newtonian broths

10- 1
H v liquJd hl1:lght

)

molar mass flux

J,

molar lllilSS fiux across gaJi film JI molar ma5S flux ilcroSS liquid film k mass transfer coeffident k, gas film mass transfer coefficient. "1 liquid film mass transfur coeffident kla volllmetricmass transfetcoefficient K OYel'all nl.:lSS transfu coeffident K

1

m m1 s ·1 m barm' mol ~ 1

m

mol m- 2,s mol m ~J ,s mol m- ~ · s ms- I ms- I ms- L 1S~1

ms- l

COllsistcncy index

power law index N impeUer rotational speed OTR OJ transfer r (eU where me reaction [3kes place. NB: Products form!!d take ah!! roverse l"Q~te. pellet.

immobil~lion

.l CroSS

8.2 I The mass transfer steps 8.2. 1 Effects of transfer limitations lf one mass transfer step is slowcr than tbe key kinetic reaction step. 1t will limit the formation of a desired product from a se1ected substrate. As a resu lr. [W() effects may be observed. botb with freeJy suspended cells as well as organisms immobi lised inside cell aggregates 01' solid partic1es: • The ovemll J'~actjo" rute Is beluw 01(' thcon:tirol maximum. und the process output is slowcr titan desi~d. This is the case in the formation ofgluconicacid from glucose by the acrobic bacte.rium, Gluconobucter o.\yda ns. Hete, tbe overall reaction rnte is detennined by thc fate O]t which O~ is transferred to tbe Liquid phase. After reLiev· ingthe limitation. rhere is no irreversible effecton this particu lar micl'o-organism. Allothel' example 1S a limited supply of sugac lo immobilised ceJ Is due to slowdiffusion inside an immobilisatlon canier. lbe overaU rate ofproduction is ofren reversibly reduced. However. the:re: are a1so examples ofsystems where me biosynthetic capacity ofa ceH i5 ineversibly damaged afterimposingan 02

175

176

NOORMAN

transfer Iimitation (e,g, in penidUin fennentation). Such processes are very St!nsitive to mass transfer limitations . • !he selerrivityofthe reacrion is altered. Forexample. 0l serves as an ele C61- lbe.oil phaseis present in [he form of smaU droplets and mass uansfe.r resistance is a e tbe side of tbe surroundíng water layer_ Also. the exrnaoge of material between a solid phase (substrate partic1es, parceles tbat coutaio microoTl:fdnisms) and the liquid phase obeys similar principies.

8.2.3 Transfer inside a single phase lnside a gas bubble. oroil dropler lhere is usuallyenough motion to guaraotee a quick transfer of molecules ro the interface with the water pbase. so the resistance is at [he water side ofthe. inrerface. lftbe d istances in tbe bu!k liquid phase to be bridged are rela tively large. a traos' port resistance can occu r in this pbase. $uch a sltuation is t'ncountered in large bioreactorswhere bulk liquid mixing is usually sll~ primal .ln industrial practice. ir is important to reaUse thar one has te live with this potentiaJ Iimitation . The.refore Its effectson the microbial reacrion system sbould be borne in mind during process developmentwotk. Mass tr.msfer Iimilatiolls imide a solid phase can QCcu r within biocaraJyst particles thar contain immobilised micro-organisms. either as a surface biofilm attached to a carricr, or dispersed throughout the carrier material. Alternatively, the micro-organism (usual1y mam en· tous) itselfmay be preseot as a c1ump 01' pellet. A substnlte cntering the particleor pelletmay be consumed so fast th;:¡t nothing enters the inner part of the particle. so that the efficiency of tbe caCalysc is below maximum. Also, che reacrion {llay be slowed down because a toxi
e

"fwo·fllm lheory: mass

tranU'era
P=Pin

reactor height

•• • ••

in the gas pitase H th~J.¡r bubbles tr.lvel up through J.l'f!acror YeS$~ .

¿,~:"¡'-I~-1r---- P == complex p

". Iinear P (-hydrostatic pressure)

p

I

I-_~_+--- p = lag mean p

.•

I

P = POllt Pin

partial pressure

operating volurnc) the bulk Iiquid pha!ie is assumed tO bt! well·m.ixed and henceCis constant throughout the liquid oHowever. in pilor planrs or prodUCtiOD scale vessels (> 100 lirres) this win nor be the case. and local concentration variations nee
MASSTRANSFER

In a stirred tank reactor (Chapter 7. Section 7.2.1) the flow phenomena are determined by the balance between.aeration forces and agitation forces, and large local variations in combination with a number of tlow regime. transitions make a precise quantification ofmass transfer difficult. Sparged gas: is usually rapidly coLlecte
l;a = 0.026 (P JVl·4. (v¡(pofp)o.~

18.12)

non-coalescing: k¡a =0.002 (PJVI)O.1 (vgpJp) 0.2

(6.13)

coalesring:

In a coalesci.ng liquid the influence of aeration is larger than agitanon, while for a non-coalescing liquid the opposite is true. (Nore that me COI"relatioru are independenr ofthe agitator type.) The energy input by agiration is a vital variable in Eqns (8.11), (B.U) and (8.13). The amount of power dr.iWD by a stirrer of diameter D with a rotational speed N is lIsuallyexpressed as:

~

IB.14) The impeller power number is a function oftbe aerationrate. tbe impeller Reynolds number (= PI N IJl¡ ¡,.t) and the impeller type (see Fig. B.5). When a brotb is aerated, Pa generally falls due ro a growing size ofthe cavities behind the blades. A typical value fer a RushtOll impeller is a factor 0.5, while for a Scaba type (6SRGT] tbere is hardly any drop.

Prochem 10

1!Xl

kWm- 3, wegec:

Bubble column; Egn (8.10):

kl u= 0.05 s-J

Stirred tank;

kl t1=0.14s- 1

Eqn (8 .12):

Assuming (C'"-C)=O.59 mol m- l -0_l0 mol m- l =0.49 mol m- 3 , the bubble colllmn OTR is 0.024 mol m -l·S, whereas for the stirred tankit is

10000

Impelle r RevnQlds numoer, Re Som~

Example The o~ transfer perfonnance oC a stirred tank reactor is generally better than ofa bubble column witb similar geometry and aeration rateoFor a coalescillgliquid ina vessel ofl00 m J reacrion volume. with tankdiameter 3.5 m, aeration rate 1 vvm (or 1.67 Nrn 3 s-1 ), head-space pressure 2 bar, impeller diameteJ: 1.75 m. and powcrinpur per unir volume P/VI = 2

'QIXl

unaflrlIed

Impeller pOWt!r numbers, Po' as a

function of the Impener Reynolds number. Re. In me turbulent now reglme (Re>4000) in the absence of aeration the value of Po for a sClndard slx-bladed Rushoon turbine Is consumt al Q. 5. ror a Sa.ba 6SRGT I'''pene.- it l. 1;3. 1.7 in 1. whereas for Newmruan media 11 =-1.

HASS TRANSFER

llxaOlple A pseudoplastie broth in a bioreactorhas the foUowing properties: e" "" 30 g 1- 1, K= 1, ti =0.4. The stir.rer speed is 3 revolutioru pers. Using Eqn (8.16), che apparent viseosity i5 0 .13 fu s. According to Eqn (8.15), k¡a is reduced to 51 %ofthe value in a lowviscosity broth. Whatwould happen ¡fthe biomass concentration o r [be impeUerspeed doublcd? (AnsWers: with d ouble biomass conC
Air off

I

tla '= (dC/dt - ql/( C· - C)

e q .. dCldt

~Iuat_d.

for example uliing ¡ Io¡lrithmlc plotof (~ -q

t

Alr on

n . ,lm.. Time

MASS TRANSFER

phase romposition will Dot be uniform afier sparging with N1(or [he gas hold-up must be built up again after shutdown ofthe ai,. flow). In any case, a rapid 0 2 electrode is a p,.erequisite for accurate results.

8.5

Large bioreat:lor

I The effect of scale on mass transfer

8.5.1 Scale-up In large-scale bioreac[ors. 0l transfer is usually better than in a labora[ory scale orpilotplant reactor. This is due to a larger contribution of the gas phase (higher superficial gas veloclty). and a larger driving force (high headspace pressure and hydrostatic head). Example Consider two geometricaJly similar. ideaUy mixed stirred tankreactors, one ofO.1 m l reaction volume. and one oflOO m 3 , 111e H.jTv ratio is 3,0, and the D{ry ratio 0.5. Scale-up is (arried out according to a constant power input, Prv;. of2kW m- 3 • aconstant relalive air flow rate ofl vvm, and a constantaverage pressure in the broth (determined by [he headspace pressure plus the hydrostatic head) of 2,45 bar. Assuming thar there ¡s no deplerion ofthe gas phase, the followingcomparison is made (coalescent brorh, C"= 0.2-4 mol m- J ar 1 bar, C= 0.10 mol m- J ): 0.1 ml : \1,

pJp= 0.007 ms- I "La = 0 ,046 S- l OTR = 0.022 mol m - l·s

100m3: v. vJp= 0.071 ms- L

lela

=0.145 S-

I

OTR ::: 0.071 mol m - J·s

What would be tbe OTR diffe.re ncc for a non-coalescing bl'Oth? (Answer: OTR= 0.07 orO.12 m ol m- 3 ,s, resp.) In a large bioreactor the OTRhas maximum limiLS due to rbe following restr;crlve conditiOn$, • Tbere may be mecha nical construcnon difficulties invery large ferme nten (> 300 01 3), Furthermore, liquid rransport and mixing will become very sJow compared to mass tr.msfer and reaction. and tbus rule tbe overall reac tion raleo Cooling limitations may become more significant. • The average power inputshould notexceed 5 kW m- ~.]fhigher, the mkro-organisms may be mechanicalIy damaged in areaswhere the value is locally much higher: in addition the energy costs and investmenr costs for rhe motorwill become excessively high. • !he pressure corrected superficial airvelocity should be beJow 0.10 m S- l. Compressor costs are restricting, and high gas bold-up will ¡ncrease ar the rost ofbrorhspace. • The head-space pressure has a maximum fue mechanicalreasons_ln addition CO 2 partial pressure will also ¡ncrease. and inbibit growth and production. • The gas pbdse canDor be consídered ídeally mixed. The O}partial presrure will faU as the bubbles travel up through tbe reactor and rhis reduces rhe driving force for mass transfer.

Low

o)(ygen

Air liquid now and DI tranger In a large biore:lctor. Most

ofthe 0 1 is transferTed In me region n...artlle impeller. In the circulation loop. Le. me path the brom travels Imm thft lmpeller; Out

into the body 01 th. reactor and back to the mpder. more 0lls consumid than cransferred and tite

0 1 conCf)mraúon wi!! decrease. In a ful.sale stlr~d t:ank reactor the l!quld circuladon loop can be as long as 10m. Wlth ~ IIquid 'ieloclty of r m 5- 1 che. me~n circulltion timI wiD be 10 seconds. As. a worst

cas.e Istimat. (no tr.msl.r at atI ouuide the impeller re¡ion).ltwlll Clke 10 s befOl'l'- the 0 1 wil become dep!eted In the /uo¡:>. lherefon=, theO l concent~tIon In me bottom Co:!mp;irtmellt sl'lould be JO tllgl'llhalloal OepIetlon. whlch can be 6elrlmentallorthe microbial state and produa.

fonnati<Jll ratl.'s~, No:!t. that Eqn (8.8) prMka thu tllfs wll r.d1lA cM mus trandu ratl blcaUK m. drMng fon:l (C"- q in che boaom part Is Iow as both Cand C will be h1;tl.

185

186

NOORMAN

lnreactoTs largertban ca. 10m3, the prcx:ess~oruquid transportand mass transfer become comparably slow. Mass transfer and liquid e~-ulatioll willinrerfere, and should be treated togelber.ln a stirred tankreactorwitb one si ngle impe1ler, most or the Oz ttansfer takes place in the impcl1er 1:One. A comp.nison of 0l rransrer in the bubble column and the stirred tank(see example in SectioD 8.3.2) revealed thatoutside the impellerzonf' the OTRmay be only one tbiId ofthc value around (he impeller.The im por· tance ofdrculation loops is described in Fig, 8.8 onprevious page.

8.4.2 Scale-down As illust:r.lted aboye, micro-organisms can experience a continuously ehanging environment when travclling around in a large bioreacLOr. This may giw undesired scale-up effcets. To avoid these problems the large scaJe should be taken as the poi nt of referenee, and the possible effects should be studied by simulado n ofthe large-scale val'iations in a sma U·scale experimental set-up (think big, ael smal1). Limitip.g factors 00 a large $Cale, such as mass transter, are thus sealed down and can be srudied and minimised in a practical and economic way. In reality, scale-down cannot be precise. beca use the large-scale con· ditions are difficult to detennine, and a lso too complex to be fully undcrstood. Several tools are available to find adequate SOIUtiOllS to down·scaling: • Two-compartmentreactor ser-ups ean be used, mirnicking (he two mosti mportantreaetor zones, and recirculation ofbroth between the zones (see Fig. 8.9). The size oftbe two eompartments and the circulation rate are e rioeal. but also the Oow type in e3eh companment, Le. rangingfrom well·mixed ro plug flow. • DeveJopment and (large-scale}verifieation ofsimple or more sophisticated m3thematical How models fuI' me large vesselcan be c3rried ou t, :m d then these used to design t he sealed-down experiment. • Wcll
Nou:l: • Ce ll dE'bris dell$ity!h.·~nds on compolitinn. e.¡¡r. lipld co nTen to

simplicity of operation a nd low tosts, vacuum filters are frequE'ntly used for clarification oí" fermenta tion broths conta ining 10-40% solids by volume and partides wirll sizes ofO.5-10 ~m . The bestlcnown are rotary drum vacuum fllter and filter press. The former type is commonlyemployed for filtration offilame.ntous fungi and yeast cells . and

is scbematical1y presented in Fig. 9.2. Resides simplicity and effectiveness offiltration . its a t tractive features a re Iow power consumption and

Cost of recovery Highest

< < < < < Lowe"

18'3

190

HATTI-KAUL ANO MATTIASSON

Washing

A rm;u-y drum vacuum f~ter. The

filtralion eI!'!ment

compriSe5 erofstages {maximum 5even} using rhe vapouJ' of ·ne stage as a heating 50Ul'Ce for the nexr. In falling film evaporamrs, tbe liquld to be concentrated flows .own long tubes and distributes unifonnlyowr lile hearingsurface as thin film_ lbe vapours fiowing in rhe same direction ¡ncrease the .near velocity of the liquido rhcreby improving the heat transfer. .csidence times in the evapOl'ator are in rhe order of minutes. Tbese vaporaton are suited fol' concentrating viscous products (up to 200 lPa), and are frequently used in the fennentation indusrry_ Plateevapratol'S, where the hcaring surface is a pIate as oppo~d ro ;:¡ rube. have relatively large evaporation area in a sITIal! voJ ume, but the posslbility ftreatingvi5cous andsolids-containing fluid s is limited. For higher vis:lsities. forred filmevaporators with rnechanica11y driven liquid films re suitable. in sorne cases producing a dryproduct.1'he residence time

195

96

HAm-KAUL ANO MAITIASSON

ranges from a few seconds to a few minutes. CentriCugal forced..fUm evaporators pe.rmit a further redurnon ofthe residence time so that even the heat labile subscmces can be concentrated under gentIe conditions. EvapOl:ation takes place ona heated conical rurface or plates over which tbe transport of the liquid takes place through the centrifugal force produccd by the rotating bowL

9.5.2 liquid-liquid extraction Liquid- liquid extraction is applied on a large scale in biotechnology both for concentration and far purification. It involves [he transfer of solute from one liquid phase to another. The efficiency ofan extraction process is governed by the distribution ofsubstances between the two phases, defined by the p.aTtition coeffic:ient K (= concentration of sub-stance in extraet phasefconcentration of su bstance in raffinate phase). A partition coefficient well removed from unity is desirable. TIle physico-chcmical properties oftheproductinfiuence thedemands on a liquid-IiCjuirl extraction process. as illustrated in the following sectioos. Extractlon oflow molecuJar weight products Small lipophilic target molecules are extraeted using a.n organic solvent. whereas for hydrophilic compounds it may be difficult to design an efficient extraction process. Extraction in organicsolventcan be done in one of three ways: Physical extractlon: The compound distributes itself between the two phases according to its physical preference. Th.is applies [O nonionising compounds and the extraction isoptirnised by screening for [he solvems thar would lead to a high K value aDd also show a max:imal difference in K for the differentcomponents present in the crude mixture. Dissociative extractiOQ,: Difference in [he dissociation constant of the ionisable compone",S i5 exploi ted tO achieve separation. and these differences are onen large enough to overcome an adverse ratio of par tilion coefficients. Exuaction orpe.nic.ilJin 3nd some other antibiotics are typical for this type ofextraction principie. Reactiveextraction: A carrier, such as an aliphatic amine or a pbospiloroos compound. is added to the organic solvent which forms selective solvatioo bondsorsrokbiometric complexes thar are also insoluble in the aqueous pha5e. Thus, the compouud i5 carned from the aqueous to the orgaruc phase. This type of extraction is advantageou5 fur compounds thar have a high solubility in aqucous medium. e.g. organic 4

3ods. In most cases, cells and orher particulates ilre removed prior to

extraction to avoid the form arion ofemulsions ar the interface. After extraction, the productis recovered from the solventeithel' bydistilling off the product in case of a higb·boiling solvent or disrilling off the solvent when this is low boiling. lf the product is heat st'nsitive. ir is recovered by back-extraction into a new aqueous phase unde.r conditioos different from the firsrextraction. e.g. penicillin 1S extracted into butyl acetate or amyI acetate frem the fennentatioll medium ae pH 2.5-3.0 and back-extracted into aCjueous phosphate buffer atpH 5-7.5.

DOWNSTREAM PROCESSING IN BlOTECHNOLOGY

For hlgh extracLion yields. multi·step extraction in a countercurrent mode is used. which provides abo savings in both solvent and time. Oifferent kinds ofextraction equipment are availabte induding mixer-settlel'S, coIurnns and ceJltrifugal extractors. Tbe latter are often used in exCTaction ofantibiotics and steroids; representative examples beingthe Podbielniak extractor. Delaval contactor and westfalia extraetion-decanter. Superoitical fluid (SCF¡ extraction has beell consjdered as an alternative [echnique to convencional extractioll which has the disadvantageoftoxicity and Hammabilityororganie solvenrs. SCFs are materials thatexistas fluids aboye their critieal teOlperatureand pressnre. re."pec. tively. Many ofthe properties ofSCFs are intermedia te hetween those of gases and liquids; e.g. their diffusivir.y is bigher than those of liquids while viscosity is lower. The atttOlctive feature of SCFs as extraetants 1S that theirsolvent properties are highly sensitive toehanges in both temo peratureand pressure, which provides the opporrunity oftailoring me solvent strength lo a given application. Superc:ri tical COz is most eom· monIy llSed for extrac:tions because ofits r elatively low critieal tempero ature (31.3 oC) and pressure (72.9 bars). Supercritical extraetíon is still rather expensive. though a few large-seale applieatious in the area of fuod processing have demonsrrated the possibility of economicaUy viable operations. By adding a co-solvent such as ethanol. in small amounts. ir is possible to modífY the properties of the system and thereby influence the extraction behaviour. In a typical extraction precESs. the compressed SCF is con meted with che feedstoek to be e,,:tracted in an extraetíon column, and tbe loade
Extnction in a'luaous two-pnas.e system. The components of die twO-pl'we synem are mblf!d dirKtly WldI the cruda hornoge/l3.te. EquiJibradon Is dorMI by ¡ende ml~"r1g alld is foll~d by phase sepoa ....t1on. The protlin prodyct partitioned ro th. top ptwe Is subsequer1tty re(o....red enner by a sec:o nd extr.Ktion nep. where it. i!l u-anderred i¡¡oo a new salt phase. or by d:rect adsorpticll'l O!'l tO a dlromnography matrix. Recyd"'i of TM ph3 Sf! componenu ¡, normally possible, mus rnil1lmlsln¡ th, matcrlll COSts.

CruOe homogenale

+ - - - - . PEG end salt - - -- - - -1

-

Equilibrium + phase separatlon

Cleaning & recycling

I ! \

PEG-riGh

LOp pnase (productl

S~lt·rich

! ! Clennlng & ! rocycling

! I

bottom phase (cell debris, prota ins,

~~_- _ n_,,";"";~''--i

PEG-dch

Salt-rich bottom phase

top ph8se

(productl

Desalling by utl rafiltllltion

I Recycling

! i i

Retenllte (product)

Permeate salt __ solution

J

homogenates having high viscosity and heterogeneous distribution of partide sizes. Otber aUractive features ofthe technique are bigh capacity(biomass tovolume ratio) and str.r.igh tforward scale-up. The process is easily adapted ro the extracrlon equipment used for water~rganic solllen[ systems. Figure 9.4 shows a schematic presentation of pl'olcin isolarion and partía] purificaríon in AUS . Por industrial scale separations. PECfsalt systellls are used beca use of their relative]y low costo lnexpensive and biodegradable po]ymers may aIso be considere
DOWNSTREAM PROCESSING IN BIOTECHNOLOGY

Type of membrane

Driving force

Applic.atlon

Microflltrauon

Hydrostatic pressure Ap 0.5-2 b.,.

Concentration of bacteria and viruses Harvesfmg of cell" O arincation orrermentation broth

Ultrafiltration

ü p2-I Obar

Fractionation of biomolecules

Desalting Production of enzymes Processing of whey Hyperllltration

.6.p 20- I00 bar

ConcentratJon of pharmaceuticals Production eflactose Part desalination of solutions

Bectrodialysis

Pervaporation

~El ectric

fi eld

llpdrtial vapour pressure

Purification of charged small molecules e.g. organic acid" SelectNe removal of solvents (ethanol. pressure acetone-butanol) during fermentation

Puriflcation of solvenls fmm azeotropic mí>..1.ures

F'erstraction

Partition

Extraction of SITIall molecules from aqueous/organic solutions

interactions between the protein surface and chargedsurfactam . and is thus dependenton pHand ionicstrength.

9.5.3 Membrane flltradon The use of me.mbrane technology for separadon of biomollX'llles aud ['anides and concentration of process fluids has expande
I'J'J

200

HATTI-KAUl ANO MATTlASSON

deposi tion of colloidal spedes, adsorption ofmacromolecuJar solutes, precipitanoo of small salutes, etc. on tbe surfat e and in the pores of (he filter leads to a deposition ofa cake which grows in thickness with time, reducing the flow through the filter. Cross-fiow or tangential How liltraCion has now completely replaeed the dead end filtration for harvesting cells on a large sede. Here. a flowofthe fL>ed stream is maintained parallel to the separation sut:fuce, with (he aim tO provide sufficient shear force clase to the membrane surface, thereby preventing particlllace matter from settling on, or within. the membrane scruc· ture. In pr.1ctice, the membranes in cross·flow filtratioo are also rubject (O fouling. but the cake thic:k:ness is Limited to a thin layer as rompared to the dead end mode. Although mast filtrarlon media are relatively merr. the formarlon of gcl layer is inevitable. The probIem of permeate flux reduction can be minimised by optimising the filter selection. operating pressure, Oow properties of feed . and frequent back·flusbing. Microfilrers and ultrafilters are avaiIable in materials sumas ceramies and steel tbat can be aggress ive:ly deaned and srerilised in place. Membranes composed ofpolymerie materials such as polyvinyldifiuoride ¡PVDF) and polyethersulphone (PES) are also used, but are more difficul t to clea n and may require chem icaLrather than steam sterilisati on. Membrane ruten are commonly pIate and frame systems. e mploying eartridge filten within which the membrane is present in a highly folded formal. This gives a large filtradon sumee area in a compact space with no dead spaces. Another fonn is lbe hoUow·fibre system, wh.ich eomprises a bundle ofhoUow capiUaries packed in a tube. TIlc liquid to be filtered is pumped through tlle central eore ofthe hoUow fibres. The permeate passing through (he capillarywalls can be drained as a pe:rmeate from one end of tbe caruidge. while tbe concentrated retentate.emerges from tbe othel' cnd. Membrane adsorbers New micro¡m.acroporous membrane matrices with ion exchange groups and affinity ligands. called membrane adsorbers . have been develaped which bind proteins from the c1arified feed pumped over them.Desorpdoo ofrhe protein is laterperformed using solutions as in chromatogl'aphy(see Section 9.6).A staekofmembranes provides a total surtace area for adsorption equivalent to chromatography gels, giviog similar high resolutioo separatioo as chromarograpic metbods. In membranes, liquid transport is by coovection as opposed to the ditTusional Oow in gels Isee Section 9.6), which increases the speed ofseparation ttemendously. Pervaporation Pcrvaporation is a membrane based process havingpotential for re
Expande
adsorvtion dlromltDgt'aphy. The

bed of adiOrbent be3ds lo a colurm ls expanded by an Ilpw:!.rd flow of Hquld. A roIbte be
Molecular recognition rorms the basis ofadsorpnoD and separation by affinity chl'Omamgraphy. One of the react"ants in an 'affinity pair' . the Iigand. is irnmobilised on a salid móltrix aud is used nnder suitable conditions te fish out tbecomple.ment'lfY structure (the ligate). The llinding

Ligand type

Biospedf¡c Iigands Mono-speciflc Receptor Arltibody Hapten Substrate/substrate analogue, Inhibito~

(ofador

Protein 1ype

Ho rmane

Antigen Ant ibody Enzyme

Group-spedfte Cofactor Lectins Sugar derivatives Protein NG Heparin

Pseudobiospedfrc ligands Triazlne dyes Metal ions Hydrophobic groups

Enzymes Glycoproteins Lectins

Immunoglobulins Coagulation fadors, protein kJnases Dehydrogenases, kinases and other proteim Metal ion binding proteins

Various proteins

is reversible and can be broken by changing the buffer conditions. Potentially, sum methods possess very high resolving power. Table 9.7 shows that a variety ofligands , with specificity for one or a group of proteins, can be used for affinity chromatography. A trend in downstream processing has been to exploit the speeificity of affinity interaetions earlierin theseparation trainso as to reduce the numberofpurification steps; however this puts extra demands on the ligands with respect 10 chemical and biologicalstability. This has led to increased imerest in the use ofpseudobiospecific ligands like dyes and metal iOlls. The chemical coupling procedure for imrnobilisation of a ligand is chosen so as to provide sahsfactoryyields. strong linkage to minimise Ligand leakage during chromatographic operatian. and minimaJ nonspecific interactions with biomolecules. !be adsorbed protein is generallyeluted undeI' conditions that minimise its interactions with the ligand oe.g. by increasing the ionic strength OI' changing the pH ofthe buffer. o. by a free ligand m olecule. In the latter case, a subsequent step would be ro separate the protein from the free ligand.

9.7 I Product formulation The commerrial viability of a biotechnological product is dependcnt on the maintenance of its activity and stability during distribution and storage. Low molecular weight pwduds such as bulk solvcllts. bulk organic acids. etc. are formulated as concentrated solutions afier removing most of the water. 'Nhen high purity is required, smaU

208

HATII·KAUl AND MATTlASSON

Type o( dryer

Mode of heat transrer

Movement or the product

Belt dryer Fluidised bed dryer Spray·dryer Freeze-dryer Drum-dryer

Convection Convection ConvectJan ConLlct and radiation Contad Convection and corrtact

IntenSNe due to gas fiow Intensi\le due lo gas flow Intensive due to gas flO\o\l None or mechanical

Chamber dryer

Slight mechanical

Nene

molecules such as antibiotics, citri.c acid, sodium glutamate etc" are crystalliscd from salution by addition ofsa lts once tbey have reachee! the required degrec ofpurity. Proterns are particularly sensitive ro 1055 ofbioJogical activity during downstream processing and subsequently during storage. TIlis could be due 00 factan; 50ch as Olddatioll. temperature, preseoce of proteases elc. Proteio products are formulated as solutions, suspe:nsions ordry powders. A variety ofnabilising additives ar-e incJuded in me rormulaoons in order to prolong the product shelflife. The choice ola suitable stabiliscr ls done empirically. Among tbe non· specific chemical additives used regularly as stabilisers in proteio formulations are salts (arnmonium sulphate or sodium ch1oride). sugan (mcrose. lactose etc.). polyhydric aleohols (sorbitol, glyccrol etc_) or polymers (polyethylene glycol, bovine serum albumi n etc.). Bulk e nzymes are cornrnonJy sold as concentrared Iiquid ConnulaDom. Il is, howeve:r. often prefe.rred ro dry the product to decrease the volume as weU as the deoaturing reactions thar are enhanced in aqueoUll saludon. Bioproducrs, ofien being sensitive te heat, require gentle drying rnethods. rabIe 9.8 lists sorne of the commonly used dryers. Depending on the mechanjsm oC heat trarufer, these may be broadly classiflcd as contacto. con\lectioo-. and radiation-dryers. Batchwise drying in rnany contact drye rs is facili tated using mecbanically moved layen:. TIte advantage is [he unifono thennal stress exerted 00 the materia l beingdried. bigh throughpu[ and possibUity fur deveJ.. opment of conllnuous processes. A common feature of convertion dryers is thar the movemenr ofthe material to be dried is promoted by a flow of gas. Drying of large stre
c¡;

concentrarían vector ofthe solution red to the reactor

A

a A¡

CER

COl evolution tate coefficient matrix ('Element
(T,,¡, w", w~

specific aU!late consumpoo n rate specific growth rolle spccifk productpn:xluaion 1'3te ~ coDStant fur derivative controllel' time corutant fur integral controller stoichiometrlc coeffident spccific substrate upul:e rate critica] speciflc subStrdte up take rale specific O2uptake rd.l e used for maintenallce speci1ic O2 uptake rale used for biom¡¡ss growth

10. 1 I Introduction In industrial production plants, process control is on everybody's agenda whe:n the cost[benefit.ratio ofa process must be improved. It is

de:sirable to guide the process aJong a path. whicb. guarantee.s the process to prod uce the prod uct in such a way thar ir meets predefined quality specifkations. This confumed. the aim is to produce this product at a m inimum of Ca$(. The detennmation of such optimal proct'SS patbs is the essential pan ofapen·loop control The two kt>y eJements of process monitoring and control are: (il measurements bywhich inforUlation aboutthecurrent process state is beillg acquired. and (B) models that dynamically ÍDte rrel.l te tbe various process variables, which are importantwith.respect to the task to be solved . Ofparticular importance are rhose variables by which the

stare afthe process ca(l be described unambiguausly.ll1ese variables. however. are not necessarily the most importam ones from [he practical point' ofview. Ofimmediate practical importance are the variabl es which describe the perfor:mance ofthe process. In arder to gel access to the performance, its relatio nships to the variables which can be measured dh'ectly and to the variables thal can be manipulated are of importance. Thus. modelling for process supetvision and control needs a quantitative definition ofme objectives afme process aud rhe particular task to be solved . For supervision and control applications in industrial environmenu. [be complexity of tbe models muS( be Jeept as low as possible to minimise me expenses ofmanpower needed te maintain them. I( only makes seose to implement complex precess conlTollers, arter it was made sure: that they will work significanlJy better than conventional simpler ones. It is the cost[beneflt-ratio thar is me final c.rirerion (or whether simpleT ormore complex contrallers are used and this must indude me cost ofprovidingthe relevan t man-

po_o lt is of advantage te formula te tbe mulo-dimensional problems of proces5 modellLng, 5upervision and control using a vector representation. TIüs nor only helps to keep tbings dear, buthelps to translate them into modern software mols which are mainly matrix based. The matrix notation \Ised in rhis artide was adapted to software products available on the maTket such as MATLAB OI SOLAB where tbe variable. x, i5 generilly assumed to be a matrix. Vectors and tbe Ul>1JaJ scalar Quantities are.considered matrices ofspecia l dimensions.

10.2

I Structure of process models

Process identification is tbe procedure of de\'cloping a process model

from prior knowledge and experimental data. The dassical approach to process modclling is the development ofa mamematical model in the forro of adynamic differe.ntial equation sysrem deriverl from rnechanistic considerations. The prior knowledge usually leads to the structure of a parameterised model. leaving me parameters assooatcd with tbis model structure to be estima ted from process data. However, suirable model structures for sorne parts of the process may not always be known. Then, 'blackbox' ideutification methods that make onJy minimal a.ssumptions about the strucn¡re of these sub-processes aTe al ternatives. The state of a biopTocess is mainly determined by tbe amount, n , (measured in mol) of irs key components. !be vectoT, n , may be composed oftbe amollnts ofthe substr:ate. bromass. productetc_ The basis of a bioproce5s model is a baJance eqllation (bar can describe tbe changes of n as a fllnction of time. Please uotc that bold cbaracters or abbrcviations such as n indicare mal che corresponding quantity is a ve
MEASUREMENT ANO CONTROL

LIV¡AI"'O where tbe components A, are usually represenred by tbeir sum formula. and v¡ are the usual stoichiometric coefficienrs. As elemental analyses sbowed. the components, Al' can be considered lO be mainly composed of a few diffe.rent elements only. Often ir suffices to consider four elements le R o N] only. lfthis 15 a5rumed ro be sufficient, tben tbe individual componenlS can be identified by tbe.ir index VlXtOrs, e.g.: H

o

~1

Clucose - (O

t2

O,

O 3

O 2

o

2

t

O

2

01' 01' 11' 01' 01'

(e 1. 2.

- (O

3. Ammonia= 10 4. Water - (O

s.

CO,

- (t

Basic elements consfdtrrd Substmte

Ox""" Nitrogen source

(LO.61

Water Carbon dim:fdt

where l... J' mcaDS [be transposed oftbe row-vector,\ ... 1. Rere and in the following texto the vedor representatian by components is adopted Erom MA11AB. Problellls appear with tbe corresponding represent3tions of biomass and otber complex biapolymers. Here it is straigbtforward ro represent them as moleculeswitb tbe same relati ve composition as the originals, but with the C-index fixed ro 1. e.g. 6. Yeast""11

1.75 038 0.25]'

Biamass

With this assumption we can write down concrete reaction equations, e.g. ror aerobic yeast (Saccharnmyces cerevislac) production we get: JI,

C, HI2 0 , +

"n N~ + v.. 02~ ,,~C H,.~ 0 1).;18 No.lS + "wH,¡0 +

V,

COl (tO.7)

Using matrix representanon this equatian COlO be refonllulated into a homogeneous linearequañoD system: E ,,= 0

(10.8)

The coe.fficient matrix. E, in literature referred to as tbe 'E1emen[Composition-Matrix', is defined by the index vectors, llsing the ddlnitions in Eqn (10.6): E= r~ Glucose - 02 ~ Ammonia Yeast Waterroll

(10.91

The problem ofsalving rhe linear Eqn (lO.8l for the stoicbiometric c~ efficienlS vrequires five equations, since ane ofthe Jo'elements, e.g. that for gIucase, can be arbitrarily setta ane. lfthis substratc is chosen as me reference component. tben "s"" 1. Then we can transfonn tbe hornogeneous equation system (10.8) ioto a non-homogeneous one

Ee v= Glucose with the coefficient matrix Be =

I~0 2 -

Am.m.onia YeastWater C0 2l

and, according to Eqns (10.7) the five component yector:

(10.10)

217

218

LÜBBER.T AND SIMvnS

(JO.11) As Eqn (10.101 contains onIy four linear equiltioas and five unknowns,

we nee
(10.13)

Too can be reformulated by a scalar product [RQOOO -l1X 1-'= RQV X 11=0

(10.14)

wbich is a new li.near equation in 11. Henee, tbe row vector. RQV, de.fined in Eqn (lO.14). can be used lo extend tbe coefficienr matrix, Ec' ln the MAnAB representation we get: 110.15)

Correspondingly. we mustalso extend tiJ.e column vector on the right hand side ofEqn (10.10): b = [Substrate:O[

(10.16)

Nowwe have fivc linear equations for rhe flve unknown stoicbiometric coefficients. JI. (10.17)

Equalion (10.17) can now be solved directly lIsing linear algebra. This can be done wirh a single MATLABsta.tement v= ECJfb. From the solulÍon. 1-', we can dete rmine theyields. which are defined as:

y = all¡ ~ 6.na MW~ [kg] = v~MW~ ~b

ano

dnb MWb kg

[kg]

1"0 MW"b kg

(10.18)

Tbis rcquires the molecular weights MW. ofthe spedes ¡nvolved ro be known. The most important yields are the biomass per substrate yield telling how muro biomass is generate
HEASUREMENT AND CONTROL

Yxs =

~~: [=]

(10.19)

and the oxygen per biomass yield

Y~= ;:~: [:]

(10.20)

For measurement and control. s(oichiometric relationships are ofien used with adV
10.3.2 Conversion rates While stoichiomemc con sideratiOJl5 provide.i nformarion a bout tb ~ reJ· ative rates by which the different components are cOllsumed or produced, the basic cOl1vcrsion rate expressions, R, are a matter ofkinetics. Sino:, the performance oftbe mkro-organism is central in tbis respecto it is straightforward to discuss conversion Tates perbiomass cOnCellrr.l' tion. These quantities
lli

I

~,

Bal.nce equetion.

Welghling 01 ANN,

x.s.p

lFeedS

under considerarion. Then the question appears: how to decide when situations are manging and which ruodel components are ro be activared. For such questions. exper¡enced process engineers usua Uy have heuristic answers. which t1H~y often fonnulate by rules af thumb. Such rules can be formulated and exploited in precess computers by meanS offuzzy rule systems. An example for the structure ofsucb ID approach is skercbed in Fig. 10.2. This exampLe shows (he combination ofsctof ANN modules describing (he rafe expressions . .R. for the individual process phases aud a balance equation into which the rates must fit for the modelling of a production process. A fuzzy expert system sele
231

232

LÜB6ERT ANO SIMUTIS

Control mode

'D

P

(IIK)TI,

PI PID

(0.9/K) TI,

3.33a

( I.21K) TI.

20,

Control mode

K,

P PI PID

0.5 Kc.cril

0.45 K,'lJt\ 0.6 K,.oit

O.Sa

" p¿ u P[ l.0

P¿B.O

characterised by simple time constants, which can be id entifif'd using f'xperimf'ntal data. In the widely applie
m(tl=K(t:+...!. t edr+ 1"Dde)

e

TI

Jo

dt

(10.35)

1be vast majority of dosed-loop controllen use
step ebaoge 1... ma ... ip\l~b1t!variable (actuamg slgnal). This graph ~hows how tO ~strnatll! the Una constants of mil! proc:ess mat are n«ded tO wlle a PlO controller ae an ullconrrolled proc:ess. TlN.'! fim: ~ctiOll ob1erved i!; that me response 1, del(I!$,ls not lble te folow m e ;t(tuuinllsiz:nal immediat.lly; Innl1ad it riSfU in a del3)'ld w ay to a leYe~ A. The oolTt!SpOnding lime con sum, T, can be esllmated lrum theaJn,dna "",Iue$ of me Inlb"secu ofthe tangetll thm ugtl turnl~ por ... t of the I'1!lf>Clnu ell~ iU'od ab!ICis!.l and tu p;! raIle l al t lle leve! finally approac:hod by thc rv$pOnse curve_

me

-

- P-

- -·¡U...set

- -¡< -- -- ·- ·J.Lset

O.,

0.10~.L-'!---'--:4_L-!6:-L-;8~.L-!.'0 Tim e (h)

Mi el

o

,

4

6

8

lO

Tim e (h)

RelllJ lt o bralned wlth 3 stan dard PIO controlle r forthe specifK: growth rate oJL. at an E. co/i cu ltivarlo n on glUI;ose , wnere m e contfolle r parameters were determlned In diffef'{!nt phases of the process. The liMt.part crI me figure w.u. obr.ained wim p3.r1metl1rs determined d urillg m e In!tia! pha.se of Dne ofthe prmollS runs of the pmten. and the right w\th panmeters determined ooring me end1lhul1 o f olle or!he pnlYious rons o fme proc;ess. AJ, can be 1een partlc:ubrly in tlle right p.!.f'1: where ,,"is strongly osdllatiog. thtI controller Is IlDlmle 10 perronn me expecled task.l\3mely te keep the specific¡rowth rate dose to me set-point pmlile.

..

--/L

Typia t U'sult of 1 PID conrrolllr tor che 5p«1fk: growth

....... /L_se! 0.8

... I&, p.. ..,¡th p.1rnllleler adaptaticn.

if

0.7

O.•

me

05

"c

"-

The improvtmtnt In (om~rison wilh Itle usual PlO controller. depicted in Ag. 10.5, bccome1 oo...ious. OllCfI apio. the dl1hed c;u ...... 1s 1M sec polnl protile, wh lte [he full !ine deplcu (antroUe
(10.38)

= 0.019 - O.00240UR v

Th.is teads to significant improvements ofthe eontroller as can be seen inFig. 1O.6. When (he process dynamics is nor too eomplex it is possible to adapt the controller parameters ro the cbanging dynamics of (he processc$ even with sorne simple blad:-box assumptions . When such simple blaek· box a pproaches do notwork. me process dynamics mustbe considered bym cnns ofan appropriate process model. Then we are speaking abolir modcl-rupported control whiffiis describe
10.7.3 Model predictive control (MPC) There are many difl'erent approaehes lo model-supportcd eontrollen. One particularIy effective example. whkh is conceptually easy a nd easy

236

LÜBBERT ANO SIMUTIS

to implement. is tbe cODcept of model predirtive controL Model predico tive control . as the name states, uses a mode'''¡'ased prediction of the variable. cc' to be con trolled, to keep the process on tbe desired patb. cd(t). Th(' predictioo aUows to determine beforeband tbe expecred react ionofthe pracess upon an envisaged change in the ma nipulable vario able. uc' From tbe possible changes, the ('ontroner .selecrs the best one and then cbanges th e manipulable variable. u t ' accordingly. It suffices lO restrictthe prediclion ofthe process behaviour to sorne time interval. {t.t + tHl . referred to as the time horlton. As compared (O simple controllers. t he model predictiVl' controlJer does not m~ly determine iu action from a deviation betwee:n the actual value. ce' ofthe control variable and the correspondingdesired value. cd(t). al.a single time instant. t. onIy. bu{ from deviation or tbe entire path segment within the 6nite time horizon (t,f + ~ l from. the desired profile. This deviation can be qua ntifi~ by t he mean square deviation (c,- cd)l) betwcen tbedesired trajecrorycd(t)ofthe control varo iable and tbat(c".it)l to be expected when tbe process is running: with the pre-defined profile segmentu,,(t) ofthe manipuJablevariable within the time hon zon. When tbe time horizon is Dor too long. one can proceed in a very simple way in order to detel:lIline the minimal deviation : a finite set, (6uc,I(t)}. ofpossible chaDges ofthepre-determined pro6.1e of tbe manipulable variables can be tested individuaUy and the best of them can be taken to determine the actual conrrol acrion. A particu1arlysimple change or correction oftbe pre-defined protile. u«t). of the manipulable variable i~ a proportional shift .ó.uc(t) ""& u ,,(t ). lbe behaviour ofthe process witbin lhe time horizon can easUY be SilDulated for a set of constants, a. Thc best of the stmuJated paths Ithat determinOO with (lb) which loo te the least deviation from the desired patb, is theo used to determine [he actual control amon. Since al every time step. tt. weonly need to knowwhatto doattbe nen time step. tH 1• the corrected value control oftb e predefined profile is simply (10.39)

A typical result oblained with a simple model predictive controller is depicted in Fig.l0.7. In the current control engineering Jiterature. several variants of model predictive control algorithms have been di.sc:ussed . The different approaches may have one or the otber advantage or disadvantage in particular applkatiolls, bowever, rhe essential point to note 1S that tbe modeJ quality is tbe crucial factor ofmodel predictive control in real applications.

10.8

I Canelusian

Sophisticated control procedures malee sense particularly in cases where it becomes necessary ro ron theprocess ou(S ides the areas in the state space where stability can be obtained in a natural way. Far example. when.a higher performance can be obt.ained neaJ' sta tes from whicb the process can easily run outofcontrol. a sophisticated control

MEASUREMENT ANO CONTROL

Modol Predicti'.ft Control Veas! Cultl\etion

TY\)ioJ elQJTlple of

20

model pmctlve cor.trol applled 10 ¡ yeut eultÍVltlon proccn. Open-

Canlrolled process

"

~

15 1

~

'•••"

E .2

SOl polnt profllo ActuII model

'-'-----'-

lO

:':'---'

,

m

O O

,

~

~

'"•
240

KRISTlANSEN

tbat much processing understanding will be gained by viewing a process froman economic ratber than scien titic point ofview.

11.2 I The starting point TIle basic assumptions for chis chuptel' are that : • The projectyou are wOl'king on seems so interestingtbatyou haw been asked to prepare a case for building a plant ro produce your producto • TIle technology works as spcdfied. • AH permits (production. effluents .md product approval) will be. or have been, granced. There can be many reasons for wanting [o start producing or ¡ncrease existing production ofa specific product: • The markct is increasing and wiIl absorb anotber X kg (01' tonnes). • Vou have a technology that allows you to compete wim existing producer5. • YOUl' existing process, or that of a rival company, is becomingout of date. • There is a dear trend that a new market is opening up. fur [he production engineer, [here are primarily two issues that must bedealtwith: (1) what will be tbe price ofyour product, and (2) whatwill be tbe productiOll volume. The price is determinM by me coSlofputtiog [Ogelberthe ha rdware to build the plant. coUecred in the capital costs, and the cost of mnning me plant to support the operations, collected ineo me operating costs and what you hope to earn. Both the capital and operating costs are dependent on the scale ofoperntion . The starting point is therefore: How much ofyour productare you goingtoproduce? Having decided this. che rest is re1atively straightforward. There are procedures forc~ ting thedesign. constructingand ope.r.ning a pradoction plant, including steps to ensure that lbe plant becomes profitable, as indic3tM in the furth~ reading listo The listalso contains reFerences to sorne personal computer(PC) based simulation prograrnmes that can be used eo do che ca lculations and design the planto once you havesupplied process details.

11.3

I Cost estimates

111e decision to invest wI1l be based on cost estimates for the proposed production process, Withoutthese, no rational decision conc:erning the investmentcan be taken. There are different methods to calculate cost estimations, lanu at"f! in general vuy simi lar, (he main difference

being me n:lUlre of!he utalyst,

whether it ~ 3n enzyme or microorganism, Compared ro gel'H!l""3 l chemical planu, biotechnology

Mo:dium

Stor~e t4nJ,:

Product isolation

---->

"'"'

1---'

• ¡.--

G lucose

Soy-flour, potassium and magnesium salts 120h Bh 61

4Skg m-3 2 kg m- ) 0.36 28 oc:

Not ..: • An inlr.l.cellula.r enzymt' •

• The genes tor fabll la$ewerr found in ~ IMcrel'ium. bllt the l'omp.lnyhas expl'ess~-rhe gelle'! inA.---' Exil gas Air filter

Other !utrients

$Gluco,a

Wat"

Holding

Blending tank

Air

co'""p-,Le'.s-o-,--------~~

0----- ------'

A,,,Hte,

¡:f .

Holding tank

,

Ammonium sulphate

Salid waste

Jo

Precipitation

Centrifuga

Homogeniser

L ----'~D~;:~~-'----~,~~,~~~~~ LlquJ wasta

U

Freeze drier

Centrifuge Di",... rn 01 da planl Ior produaion offabulase (dl'1lWn with permlsslon !mm IntellIgen Ine. New }torsey. USA).

fermenter

Fabulase

PROCESS ECO NOMICS

It""

Number

Co5t(K€)

Holding tanks Blending tanks Conoouous steriliser Production reactor Seed and inoculum readors Ho mogeniser P~ c i pitation tank Centrifuges

2

70 100 lL5 2324 240 185 700 335 1100 145 39 1300

Comp~ssors

Freeze dryer Air fitters Auxillary processing equipment

2 1 1 2 1 2 2 1

2

Total pun:hase costs

6663

It,m

e"" (K€)

Equipmenl InstaJlation cost (250% of equipment cost - indudes lnstn.Jmentation) Total di~ct costs Construction expenses (70% of direct costs) Total direct and indirect costs Contingency ( 10% of directand indirect costs)

6663 16658

Total capital costs

2332 1 16325 39646 3964 436 10

Utilities Assumed lO be IO%oftb e production casts. see aboYe. Waste treatment The assu med costs for treatment of Iiquid and salid wastes are 0.001 €Jkg and 0.01 €fkg. respectively. Venting ofgas srreams wHl in dtis case nol involve any cosrs.

Labour costs Te is estimate:d tbat t he 24 operators (three shifts of su and one st.md-by shift) will be required [O run the plantoworking a 37.5 hourweek witb-4 weeks holiday a year.Additionallabour costs will be supervision (1 0%of operator costs).laboratory (15% ofoperator coses) and mainten ance and social costs (50% oftotallabour costs).

249

250

KRISTIANSEN

CO>I(K€)

Raw materials Utilities Waste treatmerrt Labour (@20€ per hour) Administration and overheads (40% of Iabour) Depreciation (10% of opital costs) Contingency ( 2% • " ) Insurance (1 % • ) Taxes (2.5% " )

762

1173 11 2 160 864 436 1 872

436 1090

Total production cosís

11729

Item

CO>I (K€J

Capital costs Start up ( 0 $ (5% of capital costs) Total investment Income rromsales @1700€kg-1

43610 2181

4579 (

Taxes (@ 40%) Netprofrt.

17 192 11 729 5463 2 185 3278

Expected retum on investment

16.2%

Production costs

Gross profrt

Other COSt5 of sal es. R&D expenses. patent and royaJties (osts mil nOl be included. Thcse items are very product specifit with typical figures of 10%. 5% and 5%ofproduction costs respectiveJy. lhe production cost (or the production of 10 tonnes fabula se per yearisgiven in TablelL7.

I 1.8

I

The costs case - to build or not to build

To obtain information on which a dedsioll ro build. the p lallt can be based. we w iH carry out a profitability analysis. The results afsuch an analysis based on a plant lifeof15 years is given in TabJe 11.8. ln some texts, lhe tenn 'operating profif is used. This is uscd to describe the proflt generated frem plantoperations and is the same as gross prafit in rabIe 11.8 and does na t ioclude taxes, cose of capital . depreciatíon etc.

PROCESS ECONOHICS

.S E .2

Result 01' a COSl

...u. !i}-

-,

.~]

~

&!

I... Salesprice

1 0_ _--"'2_ \Il

\

4p

I ... Operatlng cosls ' ..... Investmentco~

% change in cost

Tbe expected rate ofreturn on the capital invested helps to decide whether ro invest in the process. For existillg processes. tbe generated cash flow may be a better indication of the. health or tbe company. The cash t10w is obcained by adding money spent o n che depreciabon ofthe plantta the netprofit. Forourplant. the cash tlow will be {in kiloEuros): K€ 3278 Ne.tprofit Depreciarion K€ 4134 Cash flow KE 7412 An expcct'ed retum on investment of16.2%, equivalentto apay-back period of 6.2 yea rs. is relatively low fol' the biotechnological industry and ir is unlikely that our factory will be built. However, the returo is suffidently high nOI lO be discarded irnmediately and is accordingly subjected to a cost sensitivity analysis. Hete, the efTect of changes in important cost parameters . suth as sale plice. invcstment and operating cost, are nudied. The resultof such an analysis is given in Fig. UA.

The figure shows tbat the process is sensitivc to changes in all rnl'ee parameters, although it is more sensitive to changes in rhe sale price and investment(o.ca pital)tosts as the slopes ofthese curves are approx· imateJy me same and sreeper than the slope ofthe operating costs. It may be difficult for liS to influence the sale price as this is set by a numher of external factors over wltich we have ¡ittle control and will depe.nd on such things as the number ofplayen; in the mal'ket. the age ofthe market, competing products, etc. Howevcr. the figure shows that a decrease in the in~tment costs will also lead to a higll er fOlCe of retorn, and we will therefore go back ro ourdesign to see ifwe can cut the cosrs without afTecting plaut performance. Thus we must find o ut: • Are all the processing steps required? • Can we alter the capacity ofche units? • Can we reducedowntimes (a cornmOD fault for a first dcsign effOrt is to overestimate the requirect downtime)? • Can we lIse cheaper materials of co nstruction? • Can weuse multi-purpose units? Whilstdoing this we musr I'emembe. that the new plant must give the same performance as before.lfwe can cut the capital costby10%, we wiU get a .etllrn of around 20%.1bc proeess is now beginning to look ralhe. aUractive and will warrant furth er srudy. Ir is at this point that the reader takes overo Good luck!

senmMty an¡lys!s, in whidl the ,ffect of change1 in !mPQrtant con p:trameters ane studled .

251

252

KRISTtANSEN

11.9 I Further reading AsprnPtus.AqJen Techno[ogy lne. MaJlsachusetu. ISlmulatlon $(lftware.1 Petas, M. S. and TImmerh3us, K. D. (1991). P1anWrdgn and Ecorwmlcsfor Chemiml EnginCC1'S. McCraw·Hllllnt~rnatlonal Editions. Reili Olan. H.. B. (1988).Econarnlo:Anll/y5is ofFurnentatiOl1 PrOCt$ses. CRe Press. Boca Ralon. Rorida. Seide r, W. D.. Seadtt. J. D. and Lewin, D. R. ( l993~ Process Drngn Principles.John Wiley, New 'l'ork..

Superf'roDesi'gntr, lntd.ligen lne. Nl!w Jl!rsey. New York. ISimulat ion software.1 'IUrlon. R.• Baille . R. C. Whiting. W. 8. and ShaeJwitt. j. (1998).Mnlysis, Syn thesis and lk~gn ofChcmicall'n:lassa. p ren tice Halllnternalional Series in th.e Physical and Cbemk al Engi neering Seri es.

Part 11 Practical applicatíons

Chapter 12

The business of biotechnology William Bai ns and Ch r is Evans Introduct ion What is biotedmologyused for?

Biotechnologycompan.ies. thcir care and nurturing Investmenf in biotechnology Who needs management? Patenls and biotechnology Conclusion:jumping rhe fence .Further reading

12. 1 Introduction 1

Biotechnology is the application ofbiOloglcal processes. 'New' biorech· Ilology is when mis 15 driven by systematic knowledge ofbio logical processes. In this cbapter we will di scuss tbe 'new' biotecbnoJogy industry'~ mast specracu1ar commercial maoifestation - rhe 'biotech start-up company' - and what factors contribute to the success and fa.ilure ofthe entreprelleuriaJ application ofme science describe
12.2. 1 The applications - medicine lhe majarity ofbiorechnology investJnent since the mid 1970s has been in health-care. and speciflcaUy in fue discovery ofnew drugs. An effe
'Hislolical' colleetlon

/'

•

_.r .... ,#"

lead development

Screenin9

T

\ tJ- 1 ::

s

Prlmaty

SIICondilry

8SSay$

a55aft

ni,," .hroughputl

1-

(m~

(ve/Y

-.·c.- "

~ !! 2"

disc rimina.ing)

fs oS

E-

S~

li . . ~=

~

I ..

• •

_.r.. ,#

':'-~

TMI eompcuoc!s lo $olmal modol$ of dil;fl.t,e

..

[U

Syothmlse

bona. varian!s

1

Modlclnal cnllml!rt.y

Genelala

'librar{ 01 cnBmicBI~

Mu!llwell pla.e .oohnology RobotlCI

01\1& di$covery pam. A C\Jrn:fI! rnodc:l 01 me dl'tlg

dk~overyproceu. Process Hows m:.m Ieñ: lO righL The process !uruwim genornicrdrlven di5Covery 01 a 'wget' gene, ~nd !lence proteln. ¡no:! with me ¡meratlon of a ¿!verse setofcbemials lrom combillat0ri31 librarlas or from coUettlon$ of dlemiaka.ccumuJated dIJ"ng a oom~ny's hlttor)'. The dwtmicals art1 aSS3)'ll!d lor thelr ability to block (or 50metlmeJ enhll1cl!) the Q/"gCt p!"Qtcin'$ ltrlon lnltially In I high--dlrougllput, us!L1lly biocbemical assay, Bnd t.lu~o In mora complex '~e(1)Od¡rr' unys, usullly ccllulu funcrion IU:I'f5. Thtt re~tis a KI"Nn '\ud'. Thes.a" ~d In wholl! animal diseasl! models. aOO tened for pharmacologicIJ profM!rdcs, rnd 1fnKHury modififld by dlrected med!cinal cbMlistry 10

produce a candidatlll drug.

Stage Tdrget discovery Screening Medicinal chemistry Pre-dinicat development Phase t ctinical trial ?hase ti dinicat triat ?hase lit clinical tria! Total

Ce" (M$)

35

Time (years)

J

S 7

65 60 SO

6

10 10 140 180

$uccess rate (%)

} S

}25

10

4

""'= CohllIm 1 ; .tag~ in drusd~ryar:d deteosM! reconb which are ure
penn inio n te market Ihe prod uct as a dru&.

Racord •• clinic.1 result.s

RMordi. ennic.1 r~sulls

Reoords. "'in ieal resulla

1998 R&D expcndi tll1'e on t hings that do not work. They are therefore willing to pay very large Sllms to biotechnology companies thal can provide science or ttrlmology that • enbances the understanding otthe disease (and henee lowcl's the inherent risk in this approaeh); • increases the effkiency ofthe discovcry process talld hence mcans you can do more discovery tor less coS(); • has already been proven in clinical trials to be superior to existing lherapy. This is a continuum of activity from basie biomedical te5earch to commercial drug development. and t.he drug discovcry bioreclmology industry occupies the middle ofthis continuum. Thus sorne companies are essentially applied extensions ofacademicgroups, otbers are indistinguishable from sma11 drug companies . Inbctween are companies provicHng specific rechnologkal skills o]' services, 5Uth as companies providing genomics, combinatolial chem.istry, o. molecular design technology, or companies spccialising in screen.ing. In addition. sorne c:ompanies are see.king to radieally alter the order in wh..ich these steps are done. for exa mple perfonuing aspects orthe conventionaldevelopmen! (Fig, 12.2) as par! ofdiscovery (Fig. 12. 11. Medica! diagnostks bave a quited.ifIerentdynamic. While itishard for ilJl academicresearcher to discover a ncw drug, it is relativE'ly easy to diseover a new diagnosric: 'marker' for the diffcrence between sick al\d hea.l thy people. The limitation on theil' com.mercj¡¡lisation is making them reliableand simple enough to be used on a large $Cale, and idealJy to be performed by automated machinery, thus removing the need for

THE BUSINESS OF BIOTECHNOlOGY

skillcd assay tet:hnicians. As a result, tbe diagnostics industry is dolllinated by a small number of campanies with powerful marketing and distribution abiJities, u.suaJly aUied {Q their'platform' instrumentarion - Jarge automared instruments rhat can perform a widerange oflestS. Small companies can o nly gain a foothold in tbis market by finding spedalí.st ruches. such as sp«iaJist 'over-tbe-counler' tests (forpregnancy, cholesterol ere.). or unusual medical specialties chal do DOl fit mto tlle rnainstream of diagnosrics. Genomics-drive n drug discovery m ay change this. with drugs bcing increasingly targeted a 12.2.2 The applications - food and agriculture Food and agriculture is m or e importantec:onomicallythanhealth«lre, even in Western countries, and is c\early ofmuc.h greatef concem lO the

test of the world. However these arcas havc not attracted so many biotechnologycompanies, At root, this is because a new food cannotbe sold at $1000 a mea! in tbe same waythar a Lle\V drug can be sold at $1000 a bottle. Food is price sensitive - the highe.- tbe. price. the less you sell. Abovea.certain price, you sell none (price limited). So it is hard to justify expendillg very substantial amountli ofmoneyon developing n t'w food materials beca use tbat money cannot be rt'c1aimed in 3 premium price 00 thefood. Thc main exception is in breeding. whe.re Che cost of generating a new strain of plant can be offset botb by saJes of a very large amount of seed·stock and in the premium the fanner can charge foc the resulting produce. or the savings in produaion. lo principie, dle cost of d~lop ing a transgenic erop plant that is resistant lO pests (aD exercise costing tem lO huudreds of millioru of doUars) can be re
Upecl UI recelve folowing ... venwre capital fundlng raule. The IKMIs ilUSU
THE BUSINESS OF 810TECHNOl OGY

The due diJigence proc:ess gives the VC ¡m estimate for how reliable the current science is ;llld what the market might be. UsuaUy this will differ materially from tbe sci~tis;ts' view. It is critical for rhe calcula· tion ofthe 'val ue' afthe company roday, and hence.a calculation ofthe Return on lnvesbnent (ROJ, also sometimes caJ]ed the InternaJ Rat:e. of Retum - IRR). This is the amount ofmoney they wiIl getout COmpOll'ed ro me amount they put in, and is usually expressed as a percen tage annual growth filte (Hice a bank might offe!: 8% to irs savers). ves usually lcok for ROIs of50% per annum or more: this is not greed (or not only greed), butre.fl.ects the factthat this is theROI theywill getifeverything wor.ks - usually, ofcoUl"Se, it does not aod thcygctan ROl ofless than 0% . It is also worthwhile for an entreprenellr to do 'due diligence' on thc ve to see wh 12.4.4 Grants Occasionally agencies that provide grants to 3cademics ro pe.rfm:m research will also províde grants ro biorechnology comp.anies. However much more common isother types ofgCM!rnmentgraotsupportaimed at such 'SMEs' (Small to Medium-sized Enterprises). TIl e biotechnology

THE BUSINESS OF BIOTECHNOlOGY

industry is knowledge-based, clean, rapid1y growing, and base
London Stock

Exchange (LSE)

Main London exchange ('The Stock Exchange' in London) that lists sorne larger companies such as Chirosdence . USlJally companies must have a sales record or have at least 'tINo products in cJinlcal trials lo be allowed to list Q(1 15E

Alternative Investrr1ent Mark.et (AIM)

London-based attempt te have a market for smaJler companies, in fact trades mostly in very small or ver'y young companies. induding sorne UK biotechnology compan ies

EASDAQ

Very new European 'clone' of NASDAQ, has yet to prove itselfbut. promises well. Belgian biotechnology company lnnogenetics was one of the first to list on

EASDAQ Fran kfu rt

Neuer Markt

Paris

Nouveau Man:he In order ro get a biotechnologycompany 'Usted · (i.e. llave their nante put on the list ofshares available for trad e), (be company has to demonstrate that it is suitably stabIe. ln the UK this rneans havi.ng a b'ading ~ord for severaJ years. or h aving at least I:WO products in cUrucal triaJs, o r a numberofother eriteria.Ir also rue:ms having a prospC 13.2 I Commercial use of .mino .cids Amino acids are used for a variery ~f purposes. The food indusuy requirt"S L-glutamate as a Havour enhancer. a nd glycine as a sweetener in juices, for instance (l'able 13.1). The chemical industry requires amino acids as building block.$ for a diversiry of compounds. The pharmaceutical industry l"ffluires the amino adds thcmselves in infus ions in particular the essential amino adds - or in special dietary food.And lastbut not least, a large marketfor amino acids i5 theÚ' use as animal fecd additive. The reason is that rypical fl."E!dstuffs. sucb as soybean meal for pigs. are poar in sorne essenrial amino acids, Iike methioninc. for instance. This is illustrated in Fig. 13.3 where the nutritive value of soybean mea! 1S givcn by the barre] but the use of the total banel is limited by the stave representing methionine. Methionine is added for thisreason, and considerably increases the effectivencss ofthe feed. The addition ofas little as 10 kg metllionine per tonne increases the protein quality of the feedjust as effectively as adding 160 kg soybean meal 01' 56kg fishmeal. The lirst Iimiñng amino acid in fet'd based on cropsand oH seed is usually L·methionine, followed by l-lysine, and L·threonine. Anothee aspcctoffeed supplementation is lhat with a balanced ¡¡mino add content the maDure contains less nitrogen thus reducing enviran· mental pallution. Over me years the demand fOl" amino acids has incl"t"ased drnmatically. The market is growing sreadily by about 5 to 10 percent per year. 'lbus, within 10 years the total market has approximatelydoubled (Fig. 13.4). Sorne amino aCids, such as L-lysine . which is required as a feed additive, display a particularly grcat increase. Tbeworld market for th is amino dad has increused more tban 2D-fold in the past two decades. Other amino adds have appeare
10

100

Flavour enhancer Feed additi ve Feed addftive

kpart;une Aspartarne Feed additive Food additive, sweetener Food additive, pharmaceutical Pharmat::eut:ic.tl Pharmaceutical Pesticides, pharmaceutícal Pharmaceutical Pharmaceutical

llle am;no ;¡c;d ~ wItto

Ile Se

Ala

Main use

1000

10000

100000 1 000000

Production capacity (tonnes year-') currenr worldwide demand fur the mosr relevantamino adds are given in Table 13.1 ..L--Glutamate continues tooccupy (he top position followed by L-Iysine together w ith D,l.-meth.ionine, while me o rher amina adds traiI behind at a considerable distance. There is a daSe intecaetion between the priees of the amino adds and tbe dynamics ofthe market. More efficient fermentation technologyean provide eheaper products and henee boostdemand. This in rurn will lead to production on a larger scale with a further reduerion of costs. However, sm ee the sUPPly ofsome amino acids, e.g. L-lysine, as a feed adclitive is directly competitive with soybean meal (the natural L-lysine sollree) there are considerable fiuctuations in the amino add demand depcndiog on the erop yie1ds. The amino acids produeed in tb e largestquan tities are also the cheapest (Fig. 13.5). The lowprices in turo dictatc the location oftheproduction plants. The main faetaes governing [he loeation ofproductionplants are the pricc ofthe carbon souree

2a)

284

EGGELlNG, PFEFFERLE AND SAHM

and the local market. Large l-glutamate production planrs 31'espread aH over the world, with a significant presenee in the Far East, e.g. Tbailal1d and Indonesia. For L-Iysine the situation is different. Sin ee one-third of the world market is in North Ameriea and there is convenient access 00 maize a s a feedstock material for the fermentation process. about onethird ofthe L-Iysine production capacity is locnted rhere. tn almost a ll cases, the companies produciug L-Iysine are associated with the maize milling industry, either as produeers, injointVClltures 01' as suppliers of cheap sugar. 111i5 illustrates che factthatthc conunercia l procluction of amino acids is a vigorous ly growing and ehanging Beld with mOlny g lobal interactions .

13.3 I Production methods and tools Some amiDo acids are chemkaUy synthesised, such as glyrine. which has no stereochemicaJ centre, 01' D, L-methioni ne. This latter sulphurcODtaining amino add can be added tO fee
Functional genomics Another tool whose potential is onJy now beingexp!oited is the genome analysis ofproducer strains. The availability oftbe entire sequence of the chromosomes from C. glutamicum and f . colf apens up excicing possibiliries to compare mutants and \O uncover new murations essentia! fur .high overproduction of metabolites. Fur instanee, RNA analysis using chip technologywill make it jX>ssible to dete::-

L· glutamate

Sketdl of main reaction$ In C. pm:m;cum connecte
ill'ld al mtlffilnce for L·g.Iltarnó1te proooClion. Abbrevlatlons: PyrDH. pyruvate dehydros.nase: PyrC. pyruvate cillrboxyl~SI!:

PEPe:

pbo:sphoenl)/pyruvau carbox)'lue.

ylating reactioo mus[ be presenl . lbe pursuit of this enzyme activity resulted in [he dctectionofpyruvatecarboxylase activity.l'yrC, and the cloning of its gene. This carboxylase was Ilor rletectecl by rhe original enzyme measurements sincc ir is very unstable in crude extr.lcrs. Irs detectíon ¡'equires an in situ enzymc assay tlsing carcfuUy permeabiliscd celIs. Therefore, C. glutamiCllm has the pyruvate dehydrogenase (PyrDH. shuffting acetyl-CoA ioto tbe citric acid t:yde bur two enzymes supply· ing oxaloacetate: pyruvate carboxylase (PyrC) rogethcr witb a phosphoenolpyruvare c.1rboxylase (pEpc) (Fig. 13.6). The successful c10ning of both genes togetbcr with mutanr studies shoWt.'d rhat botb carboxylases can basically replace each otber to ensure conve.rsion of glucosederlved Cl-units ro oxaloacetatc. This is different fi·omE. eoli. which has exclusively the phosphornolpyruvate cal'boxytase seIVÍng lhis purpose, or Bacillus rubtilis. wbere only che pyruvate carboxylase is presento Since C.gfutamicum possessesboth enzymes. ¡thas anenornlOUS flex:ibi lity Cor replenishing cirricacid cycle intermediates upon theirwithdrawal. 'file reductive amination of Q'·ketoglutarare ro yie.ld L·glutamal:e is caCalyscd by glutamate dehydrogenase. The ellzyme is a mu1timer. each subunithavinga moLerular weightof49100, Jt has a high specificactivicyofl.8mmol min-1'rugprotein, and L·glutamate is presentin theceU in a rather higb concentration of about 150 mM. In the case of other amioo acids, in contrast, the intracellular eoncenrrations are usualIy below 10 mM. The high concentl'ation serves to ensure the supply of L-glutamate direcrJy r~uired for cell syuthesis and al50 for {he supply ofam ino grau ps via nansam ioase reactions Cor a variety ofcell ular reae· rions. As much as 70% ofthe.amino groups in cellmaterialstems from L-glutamare.

13.4. 1 Production strains Por the biotechnological production of1.-g1utamate che intTacellularly synthesised amino aad must' bereleased from the ceU.This is. OfCOltrSe, usually nor the case since the charged L·gluramate is rerained by Lhe cytoplasmic membn1l1e, otberwise che cell would llot be viable. However, as shown by t he spec.ial ci rcumstances in discoverin gC. glutam· lCum , L·glutamatc is alreadycxcreted when biotin is limiting. This strikiug fuer is based on two essential char.\cteristks: • acarrier is presenrmed.i adug the activc excretion ofL-gluramate; • the lipid environment ofrhis canicr trigge.rs lts activity. A ~"Pecific carner is required since otherwise. in addition to tbe charged L-glutamare, otber metabolites and ions WOll1d also leak from rhe ceD. Moreover. only un active exporr enables rhe energy-dependent 'uphiD ' transport of L-glutamate from inside tbe (:e Jl (O.15 M) towards the very high concenrrations obtained in fenncntation brotbs (more chan 1 M). Ho~r, for practical purposes, tbe triggering of active export by the appropriatc molecula r cnvironmenl of tbe cytoplasmic.membr.:me is importanr. The ~wit'ches fortuning (rus enviromnenrand (hus eUciting gluramate export are surprisingly diverse: (i) growth under biotin limi· ration. (ii) addition of loca] anaesthetics. (tU) addition of penicillin, (tv)

AMINO ACIDS

additiOI1 of surfactants. (v) use of oleic acid auxorrophs. and (vi) u~e of glycerol auxotrophs. All ofthese means trigger L..glutamate excretion. Althougb.. overall. there are as yet no completely condusive ideas on the molecular changes thus caused. nevertheless in the classical biotin effuct part ofrue causiorin limitation the pbospholipid content is drastically decreased ftom 32 ro 17 nmol mg~1 dryweight. and the content ofche llosatlll. ~

Active kinase

'NA,

."¡

'NA,

A ,d

11 Inactive kinase

M'Fi,; The /y¡Úl¡d operoo-01 C. ,!utomlwm ~nd ;¡Jlosteric control or the klr¡ase. T lle second promoter withln /yse results in rorm~tIon of me f3 subunit ~on5tltutinx the reguluory suburOt of die kinue proteil of (l2íi.

uructure.

\/.IIg ....

--

...

~,.. , ,,..

At me beglm1ng of tIle l..fysioo rerm~"t:ltlon use

prevaJls 01 the dehydrogeoue variam olrer UUt orthe sueclllrla1e Irariam. whereu al the end ~h e slKcinylas. varian! "l,Ised almo $t exdw.iVely. Vari arll use 15 In

percent.

The synthase limilS flux A further important step offlux control within lysine biosynthesis ¡s,l( the level of aspartate semialdehyde distribution. The dibydrodipicolillate synthase activ:ity competes with the homoserine dehydrogenase forthe aspartare semialdehyde (Fig. 13.9). InC.glutamicum. the synthase is not regulated in iu catalytic activity as is the corresponclingenl.yme in E. eoli, for example . lnsteHcl. in C. g!utamicum it is the amouor oftbe procein whkh directly controLs the flux. This is thus different from the kinase vmere t he catalytic activity is regulated by L-lysine and t hereby controls tbe flux at a constant amount of protein. Graded ove1'expression ofthe syn thase gene, daplt. bas shown tbatwith an ¡ncreasing mnount of synthase a graded flux increase towards L-Iysioe is rbe re;ult. Surprisingly. dapA overexpression also has a second cansequenc.·e: the flux ofaspartate semialdebyde into tbe brancb leading to hom.oserine is already diminished withjust two dopA copies. Oue ro fue shortage ofthe homoserine-derived anlino acids, lhis resuJts io a weak growth limitation which is advantageous fur L-lysine fonnation , smce now more intermediares ofthe central metabolism are used fu r lysine syntbes is jn..~tead for cell proliferation. Lysine synthesls Is split whicb emures proper cell wall formation AremarkabLe feature ofe. glutamicum is its splitpathwayofL·lysine synrhesis. Ar the level of piperideine-2.6-dicarboxylate, Oux is possible eiche r via the 4·step ruccinylase variant or rhe l-step dehydrogenas.e variant (Fig. 13.9). In contrastoE. eolí. forexample , has olJ.!y tbe succiny¡ase variant and Bc:dfius müet'Mns only the dehydrogenase valiant. The flux distribution via both pathways has been quantified in a study using NMR spectroscopy and 11-IJqglucose as the substl'ate. Surprisingly. fue flu x distribution is variable (Fig.13.11). Whereas at the startofthe rultivatiOR abour threi'-<juarters ofthe L-Iysi.ne is made via [be dehydrogenase variant. at the end me newly synthesised L·Jysine is almost exclusively madI" via the succinylase rotl te. There is a mechanistic reason ror this.k kinetic charactensations llave shown, the dehydrogenase has a weak affinity towal'ds its substrare. ammonium, with a K.n of28 mM. Thus at low ammonium concentrations, as are presentat the end of the fermentation, the dehydrogenase cannol contribute to l·lysiue formation.lnstead. flux via the succinylase variant is favoUIed. \'ihere after succinylation of piperideine-2,6-dicarboxyJate, a tIansaminase incorporares the second amino group inm the final [-Iysine molecuJe. The key ro understandiug [his lux uriol1s pathway construction is provided by fue ammo acid D.l.-diaminopimelate. This amino acid is required fur the synthesis of the activatec! muramyl peptide L-AJa-")', [}{;lu-D,L-Dap, which is oneof[he Iink1ng units inlhe peptidog1ycan of the celJ wa11. Upon inactivation of [he succinylase variant, a radical change to the cell morphology bccomes appare-nt w'ith low nit:rogen supply. The cells are elongnted, aud furthermore less resistant to mechanical stress. If ehher the succinylase or the dehydrogenase variant is inactivated, l,-lysine accumulation is reduced ro 40%. Thus

AMINO ACIDS

LysE protein

Topology 01 ct1e l·lyslne exponer showlng iB flve membral'!e !.panolng helice13nd additlonal hydrophoblt segn'M!nt. ThfI formaliy dlstJnct$tcp$ ofth e o-ansJocatlon proceu drlven by {he

me

membr:an-e po~tl~ 1T1i! Induded.

lysine+ both variants together ensure me proper supply ofthe cruciallinking unir D.L.¿iaminopirnelare, as well as a high throughput for L-Iysille furo mation. The split palhway in C. gll.ltamicum is an example oCan impar· tant principie in microbiaJ physiology: patbway variants are generally nor redundant but evolved to provide key metaboütes lInder different environmental conditions. Export of L'lysin e Aroino acid transpon has long been investigated in bacteria but. principally, tbis i5 only their import. In contrast, me molffular basis tOI' amino add export was complerely unknown untill996 since a spedflc expon process appeaTed nonsensical. The breakthrough WitS achieved by the c\oningorthe Jysine exportca.rcier from C. glutamiculII. which at one blow enabled amazing diseoveries conceming the nature and relevanee or a new type of exporter. The L-Iysine carrier, LysE. i5 a comparatively smaJI membrane protein of ZSA Da. It has the transmem brane spanning helices typical ofcan 'icrs, but on1y five ofthem (Fig. 13.12). A sixth hydrophobic segment is located between helixone and thrceand may dip into me membrane or be surfacc localised. Severa! dinincr steps are involved in the translocation mec:hanism. which probably requires the dimerisation ofLysE. These are: (i) rhe loadingofthe nega· tive!y cllarged can'ier with iti substrate L-lysine togetbel' with twQ hydroxyl ions, (ii) substrate translocatioll via tbe membrane, (üi) che release.ofL-lysine and [be accompanying ions at the outsideofthe me mbr.me. and final!y. (iv) the reorientation oftbe carneroTIledJiving force fur me entire rranslocalion process is m e memb:rane pmential. 6.1fr. required for the TeorientabOn ortbe carrier. Acress lo meJysine-exponer gene, ¡)'SE, has also made itpossible to solve the puzz.leas to whyC.glutamicum has such an exporter at al1.1n a lysE deletion mutantsupplied with glucose and 1 mM ofthe dipeptide, Iysyl·alanine, an extraordinarily high intracelLu!ar l.-lysinc concentra· don ofmore than 1 M aceumulates, abolishlng growth ofthe mutant.

291

2.92

EGGEUNG. PFEFFER.LE ANO SAHH

o

lysine

Thus. lhe exporrer SeIVeS as a valve [O excrete any6cess int:r.:J,cel.lul:u· L-Iysine that may arise in the natural enviTQnmeut in tbe prese nce of peptides. As in the case ofother bacteria . too, C. glutll:micum has active peptide-uptake systems as well as hydl'olysillg enzymes giving access to [he aminoacids asvaluable builclingblocks. However, C.glllfamicum has no I.-Iysine-degracling activities and rherefore must prevent any pillng up of L-lysine. This also happl'Ils in [he Iysine produeer strains where the biosynthesis parhway is mutatro . As genome projects have now shown. ho mologo us strucrures of t he l-Iysine earrier LysE are presen t in vario us Gram-negative a nd Gram-positive bacteria. Therefore. trus cype oHntraceUula r amLno aad control by 3D expon e r is expected ro be prescnt in other bacteria. IDo. Sine!'. lhe LysE S{ructure is nor shared with other translocators. LysE a]so represenLS a new superfamily oftranslocators , which is probably rclated to its new funetion.

13.5. 1 Production strains

Aminoelhvlcysteine Aminoethyl cystam

Is;a 5lJlp~u.r-cootill ning 3nalo¡:lJa 01 L-Iysl n. ror gene l'1ltlng mutanu d.ra¡lIlltt!d i1l../ysine synt~~sis .

L-Lysine pt'Oducer strains have becn derjved aver the decades by mutage.nesis to give strains excrcting more than 170 g L-lysinc per litre. It is clear lhar these strains can y a long liSl of phenotypic characters to achieve this massive Oux directioning (Tab]e 13.2)_ Typical.ly. t he strains are. resistant or sensiriw to sorne a nalogue oflysine. A typica l feature of sorne L-lysine produeers i5 their resistance to lhe Iysine analogue S-(2·aminoethyl)-L
AMI NO AClDS

is inoculated fo r each production run and is then en!arged in severa! stages. This means that the clone is fermented for about 2S generati ons so that there is a great danger ofthe plasmid containing tbe rhrABC o peron being lost. This would of course be a complete disaster ¡fit hall' pened in the final production stage. in the presence of the isoleucine leaky mutatíon. however. cells that have lost the plasmid now are cleatly disadvantaged when nor suppüed with L·isole uci.ne. Thcir further proliferation is halted. tlte reby stabilising a cuJture where almost al! tbe celIs tbat are growmg contain the plasmid. Furtbe r engi* neeringduring strain evolution involved the introduclion ofresista nce to l-threonine and l-b om oserine. Subsequently, tdh. wh ich encodes rhreonine de hydrogenase, was inactivated thus preventing threonine degradation . To obtain very high activities of the thrABC-encodlng enzymes, the operon was cJoned from a strain whose kinase and dehy· drogenase activities are rcsiSlant to L·threonine inhibition . In addition. the transcription attenuator region W

me

I d~•• pm""f,,'"" ,""o fo r L·m reonlne producOOl> $U¡~bI.

iIM:IMn¡ undrr.c:tl ~ mutagef1esis.

Substrate uptake Since the cost of the sugar soun;:e has a decisive influence on !he price ofrhe amino acid produce
Suerosa

Sucr0$6

gene ;n)(rivatlon and use of different plum;ds.

Mecha nlsms of sug¡r uptake and pho~horyladon In E.

Permeasa ,.o.

Pye

eoli. Tr;¡n!loation 15 coupled by ph ~phoryfatie". as js thG case far the pho~hOlJ"ans"rlSe systl!m (1eftand midd1 e). o r ocwrs In sympon. wlth protOllS wlthOUt phosph
13.7.2 Production process As wltb the other amiDo acids, efferove L·phenylalanine production is

thejoint result ofengineering the cellular metaboJism aud control of the production process. Control is ne
Relative productiv"Y(%)' 100 174 397 1498

301

302

EGGElING, PFEFFERLE ANO SAHM

13. 1·0

Outlook

Although amino acid.') are now among m e c1assical products in biotech·

nology. their constant development means mat processes musr be improved . new processes estabtished and o ur understanding of m e exceptional capabili ties of prod lIcer strains deepened. Just one exa m pie of molecular researro is [he retent discovery of the L-IysillC expon c.lm er. whith opens up ID entiJ'ely new freId in me (llcubolism of amino acids in IxIcteria in general. Moreover. much information has been gathered from strain dC\'elopment in conjunction w ith fermenta· tion tedmology, with the new sciem:e o fllletab olic engineering atthe interface bet\veen tbem. ln fact. amino acid production is an outstanding example of the integration of many dürerent techniques. In [bis way. theearly japanese activi lies On the taste ofkelp ¡aid thefuundalion for rhe continuing verysllccessfuJ and flouri shing production of aro lno acids.

13.11

Acknowledgements

We would like to thank the fullowing for providing mat'erial fur [his artiele: R. Fau rie. Amino GmbH; N. Kato, Kymo Univers:ily: Y. Kawahara. Ajinomoto Ltd.: W. I:euchtenbergcr, C. Thierbath. Degussa AG: S. Rbce, NH1 Bethesd a; T. Shibasa )c:i. Kyowa Hakko Kogyo; T. Tosa., Tanabe Seiyaku .

13. 12

I Further reading

Chibata . I., Tosa. T. and Shibatani T. (1992). lbe industrial pl'oduction of opti. cilll)' al1.i\'(! compounds by immobillz.ed biocatillysts.ln Chirallly In Indumy (ColJins. AN" Sbeldnke, G. N. and Crosby.J., eds.),John Wuey &- Sons. Londo n. Eggeling. L, Morbach. S. iUld S,"\bm. H. (1997). The ftuits of rn ole
3fld under

industrial carbon sourccs such as sugar bt'.et or sugar c.me molasses is even more essenti:Jl. and is mosUy carried out by eomplexati'on with recrocyanide and subsequentprecipitation . which a lso seems ro bave;¡ beneficia] clfect on the citric acid·forming metabolism of A. nig-t!r. Alteroatively. rhe effeet ofrIace metals can be antagonised either bythe addition ofeopper. whictJ bloc.ks ffiangalle5e t ransportinto the mycelia. or by the additiOil nf Lower alcohols or of lipids which may facilitare citrie acid export frOlll tbe eells. Several diffe.rCJl( hypotheses have been offered to explain the bi~ chemical b;¡sis ofthis requircmenlfor trace metal ion limitation but no single convincing explanation can yet be. offeced. The influenee ofmanganese. ions has been most thoroughly studied. The efrectseems to be a multiple one. as it bas been reported that a Jimiting concentration of Mn 2 + lncreases the Oux of carbon lhrough glycolysis, alters the com~ sition of the A. niger plasma membrane, and impairs protein turnover including that ora component of me standard respiratory chain and hence leads te impaired respiratían. A furtber striking effeClofmanga· nese deficiency on several fungi, includingAspergillus spp., is its effeet 00 the morphology ofthe fungus : manganese-deficient grown mycelia are stronglyvacuoJated, highIy branched, eontai n strongJy en thickened cell walls and exhibir a bulbous appearanee (Fig_ 14.4). The attached Further reading list provides more detailec1 information on me existing lüernture in this area. Theinfluence ofothe.r me[a! ions on theaccumulatioD oC ritric acids by Aspergmus spp. is even less clear: sorne workers have daimed;¡ partic· ularly strong influence of Fe3-+ which is, however. not supported by others. Iron limjtation has repeacedly been claimed to lead to an inacti· vation ofaconitase, the enzymecatalysing furtberdegradatlon of citrie acid witbin lhe triearboxylic aeid cyde and wruch eontains covatent1y· bound Fe. However. this assumption has now bee.il clearly refuted .

OH Cirríe acid accumulation has been reported to accumulate in significant amounts ooly when me pH is be10w 2.5. Becau se of the pK v3.1ues for dtric acid. a pH ofl .8is aummaticallyreacbed wheu certain amou nts of it accumulate in [he medium in rhe absenee of Olny other buffering agene. and benee there is no problem with tbis point. However. sorne

OI\GANIC ACIDS

carbon sources used (e.g. sugar beet molasses) contain a significant amount of several amino adds (parncularly glutamate) whkh srrongly buffer the medium between pH 4 and 5. The [eason fur che requirement ofa low pH is nor dear at the momento butmayberelated to me forma· tion of glucose oxidase, as gluconic add accumulares at me expense of citric 3dd ifthe pH js above 4. Glucose oxidase is induced by high concentrations of glucose a nd strong aerarion in rhe presence oflow con· cenl:rations of other nutrients. Le. conditions which are otherwise typical fardtnc acid fermentarion and will thus inevitably be fonned during the starring pha.se of atric acid fermentarion and coovert a sigo nificant .amount of glucose mm gluconlc aeid. However. due to the extraceUular locarion of the enzyme. ir ¡s directly susceptible to the external pH and will be inactivated once thepH decreases below 3.5. Not all straios of A. nlgi!'f show equal1y efficientínduction of glucose oxidase under fermentationconditions and reports on the effectofthe starting pH on me fe.rmentation.yield are thereforevariable. Also. sorne strains acrumulate oxalic acid at a pH > 6, whicbmust be avoided because ofits toxidty. Its formation has been attributed to tbe hydrolysis of oxaloacerate but a possible involvement of the glyoxylic acid cycle under certain conditions has stil l nor been complete1yruled out. Other explanations for the effect of pH have been proposed: one explanation suggests that citrate efflux from thecells may occur by di[· fusian driven by agradient and onlydtrate J - may be transported.lfthis assumption is correct, the low pH would be.rcsponsible for tbe citrate gradient necessary fur transport and consequently less arrate can be secreted a~higher pH values. Another explanation has discussed thal fue effect ofpH may be relate
Ilme lhl

TlmetcurJeofil typleal IndunriaJ cltrit acld fann.ntadon showi"g citric ..dd monohydnte (- ), bio~ (---), ~d $U~t ( _._) , Typk:ally. in 2S0-280hour$. 8-12 , 1- 1biomus drywund 110-115,1- 1of citrlc Kid are obtalned from 1
(4.2.4 Applications of citric acid Due to its pleasant taste, low toxicity and excellent palatability. citric ac.id is widely used in industry ror the preparation offood and sugarcon· fectionery (21% oftotal production) and beverages (45%). Dther major applications are in the pharmaceutical and detergentfcleaning indus· try (8 and 19%. respectively). lt is also able to complex heavy mernl ioos, such ..s iron and copper. ilIld therefore is applied in tbe stabilisation of oils and fats oI' ascorbic acid against metal ion-catalysed oxidation_ ln addition. citric add estersof a wide rangeofalcohols are known andcan be employed as non-toxic plasticisers, Finally. sorne ofits salts bave como mel'cia l importmce. e.g. trisodium atrate as a blood preservative which prevents blood dotting by complex.ing calaum, or as a stabiliser of emu!sions in the manufacture ofcheese. Today. citric acid is produced in buIk amounts with an estimaterl

ORGANIC ACIDS [

worldwide producrion of 400000 tonnes per year, mos[ ofwbich is produced byfermentation with the fungusA. niger. The bu)kofproduction OCCUl'S in Western Europe (41 %) and Nortb America (28%).

14.3

I Gluconic .cid

o-Clucono-h-lactone. the simpleS[ of the direct dehydrogenation praducts ofo-glucose. and iu free fo rm - gluconie acid - are produced by a large: variety ofbactelia and fungi , The equilibrium ofthe lactone and [he free add in soludon is dependentoll pH and temperanlre.

14.3, I Biology and biochemistry of gluconic acid accumulation Microbial accumulation ofgluconic acid was first observed in cultures of aceric add bacteria, and a bacterial parasite of olive crees, Pscudomontlssavastanoi. Wicb regard to fungi .gluconic acid forma tion by A. tliger was obselved in 1922. Subsequently. glucoruc aod has been shown to be produced by severa) prokaryotic as wcll as eukaryotic mkro-organisms, such as members ofthe bacterial genera Pseudomonas, Vibrio, Acctobacter and Gluconobacter, as well as species of the funga l gene.raAspergiJlus, Prnicmium and GliocJadium. Bacterial gluconic add formation mainIy occurs by a memhrane· bound o-glucose dehydrogenase, which uses PQQ (pyrroloquinoline qttinone) as a coen~e ("Fig. 14.7a), and converts extracellular glucose into cxtracelluJar g]ucoruc acid. Anocher enzyme, an intracellular NADP-dependent gluoo5e dehydroge nase, does Dot seem ro be involved in gluconic add acc-umulation_ Gluconic add i5 not urually an endproducto bU( will normally be uansported into the ceU and be further caraboLised via the reactions of tbe pentose phosphate pathway, Howeve r, the pentose pbosphate path.way is repressed by extraceUular glucose concentrations > 15 mM and a pH below3.5 (tbe latter also prevenU the formation of2-oxogluconate), aud gluconic aod is therefore accumulated when these conditions are applied . Fungal gluconic add formation is catalysed by the enzyme glucose oxidase. Th e enzyme is extraceUular, ¡,€'. partiaUy cell-wall bound in Penlc1llium spp., but secrete
(b)

Gluconic acid

Eruymk rnctio n$ If=aclioa tQ gkKonic ad 90% 00 a molarbasis) are usuaUy completed in less lhan 24 h. Sodium glueonate has been used as a superior alternative to the ealcium gluconare precess. as it enables the fermentation of even higher glucose concentrations (up to 350 g 1-1).ln this process. [be pH is mainrained close to pH 6.5 by the addition ofNaOH. In other respects, the process is similar to thecalcium glucol1ateprocess. This process has been employed for the developmem of conrinuous fermentations in Japan, whichclaimed the conversion of35% (wJvl glucose solutionswith 95%yicld. Severa! differem bacterial gluconic acid fermentation processes bave been described but only few ofthem are actually performed on an industrial scale_ As alre.ady mentioned, a high glucose concentration (> 15%. wlv) a ud apH below 3.5 are necessary for high yields. Severa! workers havealso shown the possibility to use immobilised eells fo[ g lucoDie acid production.

ORGANIC ACIDS

Methods for product reeovery are similar for both fungal and bacte· rial fermentations but depend on the type of carbon source used and the metbod ofbroth neutralisation. Calcium gluconate is precipitated frOIn hypersaturated solutions in the cold and is subsequently released by adding stoichiometric amounts of solphuric add. By repetition of tbis step, the dear liquid is concentrated to a 50% (wfv) solution ofgluconie add. Sodium gluconate is precipitated by concentration to a 45% (w/V) solution and raising the pH to 7.5. Today. sodillm gluconate is the maio manufactured fOI"lll ofgluconie acid, and hence free gluconic actd and &glueonolactone are prepared from itby ionexchange.As gluconic aod and its lactone are in a pH· and temperature-dependellt equilibrium, eitheror both can beprepared by appropriate adj1l5tment ofthese two conditioDS.

14.3.3 Commercial applications of gluconic acid Gluconic ada is characterisea by an extremely low toxicity, low corrosivity and the ability to form water·soluble complexes with a variety of di· and trivalent metal ions. Gluconic arid is thus exceptionalIy well· suited:foruse in removing calcareous and rust deposits from metals or other surfaces, including milk or beer seale on galvanised iconor stain· less m'el. "Because ofits physiological properties it is used as an additi~ in the food, beverage and pharmaceutical industries , where it is the pee-ferred carner used in caldum and iron therapy. In severa] food-directed applications , gluconie. add 1.5·Iaetone is advantageous over gluconic arid or gluconate because it enables acidic conditions to be reached gradually over a longer period, e.g. in the preparation of pick1ed goods, curing fresh sausages or leavening during baking. Mixtures of gelatin and sodiuro gluconate are used as sizing agents in the papee industry. Textilemanufacturers employ gluconate fordesizing polyester or poIy· amide fubrics. Concrete manufacturers use 0.02-0.2 wt% ofsodium gluconate ro produce concrete highly resistant to frost and cracking. According to recent estimates, its annual worldwide production is > 60 000 toones.

14.4 I L.ctic .cid Lactic acid (Fig. 14.8) was first isolated fromsour milk in 1798, and subsequently shown to occur in two isomeric forms, Le. L(+) and D(-) isomers, and as a ra.cemic mixture ofthese. The capitalletterprefixed to the names indicate configuration in relation to isomers ofglyceralde-hyde, and the (+) and (-) syrnbols indicate the direction ofrotation ofa plane ofpolarised light. The mixture of ¡somas is called m·lactic ando

14.4.1 Production organisms and biochemical pathways Lactic acid was the first organic acid to be manufactured industrially by5fermentation(arollnd 1880 inMassacllusetts, USA). The biology and biochemistry of lactic acid bacteria have been extensively reviewed. Tradition.al1y, tbey are functionaUy classmed into hetera- and

coaH

eCOH

I H-C-QH I

I

HO-G·H I

eH,

eH,

D (.)

l (+)

m'" ackls.

) and L( +) lactic

D(

I

317

318

KUBICEK

homofermentative bacteria, each ol which in tuco can be divided accordingto t beir coccoid or Tod-shaped form .Appli:catiou of molecular genctic tedmiques ro determine che relaredncss of food-associated lactic acid bacteria has resulted in signifkant changes in their ~xo nomic dassification. The lactie acid bacteria assciated with foods now indude species of che genera CarllObacterium, fntcrOeocrus, Lactobaallus. Úlctococrus, I.euronostoc, OCIIOCO«US, Pidiococrus, Srreptococcvs, TdTClgnwcucrus, Vagococcus and WdseHa. The genus Lcctoootillus remams heterogeneous with over 60 spcdes. of which one-third are heterafermentative_ Heterofennentative ¡actic acid bacteria are. invol~ in mostofthe lYPieal ferwentations Icading ro food Oi feed preservatioll and tr.msforrnation. whereas the homofermentative bacteria are used for buIk lactic acid production. Generally, strains opcrating ata higher remperature (45--62 oC) are prcferred. to the laltet, as mis reduces the powcr requirements needed fur medium sterilisation, Lactt)b!J.cilltls spp. (e.g.L delbrueddi) are used with glucose as the carbon SOllrce, whereas1. delbrueckll spp. bulgaricus and 1. hdveti! are ltsed with lacrose-containing media (whe.y). L ddbrueckii spp. Jactis call fenucnt mal tose.. whereas L IlmylopJliJlIs Cóln even fennentstarch. Most lactic acid-producing micro-organisms produce only one ¡somer of lactic acid; however, sorne bacteria, whi ch unfortunatelycan occur as infectíorn during lactic acid fermenr:ation s, are known ro contain racemate.s and are thus able ro convertonc isomcri 45 °Cwitb gentle stirring (lactic acid bacteria are anaerobicorganisms and the introduetion ofOz therefore has to he avoided). The pH is maintained between 5.5 and 6.0 by tbe addition of sterile ealcium carbonate. A5 an alterna tive to neutrallsation with caldurn carbonate , ammoniacan be used. which al so aids in tbe l'ecovery oflaetic acid by esterification (see below). but this resulls in a more expensive process. Due to the COITosive properties of lactie acid, wood oI' concrete were use
14.4.3 Applications tactie acid is a bighly hygroscopic. syrupy liqllid whicb is technically available in variOllS grades, Le. technical grade, food grade. pharmacopoeia grade and plasticgrnde, The properties of tbese grades and theil' respective applications are given in Table 14.4. Recent estimates of the current market voJume oC lactic acid are around 50000 ronnes per annum, 70% ofwhich is from fermentation. and the re mainder from chemical manufacture.

14.5 I Other acids [n addition to citric acid, gluconic acid and lactic acid , a number of othec acids are cornmercially produced by Cennentation in minar

a.ffiOunts.

)19

310

KUBICEK

• Scheme tor rKOYt!r y 01

Pure sugar medium

1 Calcium lactete dissolved by heating

Ca lci um s ulphate precipitated

Filtering, concentrating

Heavy metal ions removed with hex8cyanoferrat

Purification by

1

ion exchange

1

hydrogen peroxide

1

potassium perma nganate

treatme nl

Concentration

14.5. t ftaconic acid eH,

11 C·COOH

1 H2C.cOOH

Mti!.! ltaconlcacld.

Itacome acid (Fig. 14.10) was originally known as a product of pyrolytic distillation ofcitric acid.ln the 19405, it was fouud that this acid could be produced by AspeTgíllus tetTl'!US in ferrnentation. Chemical1y. it is a srructurally substituted methacrylic acid, and its use the.refore is mainly in the manufacturing of styrene butadiene copolymers, whcre it has 10 compete with similar petrochemistry-derived products. Commercially. itacome acid is produced by strains ofA. trrreus Ol" A

ORGANIC ACIDS

Quality Technical grade

Pmperty

Application

light brown colour

Delirning hides, textile industry. ester manufacture

lron free

Food grade Pharmacopoeia grade

Plastic grade

Glucosa

Glucose

2D-80% lactic acid Colourless,odourless >80% lactic acid Colourle$s, odovrless >90% lactic add

Food additJve. aciduJant production of sourflour and dough

< O. I%ash

Intestine treatment hygienic preparations. metal ion ¡adates

Colovrless

Z

::>

"l

326

ANDERSON ANO WYNN

PHAsran ulu In Ra/no.n.iG f llllCptlo. .

c.t1s1

environment, in the same way that pla.ntand animal waste isdegraded. Their biodcgrarlability and tbe fact Ihat tbey can be produce
PHB acculTMJladon in

batch OJiture.l\ap!d polym.r synthesis commencf' at tIle tIma of Ce.!-Rtion of growth ¿ue te

ntrtrient extl~w.tIon.

PHA are an.alogous in fun ction to the oils alld fats producoo by yeasts and orherfungi.They accumulate asg¡-iUlutes within theceIls. The gran· ules can be seen by phase.:ybutyrate. whích is usu ally known as PHB. lt is a polyestcr composed of3-hydroxybutyrate (3MB) repeatingunits (Fig. 15.2).As a re$lllrofirs highmolecuJacwcight. eYell large amounts ofPH.B ha\'(" little effecton tbe osmotic pressure within che cell. Oxidatíon of PHB ro carbon dioxide and water yields .. large 3mount of energy. For these reasons, PHB is an ideal carbon and energy reserve for bacteria. PHB atld othcr PHA are produce
oxygen. MoS[ bacteria accumulate only a small amounr ofPHB during tbe growth pbase. and the reasons fur mis are discussed below. PHB can also be produced in c.hemosl.lt culture. in which growtb is restticted by the supplyofone essential nutrient. TIte bacteria are thus subjecred ro continuous nutdent Limitation and this allows PHB to be produced in actively grawing bacteria. The amoont ofPHB produced in chernostat culrure decreases al high growth rates beca use metabolism of the carbon SOUTce to support biosynthesis and energy generation takc priority over PH.B synthesis. Bacteria require encrgy even when they are nor growing. for example to maintain concentradon and pH gradients across their cytoplasrnic rn~brane. Degradation ofPHB (orolh.er intracellular reserve materiabl such as glycogen) can satisty this maintenance energy requírem.ent and so ajd survivaL Degradarlon of PHB and otber PHA generally requires di.fferent enzymes from those used in biosynthesis.lt is generally assumed that a1l PHA-producing bacteria are abte to degrnde meir PHA bUf (bis is nor established. It is certainly possible to produce recombinant 5tralns matean produce PHB but lack the ability ro degrade it.

+

oJ-

RI

11

_ O-CH-(CHA-C Gene~

structlJn! ot

monomer unlu In PHI\. The m01t commo~

mon0mer"5 foon!! ara l.

hydroxyadds (x = 1) whh a simple )JkyI deN ChÚl. R. Side chaios th.at

1fe brandled oc con:ain an acoll"Atic rint or h.alogen aro "l\Q

""'""'

15.2.3 PHA composition and properties PIfA are. linear polyeHers composed of hydroxyacid monomers (Fig. 15.4). 3-Hydroxyacid monomers are most eommon ana 3-hydroxyacids with carbon chain lengths from CJ-C,~ have been found in the rangeof PHA produce
(:s.hown

he~). ara 50ft

rubbus.

328

ANOERSON ANO WYNN

Composrtlon of PHA

OrganlSlTl

RDlsfortia eutropha R. eutrop/la

R "'""""" oddovorans Comamonas Akahgenes lmus Pseodomonos oIeovoltJ(ls p. olet.lvorons P. oorugillOso ROOdococrus ruber

3-Hydroxyacid monomers

Other mooorners

.. .. .. O

.. (4HB)

Cubon sourc.:e(s) ~ucose

G ucose +propiomcaód GlU(ose + -l-hydroxybutyric add Gluc.ose + -l-hydroxybutyric. add Sucrose + 3-hydroxypropionic acid O Octanoic acid Nonanoic acid Gluconic ac id

O

"

O (4HB)

o •

O

000 . 0 O

O

O

•

O

O.

Gluc.ose

.. principal monomerpresentin PHA O other monorners ~ 'lHB 4-hydroxybutyrate •

XruCUlIl! ofPHBIV. J[

consists of a rllldom seqUl!nCl>of l-hydr-oxybutyrate &lid l_ hydroxyvaJlrate mooomers, and is

therefore desCTbed H a raMom copo1ymer. HaS'!: PHA conta!n two or more different monomers in me

CH,

I

CH

O

I '

11

O- CH-CH2- C

iH,

O - CH-CH2-

~ C

polymer chain.

glucose plus propionic acid, PHBfV (Fíg. 15,6) is a copolymer of3HB and 3HV monomers. aud its composition can be controlJed by varying the conceutrations ofglucose and propionic acid in the medium during [he polymer accumularíon pltase. PHB is hard and brirde. but rhe incorporarion ofa smaJl propartion of3-hydroxyvaJerate (3HV) monomers inro the polymerchain results in a stronger and more Oexible plastic_Tbis is exploited in the commercia l productionofPHBfV(Secdon 15.2,8), In some cases. bacteria can produce PHA monomers thar are Ilor relared ro the Structure of the carbon sources provided. For example, fluorescenl pseudomonads produce PHA containing 3-hydroxydecanoate from rnany carbon sources and sorne Rhodoroccus and Nocontia species produce PHB{V (Fig. 15.6) containing a bigh proportion of3HV mODomers, again frero 3 varietyot'carbon sources.

15.2.4 Siosynthosis 01PHS Of all the PHA. the biosynthesis of PHB has been studied in grea1;esc detail. In most bacteria. PHB is synrbesisIW from acetyl-CoA in three sreps (Fig. 15.7). 3-Ketothiolase (encoded bygene phM) catalyses the condensation of two molecules of acetyl.coA to produce acetoacetyl-CoA, wbich 15 then reduced by an NADPH-dependenr aceroacyl-CoA reductase (PhbB) te yield R·3·hydroxybutryl-CoA, Addition of 3-hydroxybutyrare (3HB) to rhe growing PHB chain involves PHAsynthase (phbC), an

PHA. POl YSACCHARIOES ANO UPlOS

enzyme associated with me memhrane SUJTDunding PHB granules. lo Ralstonia futTopna, the genes for these enzymes are organised in an operon: phbCAB. The genes have been clonedana expressed in other bactelia and aIso in p lants (see below).

CH3 ·COSCoA Acetyl.coA

CoA

~

15.2.5 Regulation of PHB metabolism The enzymes for PHB biosynthesis are constitutive - tbey are prese.nt even during umestricted growth. This allows irnmediate PHB synthesis as soon as growtJ¡ becomes restricted by the avajlability ofan essential nutnent.ln natUI
Str.ll~ 01 PsettdomOlKlSmendoci
I

growong 0:1 a¡ar.

associated with tlle cen. as a capsuleorslime. or simp1y dissolved in the mcdium. TIús depends on vanous factors, incJuding the chemieaJ sttuc· rure ortbe polysaccharide, and how vigorously tbe culture is agiraled. On solid media.large slirny coloIDes may be produced (Fig. 15.12). While sorne mkTobial exopolysaccbarides . orgums as chey are gen· erally known in.industry, are welJ estabLished as commerdal products. they muse compete with plant polysaccharides. sorne of whkh are manufactured on a vast scale and at a low price. Production of mi

Pyr I I 46

'" r -

Y ~-o-Man- ( 1-+4) -~- D-G IcA-( 1-+2 )-a-o-Man-6-0-Ac I

I

Divalent cations can scronggel.

c..TOSS-link

The strUCWre 01 XlIIlthao. The extellt Qf ;H;11[)'b.tion ofthe Il'QnllO$e \l nlt ;J.djacent ro die

Oackbone i5 cornmonly

~

bol:

an be sl&nlfia.ntly lo'HI1I" or Ngher.

polysaccharide chains to produce a

IS.J .J Xanthan Xanthan is produced by the Gram·negative bacterium. XtinrhamaflÜs cnmpesbis. It is the best.-studied and most widely used exopolysaccha· ride.. Xanthan is a largc polyrner. having an M r in excess of ]1)6 daltons. lt ís a branched polymer with a p.(1-+4) linked gluca.n (Le. polyrner of glucose) backbonc with a trisaccharid e sidechain on altemate glucose residues (Fig. 15.14). The pyruvate and acetare content depend on the bacterial strnín. cultm"e conditions and processing of tbe polymer. These substituents do not have a great inflt1ence on the properties ofthe polymer. Xanthan is a polyelectrolyte due to the glucuronic acid l'esidues in the side chains. Despite bcing an acidic polysaccharide. the viscosity of xanthan i5 relative ly independentofthe saltconcentration. xanthan is the m ost ünportant commercia! microbial polysaccha· cide. aud eurre ne production is around 20000 tonnes each ycal'. Kelco, nowpartofMonsalHo, is the principal manufucturer. Xanthan was fiNt used in 1967 and approved forfoad usem tbe United Statcs in 1969. (t is widcly used ful' stabilisation . suspensioD, gelling and viscosity control in the foad industry. These propenies are a lso exploited for W
15.3.7 Curdlan

->3)-f}-O-G Ic-( ,->

.hU StruCtu~

of curdlan.

Curd lan (Fig. 15.18) is a l --t3-tJ-glllcan produced as an exopolysaccharide by A!ct:llIgenes fuecalis varo myxogenes. Similar polysaccbarides are p~ duced by Agrobacterlum radiobacter aod ;'grobactmum rhizogenl's. and RI.izobium mfolif.

PHA. POLYSACCHARlOES ANO UPlDS

,->

- >5)-0:-D-G le-( ' ...4 )-a-o-Glc-( , ...)-a-D-Gle-(

M"U

StrllCllRofpullulan,

Unlike sclerogluC3n, rurdJan is insoluble in water and forros 3. stronggel on heating ahove SS oC and this gel furmation is irrevenible. Cnrdlan can be used.as a gellingagent in cooked foods and as a support for immobilised enzymes. The properties of enrolan resemble those of the 1~3 -J3-g1ucan, laminarin, which is fouad in many brown algae.

15_3.8 Pullulan PuUu]an (Fig. 15.19) is an ~Iucan with a trisaccharide repearing unit. It is pl'odueed commercialiy using the fungus Aureo/1a.sidellm pllUuluns. The ferm entation is relatively slow (5 days) compared witb the production ofbacterial exopolysaccharides but70%ofthe substrate (glucose) is converted to polysacc.baride. PuDu lan forros strong, resilient H1ms and libres, and can be moulded. The ti lms have a lower permeabiliry to 0 l than cellophane or polypropylene ando being a natural product, the plllluJan i.s biodegradable . Similar polyrners are produced by sorne bacteria.

15.3.9 Alginate Alginate is linear polymer composed of mannuronic ¡nd guluronic acids (Fig. 15.20). lt is produeed by the Gram-negative bacteria A2otobacter vlnelandU and Pseudvmonas species. The bacterial exopolysaC'Ch:ll;de is similar ro algal (seaweed ) alginate, except thatsome ofthe mannuronic acid residues areD-aeetylated. l'he relatiw abundance of mannuronic and guluronic adds and tbe degree of acetylation depends on the organism and growth conditions . Polymers conraining a high mannuronic acid contenr are elastic gels, whereas those wilh a high guluronic acid content adopt a different conformation and are strong. brittle gels. Alginates are not random co-polymers ofmannuronic and guluronic acids, and regions containing a single monomer (Le. -M-M-M-M-M-M- and -G 15.4

I

Xanthomonas campeSlrls fermentation

•

Pasteurisatlon af culture

•

Alcohol precipitation of polymar

•

Orying, milling, packing

'iU

~an.

Prodl,lcnon of

Microbiallipids

1504. 1 Structure of li pids What are Iipids1 Putsimply the lipids (his chaprcrwill be primarily concerned with (j.e. triacylglycerols) are composed of three futty aóds attached to a rhree carbon (glycerol) backbone (see Fig 15.24). Although al l triacylglycerols share this common strucrure tbeir physical propertiesvary enormously_ from hare! waxy solids ar room rcmperature (fats) to tr.mslucent Liquids (oils).lt is the structurc ofthe fdtty acid molecules (more correctly fatty aeyl chains) attached lO the glycerol backbone that a((ounts for the properties oflipids. The oils that this chapter will focus on art!" the so-called singl e ceJ] oils. Single ceU oils are oils derived from m.icrobial sources, produced on a (ommercial ba.sis, and which are destined for human consumprion.

R,

15.4.2 Fany acid nomenclature The n ami ng offaUy acids can appear (onfusing as in most cases a single fatty acid can be assigned any one ofthree names, depcnding upon the personal preference ofrhe a ut hor. The three names can be thought of as (i) a sysrematic name, (it) a trivial name an d (m) a numencal derignation, The thre~ di Fferent names for sorne common fatty adds and tbe fatty acids that have been developed as single (el] oils are shown in Fig 15.25. The systematic DHmes. although precise. are often long and con· fusing to those unfumilial'wirh lipid chemistty. As a l'esult rhese names

Structl,lr"l;j 1adllC~ nalc

ocld

16;0

acld CH,(CH,).ICH_CH.CH,),¡CH,hCOOH

AII cls-6. 9.12· oct8lr;enoTc Bcid

Arachidonic acid

CH:.ICH.).(CH..CH.CH,j.¡CH,I,cOOH

AlI ci9-5. 8. 11, 1"eiCos81etraaooic

O",

CH,cH,.(CH~CH.C¡'¡') .C¡'¡'COOH

AII cis-ll , 7,10,13,16, 19·docollahe>1aerlOic acid

l"Llnolenic Bci d

18:3ln-~)

20:4In-6)

. ctO

Overall schftml8 of modifH:ations made to facry ackls after de novo syntllesis. Etongases serve to Increase the f¡¡tty acld chaln length by addltion of ¡¡ el unlt (acetyl-CoA). Desaturases.lndlcated by /l. intrOduce a double bond between two ad)acent e atoms. Onlythe posltlon of tIle first C atom Is given and trus Is indlcated by che number; mus en carbons nine and ten in me acyl cbain (carbons numbered from the carboxylic acid group). Subsequent double bonds can be inserted at a number of sites giving rise to rhe. n-3. n-6 and n-9 series offatty acids.ln polyunsaturated fatty acids the double bonds are a1ways metbylene interupted. so that double bonds have a saturated carbon between them (i.e. -CH = CH-CH1-CH = CH-).

(fans do ubio bor"ld Strl.lCture of ciS and ¡ronr double bomb. ~ble bonds 'Iodc' me fatty add 5trlKWre and lead w the eltlnence of cis and Ir<mS I$ome~. Fatty 3tids in biological $ysums ue almost eltclusively t hlt cis ISM\Itr. RI and R:t

atr'

rllpNl5enT chaln" In a fatty acld mclatullt on. wl. ponen tohe

Ulrmlnal m. thyl (eH¡) ¡roup whht me omer wll! pouen the tarooqUc. ¡\Cid (COOH) ¡roup.

H I

342

I

ANDERSON ANO WYNN

:

Aschematk Cell Jipid

represenation of me tlming of Ilpld accumu(¡¡tlon In oleaginou5 micro-

organisms durlng a bau:h cultivatl on.

~--,

Biomess

Glucose in medium

Nitrogen in medium

Time Tropophase

•

Idlophase

OleaginoU5 yeast

15.4.5 The cellular role of lipid TriacyIglycerols are generally storage compounds. accumulated in eukaryotic cells under conditions of carbon exccss when grawth has ceased due to the exhaustion of some orber essential nutrient. llSUally nitrogen (Fig. 15.28). When produced in substantive amounts, as in sorne micro-orgamsms (termed oleaginous). tbe accumulated oH coalesces to form an ai! droplet(s) which can occupy a significant portian afilie cell volume (see Fig. ]5.29). Stored ttiacylglycerols act as a carbon 5tOfe to maintain cssential metabolic processes in the event of subsequent carbon staIVation. Another sl1ggested function. albeit restricted to marine micro-organisms, is tbatthe accumulation oflarge lipid droplets in the cytosol acts as an aid to buoyancy. Of greater metabolic significance is another class oflipids. the pbospbolipids. Phospholipids diffe! froro triacylglycerols in thatlnstead of three fatty aeyI chains attached to the glycerol backbone they only have

PHA, POlYSACCHARIDESAND UPlDS

two. The third position. on the.glycerol backbone, is occupied by (as the name suggests) a phospho-group. The phosphcrgroup can be phosphate itself or more commonJy conrains a bydraxyl
Glucose

¡

Pyr\Jvate 10

Malate

¡.

Oxaloacatate

C."" -1 ¡

6

Acetyl·CoA 7

Malonyl-CoA

, Palmitoyl-CoA (16:O-CoA}

MaJonyl-CoA is itself derive
involved. Disorders such as rheumatoid arthr.itis, multiple sderosis, schizopbrenia and pre-menstrual syndrome have becn aH reported ro (aH ioto this category, and their symptoms can , in sorne cases. be eleviated byan increased dietaryintake ofPUFAs, At tbe moment. the dietary importanee of polyunsaturated fatty acids is he.eoming apprecia ted botb by governments and the general public. 1n particular the importante of PUFAs in tbe de:velopment of new bom babies is generating m ore interest. Two polyuruaturated fatt)' adds. arachidonie acid 120:4(n-6)] and DHA 122:6(n-3)1 in particular llave been scrongly implicated in the developmenr ofbrain and eye fundioo in new-born infants.

15.4.8 Micro-organisms as 'oil factories' Cornmercial oils (used in'cosmeties and foodstuffs) are usuallyobbined from plants or animals. Han oH from a microbial SOUTee is to be prc; duced on a conunercial scale it must compete witb oils from these 'tra· ditional' sources. As fermentation teC:hnology is apensive single cell oils can only compete with the mosr expensive spedality oils. These are

3045

346

ANDERSON AND WYNN

Relative % (w/w) of fatty acid ir. total cell lipid

Organísm

16D 18:0 18:1

18,2 1BJ(n-6) 18J(n-3) 204(n-6) 2(;5(n-6) 226(0-3) Other;

17

12

55

8

16 M ortierella alpino" Crypthecodinium 25 cohnii" Throustochytrium 10 Qureurrf

14

14

10

2

12

10

30

Apiotrichum

B

curvatum

5

4

2

7

35

B

40

21

15

20

....ote: • Or!?"f~m U\ro fuI' mmm~rcial pmd\l ~rf .," nf.ingl~ ",,]] ni\.

inevitably the very long chaio polyunsatlll'ated fatty acid rich oils destined forhuman consumption for which no collvenient plant or animal SOUTee current1y exists_ Although plants produce a number of unsarurated fatty acids including the essential fatty acids,linoleic acid 18:2 (cornmon sources being sunflawer and rape seed oils) and a-linolenic acid 18:3(n-6) (eamman SOllrees beingflax and Linseed oils), they do not producevery long chall} polyunsaturated fatty adds (> 18 carbons long)_ In comparison. animals (indudingtish ) are a source of anumberofvery longchain, highly unsaturated fatty acids [up to 22:6(n-3Jl. These fatty adds tend to be prcsent in animal or 6sh oils in rclatively small quantities, however, making processing the oil to enrieh the desired fan)' acid dif:ficult and expensive. Forthermore, fatty adds frorn animal sources are unacceptable to a significant portion of society on moral andfor religious gl·ounds. The possible transfer of disease causing agents (prions) in animal oils and the risk of pollutants persisting in fish oiIs is also a potential problem. Likewise, plant oils have fue porential to contain residues ofthe pesticides and hcrbiddcs uscd in the rultivationofthe oil seed plants. Eukaryotic micnrorganisms have the advantage over ·traditional' sources in that they nor onIy produc:ea wide varietyofPUFAs (the fatty acid profiles ofsome selected micro-organisrns are shown in Tablc 15.2) but also sorne species accumulate large quantities of single PUFAs in theil' ceIllipids which simplifies oi! processrng. Microbially dcrived oils also -present .no problems for the consumer on ethical or religious grounds and, moreover can be essentially gual'anteed to be devoid of unwanted and potentially harmful contaminanrs. Although expensive, fue use offermentation technologyin the production of single cell oils has the advantage that a high degree of control over the process can be maintained.As a result the quantity aud

PHA, PQLYSACCHARIDES ANO LJPlDS

quality of oil produced can be controUed far more predsely than is possible with oilsfi"om animal and plant sources.

15.4.9 Current applications for single cell oils To date only three PUFAs haYe been produced cornmercially using micro-organisms. Thc firsr single ceU oil was rich in .,...linolenic acid 118:3(n-6)1produced using thE' fungus Murordrtind!oídes, a process developed by rneLipid Research Group at the UniversityofHull, This oi l waS produced in the UK between 1985 and 1990, by] & E Sturge Ud ae Se1by in Yorkshire. in competition with the 'traditional' oil of evenicg primrose. The process was discontinued in the fa~ ofa decreasing price of 18:3(n-6)when alternative agricultural sourcescaIDeonto tbe market in the forro ofstarfloweroil and blackcurrantseedoil The othe.r singleccll oil s, rich in eiroer 20:4(n-6) or 22:6(n-3) continue to be produced como m erciaJly in the absence of any real competition from oils frem rradi· lional SOUl'CeS. Arnchidonic add. 120:4(0-6)1, is produced using the common soil fungus . MOrtierdlaaJpfna. which has thcabilityto accumulate up [O 50% (wfw)ofits Clryweight as Jipid ofwhich as muchas 40% can be 20:4(n-6). Proccsses u sing tbis organism havc been developed by DSM-Gist in tite Netherlands and Zeneca-Roche in me UK although the future ofthese pl'ocesse!i (beyond 2000) 1S uncertain. Docosahexaenoicacid IDHA, 22:6n-3l. is produced commercially by Martek Biosc:iences, Maryland, USA and Omega-Tech (in collaboration with Monsanto). Baulder. Colorado, USA both utilising marine algae. Altbough fish oils are a patenriaJ sourceof22:6(n-3) theycannot be used to obtain chis fatty arid foc inclusion in baby rnilk fonnuJa because fish oi! also contains anotber fany ac:id 20:5(0·31 which cannol be separared from 22:6(n-3) using convemional oil processiog. and which should noe be given to inf;:¡nlS. The marine micrD- 34

3 15.5

I

Further reading

Carbon. S. E. ('1995). The role orPUFA in inftmt nutrition . 1I1form 6 , 9-40- 946. Doi, Y. (1990). MicrOOial Pvlyertm. VCH, Weinbeim. Gíll, r. & Valivety, R. (1997). Polyunsaturated ratIY adds, pan 1 :Occurr~ce, biological activities and appuciltions. Trt'nds Biot..'Chno/. 15. 401-409. Madison, L. 1.. and Huisman. G. W. (1999).Metabolicenginee.ring of poly(3· hydroxyalkanoates): Frorn DNA to plastic. MicrobIo!. MoL Blo!. Rt.'V. 63, 21 - 53. R.atledge. C. (1997). Microbiallipids. In Biolethno!ogy, Vol. 7(Re:hm, R·J. and Ret!
Chapter 16

Antibiotics David A. Lowe Introductian Biosynthesi.s

Strain improvement Generic engineering Analysis Culture preseIVatioll and aseptic propagation Scale-up

Fermentation Ptmicillins Cephalosporins New ,B-lactam technologies Aminoglycosides Macrolides Ecollomics Gaad Mauufacturing Pra.ctice:; Further readlng

16.1

1

Introduction

Antihiotics bave changed me world we Uve in. Their wide-sca1e ¡nnoduerio" in the middle oftbe 20th century loo to new standards ofhealth for bilLioos of prople. Many DI the life-tltreatening infechons ofpTeVious centuries are now convenienUy cured by oral medicine. Penicillin was the first major aotibioticfrom a microbiaJ souoce to becornmcrcialised. In acceptance and .mecess led ro the search and identificanon of thO\!sands o f nove.! antibiotics. many ofwhich are now available for therapeutic use.AntibioDcs also have applications as fced additivell , growth srimulants. pesticides and wider agricultural uses. The discovery of major antibiotics, such as penicillin, cephalosporin. streptornycin. terracydine and crythromydns. and their subsequent development, have beeo we.1J documented . Their cornmercial development aver the past SOyears serves as an cxcellent example ofhow the applied research has canaibute
35

352

LOWE

Resistance

Possible eITect

Analogues of amino acids. sugars involved in biosynthesis Antifungal agents e .g. nystrtin Toxic metals e .g. Cu , Cd, Hg Toxic metals Fe, Mn Selenomethionine, ethionine Selenide . methyl selenide Deoxygluc.ose Carboo dioxide High phosphate High salt NitrophenoL azide Polypropylene glycol Water miscible solvents Peroxide

Remove fee dback control A1tered cell wall compositio n increased, permeabllrty Increase in thiols, glutathione Impraved sporvlation Increase in sulphate metabolism Improved cysteine synthesis Reduced glucose regulated feedback Toler

" •E

~

8 Batch fermenter 2500 litres

6

•

Batch fermenter 50 litres Banle 1-2lhres

2 Shake flask 100 millilitres fmm frozen vial

O

g;Uctivit~.

~'.i'

Pmductlvity == Y/t + Tank turnaround time

Stirred tank

-Tlme{t)

plant stage where resources are limited and evaluations expensive. lt i.s always desirable tonave new cultures that easily.lit into tlle existing ferme.ntation protocols without furth~ development worl
ANTIBlOTlCS

@,.g ~rlso"

Batch

Tropophasa

Idiophase

Production

Growth

Fed batch

Pro duction

Growth

maximum production rates. conditions are created tbat can provide Lapid. early. antibiotic production with continued cell growth. Supplementcd raw materials are soluble and rdpidly utilised . Suitable carbohydrates are sucrose. glucose or enzyme-hydrolysed com syrups. Other carbon sources can be used (Table 16.6). If nccessary they can be supplemented witb soluble n..itrogen from corn S(~p Liquor. Thc ditigent feeding of a soluble. readily utilised carbohydrate such as glucosc can prevent ca tabolic rep.ression (scc Chapter 2), as the concenttation afthe sugar will ;¡Jways be very low. Oil as triacylg.lycerol, can be fard oil. soy oiL, palm ail. pt!anu t oil or r.lpe. seed oil, the final choice often dictated by local availability. OiJ addition has the additional benefir of controlling e,¡cessive foaming and air hold-up. Antifoam s, such as silicone-based products 01' polypropylene glycol. can be u sed to supplement or rep lace oil feeding. It is impol·tant rhat antifoam addition is available on an as-needed basis and nor simplybatclJed iota the surting medium due to the toxic na tureof sorne antifoams. 111e llletaboJism ofthe proreinat:eous nurrients from the complex raw materials can creare foaming, ofien ar unpred.icted

01 b:uch

359

360

LOWE

:!:!irbii1~"t¿n:F17o--:-~~~!::·t.l ·~~}JF.::::!=::~~:=:::~:;!;!:, ::i~:r:! ~:;~~"'''.';~'': .. __ . !,,::~!!~!!'!!?:~h' :!!;::::::::E:iliHmt~!~!::":::':: Beet and cane molasses Glucose Citric add Com syrup incompletely hydrolysed Com s)'rvp fui!)' hydrol)'sed Dextrins Ethanol Glyceror

11altose syrup Methanol St=h Lactose

Cortonseed oíl Lardoil Methyl oleate Palm oil Palm kemel oil Peamrt oíl Rape oil (Canola) $oyoil Tallow

times, thus itis importantto llave automated feed-back control foreffective antifuarn addüion te pl'ovide sufftcient control without excess usage of these agenU . Excess use can cause processing diffitulties on downstreamrecovery. Control offoaming and the minimisation of air hold-up are importan( factors in obtalning the max.imum volumetric outputfrom a fermenrer. Typically. tbe final harves[volumes should be in the range 80-85% ofthe total fermenter capadty. The added volume ofsolublenutrientfeed can varydepcnding lIpon its eoneentration (typically 30-65%). At lower sugar concentldtions early partial harvcsts may be necessary ro decrea5e the merease in broth volurne eaused by t he high volume of fecd addition. This addition of dilute solutions has che added benefir of lowering tbe viscosity of the broth, typica.lly a problem with filamentou s cultures_ EarJy, partial harvests, produce large volumes of dilute anribiotic fur product recovery. With correet handlinghowever. 5uth protocols can be ve.ry productive a5 the maximum produetioll rate of [he fennenta.tioD can be maintained ror longperiods. TIte pB ofthe broth can be controlled to within 0.1 pH uruts by me addition of acid (sulphuric) or base (ammonia or causdc). Often ammorna gas can be added through che ajr i.nput. The pH can abo be controlled by using me culture's own metabolism of sugar. Excess feeding of sugar in some conditions will produce aeetic acid , which will lower the pH, Conversely. a c:utback in the sugar feed-rate can raise [he pH.

ANTIBIOT1CS

•. PressUnI probe

j"'*+"O'

J _DO_ 1.··-Air/aglla\lon

Ai'..>dlb.us

~J •.

,~

probe

1",+_

1 CcoUng

Lave! proba

Alltiloam

...,--,

• • •

,...,

DO

SIl9BtI

..

""""""

Ph04Qtlate:

Sulph8le

PIOdUets

_

..

I SugafIOiI

$am !!le AnR!n.!1

pH

P~U/SOI5

C«lIM1INtlO'1 ~

~

==

Dissolved 0 1 DO Jevels ;ue critiC
16.9.2 Biochemistry and fermentation Penidllin smin improvement programmes have bee.n in existe nce fOT over so years. By using convention mutanon and selection [he original titres of less than 0.1 rng ml- I have been increased 40().fold . Furthcr gains have been realiscd by media rnodificatton and engineering developments (Fig. 16.9). The. biosynthcsis of this molecule togerher with the biosynthetic enzymes and associated genes are well characterised (Fig. 16.10). Rate limiting stcps in tbe biosyntbesis have been identified and attempts made to inerease the production oflimitiug enzymes by recombinant technology. For example extra genes coding ror
V \\

. ... .\\ '\---f?é.::!:::::S=;;;:;¡:::::::::::j ... -...-._. .....-""_ ...\

CO

'\....

,/'

Sugar feed rate

~~

.

----

----.:::::

_

rr-'./-:.......--...-.._---.. .......

-

Arn mo nia N

.. .............. ...J. .

Ti me

" ":r.¡;r.; :f6' 1ili;¡¡,¡,,:,::r,: :"-:;.;;.::;" ,"";¡:¡::: i;;¡,ji¡:: :::;; :;;::0 , : " : : : : : ;; ;::;..... ::: :.i .:., ... -:.~t~:? !:'! ~~:~ : ~~~:~~';7:. .~~ .~ . ~,~~;~ :::!i:~~: ;: : : ; ;;:E 8 lmin¡rtJon of chlorinated and all:ohol soIvents Elimination of solvent rel:overy and sotve nt/odour release Eliminatian o f hazardous chemical reagents Elim ination of liquid nrrrngen for cooling EIImincrtion of hazardoUS" and toxic waste products and their dis po '>al AlI aqueous reactJons. neutral pH and am bient temperatures Good control and monitoring of reaction through pH measlwe ment and adjustment Q uid< removill of o;oluble reaction products from immobilised cataJyst Re-use of immobilised enzyrne catalyst Easy recovery of side chain for re-use Pleasant working environment for aJl personnel Improved product quaJity, less impurities Improved yields and manufacturing capacity Decreased (ost o f manufacture

16.9.4 Production of 6-aminopenicillanic acid Over the last 10 yeaTS the industry has switched from chemical hydrolysis of penicillins to enzym e hydrolysis to decrease cost and anOlio enviraumentaJ benefirs ('rabie 16.7). SpedfLc. immobiLised penicillin amidases bave bccn developed fol' penicillin e and peniciJlin V.bydrolysis. lmmobilised enzyme can be made in-hotlse o. putthased trom third parties. From the. thermadynamic equilibrium o f&APA and the side chain. hydrolysis is sornewhat greater for penidllin V than penid llin G. However penicilün G is a. m ore versatilc prod uct due to its application in rillg expansion owhich partially e xplains its fc rmen tation vol ume d ominance over penicillin V.

ANTIBloncs

Ihe final choice between either proces5 is afien directed by lhe company's own rustoricaJ deveJopmcnt success . In conventional Splitcing rechnology. the penicillin salr is used at 12- 15% (wfv) for enzymjc hydrolysis by tbe appropriate irnmobilised penicillin amidase system.This yields mixtures af&APA and the precur· sOr acid. During the hydTolysis me pH is mai ntJilled between 7- 8 by the addition ofbase. either caustic or ammonium hydroxide. Tbe product 6-APA can be rt'Cowrcd by precipitation al pH 4 in the presence of a water immiscible-solvent rhe convenient removal ofthe precursor acid . lnoperations that have both penicilLin re rmentatian and splining processes. the reCúVe.red pre 367

368

LOWE

16. 10.3 Production of7.aminocephalosporanic acid CephaJosporin e is recovered from broth filtrates by a variety ofhydrophobic and ion exchange resins. Thc column chromatograph y is dcsigned to separare cephalosporin e from related intermedia tes and breakdown products. The t;ch frac.tions are either treated with zinc acetate to precipitare the low solubility zinc salt, or with sodium or potassium acetate followed by a water-miscible solvent [O prccipitate che saU complex o lsolated cephalosporin e is efficiently bydrolysed che.mically to 7ACA. Unfortunately. the process uses similar reactants and solven.ts used in the chem.ical hyelrolysis process for peniaUio, wlth [he familiar d.rawbacks of hazardous material handling, solvent me and negative e.nvironmental isslIes. The switch to enzyme hydrolysis has proved to be difficlllt due to the inabili ty to ¡deno fY e.nzymes to directly bydrolyse off the side chain. the unnaturnl D-aminoacid D-a-aminoadipar.e.lndirecr enzyme systems, though, have been developOO which rely on tbe sequeotial use oftwo eozymes (Fig. 16.12). 111e firsl enzyme, a D·aminoac.id oxidase, removes the chinlity of the side chaio by oxid adve dearnination lO produce a keto acid which, in the presence ofthe c.o-produccd peroxide, is conveniently decarboxy¡atoo to che glutaryl side chain. Tbe :yE'asc, Trigonopsis vatiabllis. is a suitable SOlloce of this enzyme. lbe second enzyme was d.iscovered in Pseudomoruu sp. and can directly hydrolyse che glutaryl side cbain to produce 7-ACA. In a similar manner to penicillin hydrolysis these two c.nzymes are now available. from recombinant sources and have been immohiliscd. Mostofthe major industrial producers of7·ACA are DOW switching over to me eluyme proce5s.

16.11 I New ¡3-lactam technologies There is interest in devcloping alternative ways to make [he ce.phalosporin intermediares, 7·ADCA and '-ACA. using the P. chrysogenllm fermentadon. The.availability ofbiosynthetic genes has been usOO to this purpose ro design new biosyntbetic pathways. The expandase enzymes h ave a strict substrate preference fur pen!. cillin N-like molecules aud wiII not expand penicillin ~like molecuJes. It has been dernonstmted. however, [hat adipic acid can serve as the precursor to adipyl-pen!dllin in P. chrysogenutrl , On tbe insertion of the gene. (eJE (expandase from Strepromyres davullgmlS), intoP. chrysogtnum. the transformants prooucro adipyl-6-APA. and adipyl-7-ADCA. Tra ns· [onuants witb the genes ceJEF and cefG (acetylttansferase) produced adipyl-7-ACA ln addition ro me abow (Fig. 16.13). The adip)'1derivatives do have the advanrage ofbeing solvent-f!xtractable and rheir bydrolysjs has been demonstrated using- glutaryl amidases fromPseudomonns 5p.. enzymes known to have sorne affinity for the adipyl side chain. Similady directed synthesis has becn carricd out using carboxyrnetbyl· thiopropionate. a lUoleculeofsimilar structtlre to adipic acid.

ANTlBIOTICS

M'·Mi; EnzyTTlic "trlrclpls 01

H'N"'.~~'r---f J-~ .= . &S COOH

O

o

cephalospo
Kanamycin A R=OH R,=H R2 =OH

Amikacin R = OH

R,.

o~

~

,NH

Rl =OH y'V

OH

Sisomicin R=H Netilimicin R = CH2CH l

o

NH~~

~\~N~ OH

Gen"m;c;n el

2

AmM¡ly«uides.

J7

l72

I

lOW,

El.8fi

Tetnqdinn.

R R,

""-,

OH

R, ~J

O

Chlorotetracycline

Oxytetracycline TetracycJine

Doxycycline Minocycline

~(CH,),

OH

OH

OH O

CONH2

R el H H H N(CH,),

R, CH, eH, eH, CH, H

R2 OH OH OH H

H

R, H OH H OH H

Due to tbe general basic nature of aminoglycosides. (hey are generally recovered by a combination ofresin colurnn treatments, e.g. weak cationiclRC 50. non-ionic XAD, or alumina. Activated carbon treatment is often necessary alld rhe final product can be precipitated as the sulphat'e salt.

16. 12. 1 Tetracydines Tetracydines were the first group of antibiotics recognised ro have bread spectrum activity. They ael bypreventing Pl'Otein sy.mhesis at the 30S ribosome ¡nteraetien with tRNA. They are use
'"o

Erythromycin R", H Clarithromycin A .. CH 3

Ei.".!

Macrolides.

373

374

lOWE

Raw matenals them~lves contribute 30-45% oftbe final cosroftlte recovered antibiotic. with utilities at 10- 20%, fixf"dcosts, i.e. plant overheads. at 20-30%. Recovery costs can be 20-40% ar a recovery yield of 85% plus . Fixed costs vary depending on the quantity of product pr~ duced and on rjle scale ofoperations. Cornrnercialisation of antibiotics has produced il competitive worldwide madret. The difference be.t\veenmanllfacturing cast and sale price is dependent upon a variety of fluctuating factors. Main factors are the annual volumeofthe operdtion. qUallty oE tecltnology available. local cost of tbe manufactured producto establishment of long-term con· tracts. a nd ClIrreocy ftucruations between the majar developed CQUll' tdes. Manufacnning costs are conOnuaLly being lowered through technical development, improved efficiencies, increases in prodllction volume and i.ncreases in ma.r.ket sbare. far a best case situation . a company should producesufficient mate ria l tOSllpport ¡tsown inte rna! caprive demands for furtber proc:essing to m ore cxpensive products. lt should a]5O have long term comracts lo supply third parly sales prefer· ably tomore than one customer, and hawan active sales force toseU any remaining capaciLy to otberthiTd paTries, 111is is not always thecase as over the last 10 ycars scverallarge pharmaceurical companies in mE': US and Europe have stopped makingpenidllin , Thisbasbeendueto anee
16. 15

I Good Manufacturing Practices

Over balfofal.1antibiotio manufactured today arefor human use. Their extensive use necessitates Chat t he consumer should have confidence that [he product is safe. consistent. dean and pose n o additiQnal adverse health condjtions. Govemment autborities have establisbed a pbiJosophy and guidelines (oensure thar products for human consump-con are made underwell controlled cOllditions. These are referred to as Good ManufaccuringPractices. Regulations and guidelinesare in place to enSUTe that correct procedures are followed tbrougbout the many st:1ges ofproduc( manufacrure. Rigorous roxicology tests and detailed dinkal trials have to be performed before a productca.n be considced for full·scale manufacture. Raw materials have tu meet certain preestablished quality criteria and consisrency. All manufacturing and pilot processes bave to be detalJed as standard operating procedures. All ana1ytical procedul'es have to be validated ro enSllre tbat tbey always givc tl'ue results under a wide variety ofconditions. These procedures

ANTIBloncs

have been established ro ensure that [he produLt manufactured is oh conslseent high quality. Any changes ro a manufacruring process couid resultindifferences in the final productso ilis extremely important te adhere to aU established operadog procedures. Quality chedcs are a1ways perfonned at suitablc stages io the manufucturing process. Tbe puri[}' oC i.nlemlediates and tinal products cannot be cxpected ro be 100". However specific¡¡tious have to bescr to ensurc productuniCormity. The produce should be ofa s highest purity possible at ao est.ab-

Lished accept31lCe leve!. The presence and jdentity of all impurities shouLd be known and shou ld nol exceed set limirs. The mxicityofthese impurities should be known. Analytical procedures should be in place ro ensure [he recognirion and idenlification of any new impurities. To Collow Good Manufa cruring Practices, al] procedures bavc to be dorumented and working copies available for operators to follow. lnstruc tional sheets have lo be !>igned at the completion of each stage and the record s checked by management and rNained as the batch record . These recoros are available to any inspections by Government regulatory bodies. Strict adherence to (hese polides will satisiY thc regulatory authorities, and will ensure confidence in the general public that their medicines are safe.

16. 16

1

Further reading

Elander, R. P. (1989). Bioprocess technology in..industlial fungi.ln FcrmCTJ!Qtllm l'roo:ss Ot'wlopll'lcnt eifIndu5trial Orgemisms. U. O. Neway, ed.). pp. 169-219. Maree! Delckl!r, Ni! w York. Hersbach, G.J. M.. Van Dl!r Beek. C. P. and Van Dick. P. W. M. (19M). TIle ¡x'nicil· Uns: properties. biosynthesis . and fi'1:mentation.ln HiotechnoloKY oflndustrlal Anfiblo'fa(E.j. Vandamme, ed.). pp. 45-140. Maree! Dekker, NewYol'k. Lowe, O.A. (1986). Manufacture ofpeniciJlim. In Bcta·LactamAnt'lhioNcs for Qinfral U~(S. F. Queener, j.A WebberandS. W. Queener, eds.), pp. 117-161 . Mareel Dekker. New York. Paradkar. A. S.. Jensen. S. .E. and Mosher. R. H. (1997). Comparative genctics and molecular biologyorbeta"lactam biw.ynthesis.ln 1Ilou,hnology of AntChiollcs, 2nd l!ditum (W. R. SlrOhI. ro .). pp. 241-277. Mared Dekker. New York. Queellt'r. S. a nd SchwarlZ. R. W. (1979). Penicillin.s: biosynthetic and se mi· sytl(hctic. [n Eronomic M¡crobiorogy. Vol. 3 (A. H. Rose. 00 ,), pp. 35-122. Academic Prcss. l.ondon. Smith. A. (1985). Ccphalosporins.ln Compreht.'J1sü -e 8iout:hno.logy. VoL 3 (M. MoaYOUllg, oo.), pp. 163-I3S. Pcl'gamon Press, N~ York. Strohl. W. R. ('1997). Lnd ustrial antibiotics: roday ólnd the future. ln f!wteochnology ofAll1ibiotia, 2nd Edition (W. R. Strobl, ed.). pp. 1-47. M;¡rce1Dekker. New York. Vandamme. E.j. (1984). Mtibiouc searcb and production: an overvi.ew. ln IllouchnologoflndustrlaJ Antlbiotict (E.J . V;mdilrnme, ed .), pp. 3-3 1. Marce l

Dekker, New York.

3~

Chapter 17

8aker's yeast Sven-Olof Enfors Nomenclature Introduction

MC!dium fur ba).:er's yeasr production .Aerobic ethanol formation and consumption ll1e fed-batch techniquE' used to control e:thanol production Industrial process control Process outline

Funhcr reading

1

C, C,

e, OOT

oor F 11

K," K. ,~

'" ""'" q.

'.

,~

''''''

'... '.... S I

V X

Nomenclature EtbaDol carbon concentration Sugar carboo concentratlon CeU carban concentratlon Dissolved oxygcn t.e.nsion DOT inequilibrium with gas Substrate fiow rate Convenioo COllstant Oxygen transrer coefficienl Saturadon constant Spedfic rate of ethanol coruumption Spedfic rate o r ethanol production Maintenance coefficient Specific rate of oxygen consumption Specific rate croxygen consumptioll fur sug;rroxidation Spedfic rate ohugar comumplion Specifk mte of sugar ro anaboHsm Specific rnteofethanol con.rumplion wnen over:fIow metabolism seu in Specific rateofsugar 10 aerobic ellergy metabolism Maximum<Js Sugarroncentraóon Tim, VoLumeofmedium Biomass concentrntioD

(kgru- l ) (kgm 3) (kgm J)

(% air saturatioo) (% air saturation) (m3 h- 1) (% air saL kg- l· m J) (h- I ) (kgm - J )

(h-1 ) (h-1 ) (h-IJ (h-') (h-') (h-')

(h-') (h-' )

(h'"' )

Ih -') (kgm-ll (h) tmJ)

Ikgm-')

378

ENFORS

Yos y~ Y:w Yxs

Y"",

YieJd c'oefficient exclusive. maintenallce Coeffidentofoxygen per sugar Yield coefficient of cel15 per ethanol Yield coefficientofcells pe.r oxygell 'úeld coefficientofcells per sugar

Il. JLait

Spédfic growth l
up bythe cel. Malwse ¡, fht tnmported ¡mo the cell and lhen hydrolysed In dle cytOplnm ea ghx:05e. An importal'lt qulllry aspe« o, b.aker'1 )'eUt i1 that !he

a·glueosidsS8 - --=-= ==='---- .",. ",,,,,

Glycalysis

malto,e comumptloflgt:nr$ a~ 'l,Ibjected to g!uco,e repfeulon

,

and me enrymu Involved are very ullJable wh ich mean, tIuot the

TCA-

J NAV '

CO

z

----- ,1

Acetaldahyde

NAo.----1 NADH----1 ,

ablUf)' tO utlllse ma!tose 1, variable.

re

NADH

NAO'

>

IUO;"

NADH

,

L

02

Respiration ATP

ADP

Eth8nol

fed·batch technique is employed to control the sugar concentration; in other fermentation processes it may be other substrates or miere> nutrients to keep them at conceotrations wbich will pe.rmít optimaJ metabolic activity in tbe cultivated micro-orgarnsms. The uptake and energy metabolism ofthe main sugars utilised by baker'S yeast i5 shown in Fig.17.1. Baker's yeast is composed ofliving cells of aerobicaUy grown S, cerevtsjae.The commerdal producers use valious strain.s ofthis species. They differ from the strains ofS. cerevislae used fol' beer production mainly in rheir panero of utilisation of medium components. The product is cimer deUvered as a dried powder(dryyeast) with about 95% dry weight or as a cake with about 25-29% dryweighí. containing onlywashed cells and residual water. The yeastis used to r.üse Che dough in the baking process and to give special texture and taste to the bTead. Oough raising is caused by the production of CO 2 during alcoholic ferrnentation of sugaTS available io the dough. These sugars are mainly maltose and glucose. produeed from the floor starch by the a-amylase aetivity in the Rour, or sucrose if added by the baker. lbe majn reactioo ofthe dough raising can be considered as anaerobic fermentation ofhexose to C0 2 and ethanol: (17.1) The carbon dioxide i5 entrapped in the dough and causes its exp:m sion. Tbe erhanol, even though it evapora tes in the oven , concributes (O formation ofesters. However, there are rnany other, le5S well characterised, properties of the yeast thar are important for the bread Quality, as evident from tbe difference between yeast fennented bread and bread

]79

]80

I

ENFORS

produced-with bakingpowder. that aIso evolves COl"Thus, baker's yeast should be considered as a package ofenzymes.ratber thanjustb¡omass. The: composition af lhis e nzyme package is subject ro opti.mJsation by slra m developmenr and control ofthe fermentatioR process.

17.2 I Medium for baker's ye.se produceion The stoichiometlyfor production ofbaker's yeast can be summarised as 200 g glucose+ 10 gNHl + 100 g Oz + 7.5 g salts~

100gbiomass+ 140gC02 +70g HP

117.2)

TIlis results in me following approximateyie1d coefficienls: Yxs "" O.5kgkg-t y ro = 1.0 kg kg-I YJ(N= O.lkgkg- I , 111e production is an aerobic fed·batch process on a medium af motas· ses. aromanía orammoniumsalts. phosphates, vitamins and antifoam. Which specific vitamins and additional sal ts have to be ineluded in rhe medium depends on the strain, (he quality ofthe rugar source (moJasses) and the quality ofthe water. S. cerevislae has a rlemand formany como poncnts. as evident from the complexity of a detined med.ium for its growth (seeTable 17.1). For cornmerdal production. howeve.r, rhe mol..lsses and the process water fur.nish mos[ ofthese components, Molasse5 ofboth sugar cane and sugar beet can be used rol' baker's yeaS( production. 1b.e sugar content of the commen:ial moJasses is 45-50%. A major difference becween che two types of molasses is th3t 5Ugar bf:.et molasses contains mainly sucrose and Htt1e hiolin, whlle in sugar come rnolasses the sucrose to a large extent has been hydrolysed to glucose plus fructose, aud ir is also richer in biotin. Furthermore, motasses contaius other fermentable sugars and amino acids that are udlised by the cells. A problem with the beet molasses is that 0.5 te 3% oftbe sugar is r.tffinose, a trisaccharide (fructose-glucose-gaIactose) tbat is only partiallyhyd.rolysed by baker's yeast that does not baw a-galactosidase activity. This results in a substantial emuent ofme.libiose (glu..gal). Brewer's yeas!, on the other band, often.has a-galactosidase activity. a nd doning the gene coding for this enzyme into bake.r's yeast is tberefore aD obvious possibiliry to improve the yield and de
's

Glucase ÚlI-')

0.2

,no

AP'

The bonJe. neck mod~1 ilfunroued as Memoo plocs ofthe speclrlt rones ot supr (qJ and oxyg~n (qo.) cOfl!umption. growth (p.) and ethanol production (qq) ¡nd Ulf"d1nlP00n (qJ. ~ long u tlle wpr concentl'atlon incre~s~s at concentratlons below lhe critical y,¡lue, me specific o>
JI

3!W

ENFORS

17.4

The fed-batch technique used to control ethanol production

Ir-is difficult to reach high concentrations ofbiomass ofS. Crn!vtsiae. in a batch process since tbatwould require a high initial sugar concentra· tion and, due to the overflowmetaboHsm thiswould !"emlt in inhibitory high ethanol concentrations. Furthermore, rhe highgrowth cate would res1.Ilt in ahigh oxygen consumption rate.As industrial bioreactors have quitemodest0 2 transfer capacities in che rangeofl 00 mmoll~l · h _ hig h concenlrations ofbiomass cannor be obtaint!d withoutcontrolling tbe oxygen consumption tate wirh in the range ofthe bioreactor capacity. 80th chese goals, themetabolicconlrol orthe overflow metabolism and che oxygen coruumption ¡-;:¡te control can be achie\'ed through the redbatch technique. There: are no simple analytical saluaons af the mass balance equatians of a red-batch process as in the case of rhe chemostat. Instead. numerical solutions are applied from given initial conditions sueh as concen trations ofbiomass and subso
(17.12)

where Ks is rhe saturation coostant rOl" sugar uptake. l1tis initial substrate concentration can be kept constant by application ofan exponential feed rareo P(t). calculated from : F(r) = Foel'o"ltl

(17.13)

The inirial feed rate, Po' is esnmated (roro:

,,,

Fo =s""y' (XVI,

(17.14)

where (XV)O is [he ¡nitia! biomass (kg) and SI is tbe iolel feed sugar concentration (kg m - 3). ln practice, rhe coefficients in this modc1 are not consrantand tberefore the concentra han ofsugarmay not be absolutely slcady. Alag pbase with respect: (O sugar uptake i5 often observed and then the sugar concentranon may initi.uJy rise, but this is later on compensated by an increased growth rate, so eve.ntuaUy the concentratioo stabilises at the control vatue as isvisualised in Hg. 17.2. Exponeotial fee
3 e~

386

ENFORS

that the O~ consumption ratedoes notdecline until all ethanol has been conrome
17.5 I Industrial process control The process lasts for abonr 15 hours a nd 50-60 g dry ceUs per litre will be reached. Themoculum size is al>out 10% (vJv). Theinitialrate ofsugar feed musrbe low [O avoid accumulation ofsugar and excessiVl" ethanol fonnarioa ilS the critica! vaJue aboye wbich ethanol is prodllced by over.Dow metabolism is only about 100 mgo 1- 1 slIgar (present as glucose and fructosc at a ratio about1 :3 due ro the rupid hydrolysis ofsucrose). To use a constant feed rate then, correl>1>Onding tothe critica} consumption rafe of tbe cells, would make the biomass productivity too low. Thcl·efore the principIe of exponential feed is applied to locrease the sugar feed rate ar the same rate as t he biomass is increasing. In the baker"s yeastprocess, a spedfic growth rate ofapproximately 0.25 h- ! 1Sused dw:ing theexponential pllase, sim:e a higher growth.rate would give too much ethanol by overflow metabolism even thougb the productivitywould be higher. Iftbe feed flow profile is selected ro give a growth rate at or below I-'¡rjt no ethanol is formed and tbe biomass yield would be the m aximal, but the productivi ty ofbiomass wouJd be lowcr. Thus, sel e
Production ofen:tymes has greatly expanded since the 1960s due to

PRODUCTION OF ENZVMES

the widespread introduction of fermentation rechnology and more recently from the introduction of genetic cngineering. Recombinant m icro-organisms are now becoming tbe dominant source ror enzymes for a wide variety oftypes. This trend wilI ¡ncrease in tbe futnre due ro the ease ofgenetic manipulation and the wide variety of enzymes available from micro-organisrns found in diverse and extreme environmenu. Many microbial enzymes have becn found and developed to replaceexirting enzymes from animaland plant origino

18. 1. 1 Commercial considerations Curre ntly. enzymes aTe produced from a wide range of biological sources. An approxim.ate breakdown of the SQut(es for bulk enzyme source is;u¡ foUows: filamentou s fungi . 60%: bacteria. 24%; animal , 6%; plant. 4"; yeast, 4 %: streptom)'4:es. 2%. 11rree general approaches can be taken to ¡ncrease market share of bulk enzyme sales: • develop less expensi.ve manufacturing processes to reduce final con and the sale pdces: • identi:fy and develop new enzyrnes from new sourres and seek new applications: • find new uses forexisting en zymes. Many producers prodllce more than one enzyme from the same source. This is because market changes can produce a s.hortage in one type of enzyme and over-production of orher e nzymes. These imbalances can produce price variations. Bulk enzyme producen usuaIly make more t han onegrade ofeozyme with lhe higb. quality foad grade dominating due ro me high production volumes for lhis type ofproduct. Manufatturing processes for enzymes vary a greatdeal and are gavemed by the required quality. applicatioll. cost and market volumc. Bulk cornmodity en~ymes have an inherent low value and therefore necessitate a low cOSt manufacture witb minimal processing. Al the other extreme, high 18.3 I Enzymes from microbial sources M.icro-organisms are tbe mos[ eonvenient source of enzymes. The numbee and diversity of enzymes is proportional to the number and diversity ofmicro-organisms. Microhes have been collec(oo from envil'Onmemal extremes 5uch as hor springs. r.he aretic, rain forest and deserts. Eacb biologicaJ species has 3ssociated specifie microbes and tberefore the potential spectrum ofenzyme activities islarge. Cenetie engineering techniques have en¡¡bJed (he enzyme indusn-y to ¡ncrease the fennenCltion productivity ofthese enzymes byrnanyorders oC mago nitude. Even the propei'ties of these enzym~ can be altered and improved by pr:otein engineering. Many enzymes are narurally repressed and can only be expressed under certain culture conditions. These enzymes can be both intracellu· lar, typic-.t11y in the case of E. eoil. and extracellular as in Baci!!us species. Comm.ercia1 strains of non-recombinantbacilli and aspergilli are Imown [O produce enzymes up (oa concentradon of20 kg m- l. Recornbinantcultures can also produce enzymes al (hese productivities.

18.3.1 Species-specific enzymes A number of AspergfIItLS strains are prolific producers of many types of enzymes. Duf' W well-developed rermentatLon proccsses. A. niger is airo w:idely used as a host fur expression of rerombinant enzymes (s~ also Chapb.'.r 5). This species is known to produce over 40 different rommer· cial enzymes, the most common ofwhich are shown in Table 18.7. Similar enzymes can also be produced from different mkro-organ· isms, ror example thCl'C are seven different microbial sources fur glueose isomerase and eigbt for u-arnylase (Table 18.8).

39

396

LOWE

U""'" ArnyIogluwsidase Pentosanase Prote"", (l'·amy!ase Phosphalipase Phytase Glucose oxidase Pectlnase

Pectin esterase Cellulase Catalase a -galactosidase Inulinase ,8-glucanase Galactomannase Arabinase

a·amylase

Glucose isomerase

Malted cereals Animal pancreas Asperglllus oryzae Aspergi/lus r1/ger

Actinop/ones m!5.sounen.sis Baci//us coogu/ans Mycoboaerium arWrescens Streptomyces murjns Streptomyces oliVOCeDUS Srreptomyees o/ivochromogens Strepoomyces phoenics Endomyces spp.

Bodllus amyloliquefadens Bbcillus lichenirormis Badllus steorothermophilus Bacillus subti/rs

AAjzopvs oryzea

18.4 I Large-scale production The cultivad on of micro-organlsms is economical on a large scale due ro the use. of inexpensive media and short fenn en ta tion Lydes . The.physio· logit:al sta re of the micro-organism in a ferm entad on pl'ocess can be well controll ed and the uniformity of each batch ensured. The harvest can be conveniently scheduled to tit in with t he downstream processing. The choice ofenzyme to be fermented i5 easyto schedule and different enzyme production campaigns ean be plan ned and adjusted {omeet sales dem and s. En~ e. produetivüy ea.n be inereased many-fold by both conven tion al strain improvement and fe nne.ntation process deve]· opme nr and o witb (he additional use of genetic engineering, several orden of magnitude ofimprovement can be realised within a relative.ly short perlad orrime (one to two ye.ars). Recombjn anr DNA reehniques have also opened up opportunities for the m ass production of enzymes from other microbial cultures, which conveotionally were fastidious growers, req uired expensive media orinducers orwere potential pathogens. Enzymes from extremophiles (growing at!:he extremes oftemperature, salinity, pressm e. aIkalinity) can now be conveniently grown in mesopbilic cultures, yet produce enzyrnes with the beneths oftemperature resistance or high salt tolerance.

PRODUCTION OF ENZYHES

18.4.1 Recombinant E.. co/i fermentation Enzymes originating from prokaryote sources can be conveniently produced on a large scale ar high productivities in recombinantE. coli hosts. These fer mentations can be carried out at a 3000 to 60000 litre scale, and do notrequire complex inoculum build up. Atypical fermentation protocol could be as foHows: The E. coli host \Vould harbour a plasmid with the DNAcoding fur the required e nzyme, togetherwith a suitable antibiotic resistance rnarkcr such as a mpidlliu or neomycin, and an inducer such as TAe (codon speciflc (O lactose or isopropyltbiogalactose induction). Satisfactory h igh production can be achieved by a fed-batch fermentation whe:re the ¡nitial batch medium contains components for initial growth e.g. glucose (2%). yeast extract (1 %). phosphate (1%) and otber salts togelher with the chosen antibiotic. After the initial growtb has been esta ~ lished , furtber nutrients are fed at pre-detennined rafes (O provide a readily avaiJable supply ofcarbon and nitrogen . Carbon is conveniently supplied as glucosc. aDd DitrogeD can be eitber ofa complex nacure e.g. yeast extracto casein bydrolysate or roen steep tiquo. (aU provid ing amiDo acids). or as a simple arnmonium salt or urea. GrO\vth on amino acid mixtures provides faster growth aDd enzyme producrjon , howc\'er the use ofsimpler, more ddined nitroge n, aJthough it may require a longer fermentatlon period, can support similar high ceU growth a nd enzyme productivities, Use of define
397

398

lOWE

expensive nut1rents which can bave benefits in downstrcarn proc~sjng. Ferrnentatians are typically for periods of4- B days. Enzyme eJl:pression in excess aflO g 1- 1 havc becn reporree! at the industrial scale. Enzyrnes are usuaUy produced extraceUuJarly which is a hendir as mecbanical ceU breakage is nar nceded.

18.4.3 Microbial enzymes replacing plant enzymes Some industrial enzyrues continue to be extract(>() from bovine sourc.es which pose the danger of conta mination with bovine spongiform encepha lopathy (BSE). Por example rennin. obtained from the rtomachs ofnewbom calves. continues to be used for cheese making. Ir remains to be seen.whetb.erthis presenlS any health l'isk to consumers. however, recombinantcalfrennin can now be produeed by microbial fermentations. Alternatively. with the convenient isolation cf enzymes from rnicrobial sources. and the use of appropl'iare screening technology, many new anim
PRODUcnON Of ENZYHES

only beproduccd in (he presence ofan inducer. normallyits substrate. The level ofinduction can be vclystrong(a more than 100o-fold increase over non-induced conditions) and aets by interfering wirh the control· ling repressOf. Many eatabolie enzymes are inducible. For example: • sucrose is lleeded for invertase production ; • st:arrh for amylase production; • g;:¡lacrosides for {J-ga.laetosidase production. lnsome instances a produc[ or intermediare can aet as ao induc~r: • phenylacetate induces penicillin G arnidasc; • fatty acids induce lipase; • xyiobiose inducesxylanases. ProcIucrinduction is cammon in che synthesis ofextraceIlular enzymes tequired fOf the hydrolysis of large polymers tbatothenvise would nor have the ability to emee the ceJI and cause the induction. Co-enzyrnes can actas indu cers. i.e. pyruvate decarboxylase is induced by tbiamine. in addition lO being efuctive in enzyllle production. induction can be useful in controtling the timing of e nzymc production in the fer· menter. ¡.e. a late rapid induction fur an enzyme that is ullstable under fennentation conditions. Howevcr, in praetice. induction does ofien necessirate the bandJingofexpensive.inducer compounds. which bave ro be sterilised and added aC spedfied times to established fermentarions. To avoid tbese problems regulalOry mutants can be produced in which the inducer dependence has been eliminated and are [bus cal led constitutive mutants.

18.5.2 Feedback repression Enzyme synthesis is also controlLed by feedback repression. This occurs particuiarly in enl.ymesinV{llved in tbe biosynthesis ofsmall molecules where ti)e accumulation ofthe final product can cause the rcpression ofthesynmesisofpartirularenzymes, normaUy the fustenzyroe in the biosynthesis route. Mut3nts lacking feedbac:k Tcpression can be obtained by selccting forcultute$ resistantto the toxiceffects ofan analogue ofthe product or intermediare. Th~e SUlVÍvors have lost [he feedback sensitivity towards the product and its tone mimic. Similar mutan{S can beobtained by isolatingnutritional auxotrophs wherethe culture cannotmake theflnal product, butinstead dcpends on the addi· tion ofthis compoulld for normal growth. The controlled fcedillg ofthis "uuren( willlimit lhe intracellular concentJatlOIlS ro below feedback repression levels.

18.5.3 Nutrient repression Enzyme synthesis can also be controlled by nurrient repression typi· eally by carbono nitrogen. phosphate Of sulphate. Tbese mechaniSffiS existto conserve the production of ullnecessary enzymes. Thus the cell only producES enzymes for the assimilation ofthe most easilyrnetabo· lised or mos1: readily available form of numenl. The best known exampLe is the. control caused by thc presence of glucose w here this carbohydrate- can effectively shut dmvn the production of enzymes involved in the me labolism of other related and non·related

399

18.6. 1 Site-directed mutagenesis Enzymes which have known amino acid sequences and three dimensionstructures can be altered bysite-directoo mutagenesis (seeChapter 4). Amino acids in tbe active site or other important areas can be identifled as targclS for cbanging to other amino acids, This can be

40 I

102

lOWE

performed easily by changing the specific trinudeotide codons in the enzyme gene and expressing the altered enzyme by conventiooa l cloning. Once the benefici::ll change has becn selected the process can be repeated to Cl"cate further amiDo acid changes in t he enzyme. 'rhe new enzymes are easy to scale-up using rhe same production proccdures developed for theparentwild l)'Pe as these aminoadd changes have no effect on the growth ofthe host or protein expression.Well-known such amino acid changes are increased stability, changed substrate preference, resistance to oxidation, tolerance to solveots and alk.ali, nnd changes in chiral activity.

18.7

I

Recovery of enzymes

Enzyrne recovery and purification are as important to the e-conomics of productionas the fermentation stages. The main challenge in che recov· ery steps is ro rninimise losses in enzyrne activity. Many of the steps employ conventionaLrecovery and purification units that are dl"Scribed in detail in Chapter 9. The foUowing tut will concentrate 00 enzyme· specitl:c issues in the recovery and purificatioo ofenzyrnes.

18.7.1 Recovery of extTacellular enzymes ExtrClcel1uJar enzymes are relatively easy [Q recover and pllrifY. Theycan tepresent a majar portian of tbe total extraceUular protein and often simple cell removal and concentration ofthe active saludon can yie1d enzyme preparations di~tly su.irable fur sorne applicatians. Relatively dean eozyme preparations can be obmined by cultures growi.ng on simple define
18.7.2 Recovery of intracellular enzymes Por preparatian ofintracellul,uenzymes from animal or plant sources, che tissue has to be. disrupted ta re1ease the enzyme. Dett'rgents or surf.:1ce-active agents may llave to be used to dissociate enzymes that are me mbrane bound. Tissue dcying can be a convenient method of stabil· ising and disrupting animal and plant fusue. Freeze..drying is the least disruptive and avoids pratcin dCgr.ldation. although it is proh.ibitively expensive 00 a large. scale. T~sue cao be airo or vacuum-dried. or SlLlr jected {O water-miscible solvent precipitatia n. Enzyme extraction frOID dried matcrials can be as simple as rehyruatioll in an appropriate buffered solution. Simple freezing can break some fume althougll t.his is nor a convenient mechad forscde-up, Sorne plantand animalrnaterials require tissue homogenisation where rhe tissue i5 shredded and blended by a me-chanical means to break opeo the ce1ls. Once the enzyme has been solubilised, the residual cell debris can be con ven· iently removed by tiltration. Low speed cenrrifug;¡rlon can also be used. In any tissue with fat presento t he removal ofthefatlayer can be problematic for cermjfugation. fany materia l c
18.8 I Isol.tion 01 soluble enzymes It ¡s critical to separare the soluble enzyme quick.ly and efficiently from the. remainillg cell debris. Chilling, use ofaD appropriate buffer, and the presente ofellzyme protectants such as mercaptoethanol may be neces· sary (O stabilise sorne enzyme preparatians. Ofien pratein inhibitors have ro be added to reduce me clestructivc. effect ofproteases. Soluble enzymes can be collectcd by membrane .filtration ar by cenoifugationthe latter has been usoo since che turn ofthe century. It is relativelyeasy to perfor:m at a laboratory scale. where high centrifugal forces can easily be obtained. The same high g forces caunot be auained on large-scale equipment. Large centrifuges are afien constructed from expensive tila nium aUoys ro withstand tbe high g forces, bUl even lhen onJy

403

04&1

lOWE

moderately high g forces can be obralned. To combat this problero , prorein- and nuc1eoprotein-precipitants, such as polyerhyleneimine. can be added to the ceJl homogena res to f10cculate unwanted materials and aid the settling rimes . Filtration, with the use of filtr;¡,tion aids, such as a diatomaceom earth. can be a convenient way to obtain soluble enzymcs_ ScaJe-up ls easy_ Additives can be used {Q speed up the filtratioD_ Often several precipitating agents can be used lOgether, in order ID reduce subsequent processing steps_ With wasbing, product re=0

A

Baker's yeast

~

+

H ceOA

X~OR B

B"'!.I/"H

~\ A ~

+

OH

..::> kJ and me lower monoeuer (1R.2S) wlR be eonsumed fasl"!. Hcnce both st~ wllI k;.ld w an Increase ofme u ppe1' enantlomerat Ihe monoenlr nage. lr the reOt.her meso- 19.3

I

Chiral building blocks roe synthesis

Chira l building bloeks fOr syn thesis of complicated organic molecules can be provided by three basically different methods: • che mkaJ tr3osformacion of enantiopure natural products: • asyrnmetric synthesis from prochirnl substrates; • resoluDon of ral.""emk mtxtures. Enzymes as ehjl'al caralysts play a role iLl all three methods.ln na.ture. enzymes catalyse production of chiral compounds. Enzymes mayalso catalyse asymmetric synthesis, as well as resalve racemates. \Vhich me thod is chosen in different cases depends 011 severa! factors. like pl'ice ofsraning materials, number ofsynthetic steps, available produc· tion technology. know·how etc.

SYNTHESIS OF CHEMICAlS USING ENZYMES

19.3.1 Asymmetric synthesis Asymmerric synthesis is tbe terro used w hen a prochiral substrate or a meso-substrate: is con verted mro an unequal amoumofchiral product. A prochiralcompound is a compound thatmay be converted in[o a chiral compound in one step_ A IIIC'SO-Compound has sten'Ocentres bllt rhey are organised in such a way that me compound as a whole is achir.a:1. The product of an asymmetric symhesis is characterised by che enantiome rice.'"cess, et. For instance ¡fthe prod uel mixtu re eontains 95%of one enantiomer and 5% ofme other, ee=90%. A raeemic mixture which contai ns 50% af eaeh enantiomer, has ee =O. The thearetieal yield of 100% afon e single enanriomer may be obrained ifall ofthe starting material is con verted into Dne single isomer. If the two possible produets are enantiomers the reaetion is enantioselective. Typica! examples of enzyme-catalysed enantioselective asymmetric synthesis is -reducdon ofa non-symmetrical ketone(Eig.19.3d) orhydrolysis ofa prochiral diesrer (Pig. 19.3a). The starting material may also be a meso compound as in Fig. 19.3b. In asymmetric syntbes is. me enantiomeric excess of the product will be consrant throughout tbe reacrion and ¡twill depend oruyon tbe tl6.ct of the two possible courses of reaction. Reaction between me prochiral substrate nn d the enzyme leads to two diastcreomeric tr.rnsition states with different energy. The difference in free energy ofactivation is re!ared ro tbe ratio oftbe two rate CODstants ofreactions. Ifthe measured ee ofthe produce is 90%, the reladve rateo constill1tfor formation of the isomers is 95{5 = 19 which correspands to ó.ó.ct= 7.3 kJ llloJe- 1 (6.6.(;1= - RTInK= - 8.31441 x 10- ) X298 X 19) This is a smaJl nUlllher as wmpared to the total6.G1 for the reaetioD whicb may be in the order of60-80 kj m ole ' .

19.3.2 Resolution by hydrolysis: irreversible reactions A racemic mi1'turc (rncemate) of a desired building block may be produce
1n[1 - c(l + eep)] In[l c(l ~ep)]

The degree of conversion under mosrcircumstances (equal amounts of enantiomers at the beginning of the reaction, no side reactions) is related to te" and ee, by:

u,

,~---

ce, + re"

Hence, another expression may be used ro calculare E:

LnI"rl1 - « ,)1 H

(l't',+ eeJ

In1eep(1 + f'cJ)1 (f'C, + et;)

Ihe advautage ofthis expression i5 that ie does not involvec which may be difficultto measure accurately. As. opposed to ee, aud f'C,. which are relative quantities. e is .ln absolure quantity. The most accurate way. however, is to use a computerpl'ogram te fit many measured data poinlS from several conveísions to ealculated cuJVes fOí differentE-valucs. In a resolution ee oC the substrate fraetioa is zero when fue reaction starts. Provided tbe enantiomeneratioEis high . the product fraetion will have high ee. Forinstanee ifE"" 19 (95 :5). eep will be 90% atilie startofthe reaction. As me reaetion proeecds. the eoneentrations ofthE': enantiomers change and also ce" and te,. Tbe relationship between ceF• ee, and e fuí three different values ofE is shown in Pig. 19.5. Tdeally, ifE is very high lE > 100) both eel'and ce,will bec.1ose to100% at 50% conversion and the reaetion virtually stops. Even reactions with moderate E-values can give the remaining substrare with very high ee praviding that yield can be sacrificed . A resalution tbal proceeds witb E = 12 will bave ef. of 100% at 75~ convenion . However. half ofthe theoretical50% yield islost.

19.3.3 Resoludon In organic solvents: reversible reactions Hydrolytic en%)'J1'la may be use
SYNTHESIS O F CHEMICALS USING ENIYMES

% Conversion

Enantiomeric exceu 01 product (ee" lullllnM) and nnnalnln¡ substr.ue (ee., linel) n. degree 01conversion calc:~lated for dv'u dlfflrlnt ,,~ I ~¡ 01 lhe en~tiomerit" ",00 E {oran rreversib~ resolutlon, Ideally, ¡fE ts ~'1 hl¡h (E> I 00) both ee. and te, wiB be dos-e to 100% at SO"~sJon Ind the react\on vlrtUally nopl. The ~V5, conven.ion CUNe$ lor the dilJerem E-vaIuet ¡nfer chat ",Is at tu maxlmlJ'Tlln th, begiming oflhe rnlOction white ee,~hes mvdmum at" bllr staC', ThIJ has an mponant conS(!r this reuon it mOl)' be easier toobt:ilin the ~maiJlin& ,ubttrate with higber te. s~pled

catiOD or better transesrerification in non-aqueous media (RCOOR. + R.pH = RCOOR;¡ + R,oK) is perfonned. A starting escer ís needed: the acyl donor. RCOO~. lt reacts wim rbe enzyme [Q fonn rhe al:yl enzyme which in rum rearo with the racemic alcohol. che acyl acceptor R;¡OH (see Fig. 19.2). Since the enzyme shows lhe same stereopreferenee no matter hydrolysis or transesterification. eirher tbe ester or the alcohol may be separated as the remaining subrtrate. butwith t he sameconfiguration. Itthe (Srester is me remaining substrate in hydrolysis the (S)alcohol will be the remain ing substrate oftraruesterification (Fig, 19.6). The mat hematical expressions presented in Section 19.3.2 are restriete
Hydrolysis

H OCOR

PhO~ R

H OCOR + PhO~

..

H,O

S

H OH Ph0vX. R

H OCOR

PhO~ S

+

Remainlng substrate

Product

Tra nsesterification

Acyl donor RC0 2 R1

H OH Ph0vX. R

H

OH

+ PhO~ S

U

HOR 1

H

OCOR

PhO~ R Product

' . Hydrolysls of iI r¡¡cemlc secl.>fldary este.. or tr.ln~nterlf!Cation 01 the corrulxmdlng ~ondilry alcohol (l·pMnoxy-2·prop3001) wlth ~

H

+

OH

PhO~ S

Remaining 5ubslrate

Computer programs fuI' ping-pong bi·bi kinetics, whicb use l'!~va.Jues mcasurcd at severa! degree§ of cODversion, are available.lfboth en::mtiomers can be provided in p~forms , it is also possi.ble to determin'e E and K... from initial rate measurements.

buuoolc acyl dooor ¡nd CALE u

19.3.4 Problems with reversibility

catlllylol. bQV1 yield lhe $ilme e nanll ome r u prodUC:L The p!'OdUC:l of hydrolysjs 1$ ¡he (fI)· alcc:>hol whlle lhe p~u c:t 01 transelolerif'ocation is tno (R)-estegres$ cUrYlO$look lil«! the examples of Flg. 19. 5. For reaCl.lo"s wlth sma ller K.,¡ val~ a dr.lrnatic effect i$ observed for ee,. The curve reaches a rnlXlmum. a1 the n!2.ction progr'eS5es furcher. tt, is reduud and ~he curve Ilt.'ver reaches 100%as le alw~yl does in cm. Irre .... rslbte cue. The e!fea of reverslblUty If noc U dr;¡matlc 011 fil~. The CUr-il ¿ips down al an urlier oogree of conve rt lon wnen Koq Islo_red. An obvloU1 way lO proued is 10 pum lhe (euuon towards the pl"Odu
n..

cwo mechu\hml of atdobses. GtClup I emymes from

animals and hI¡htr planu U~ an amino group In the enty~ lO kmn

a Sd'1iffs base Incenntdla!t CCl ac:civacetht aldol dOl'lClrs. Gr~ 11 .nl)'mes "om ICl'fier Clrpnltms. us. ¡ meco! Ion, IJslJ (white bJood cells). The ligand that E'selectin recognises is me tetra· saccharide sialyll.ewis X (SI.e"). Since Sl.eX competes with white blood cells for binding to E-selectin. [hus inhibiting the adhesion process. it may useful as an anti·inOammatoryand anticancer agent. Non-en"l)'IDic synthesis ofSUr" involves a large numberofprotection and deprotection steps which are not suited for largescaJe..production. However, enzymic processes using transferases have been developed with great 5uccess. Thecrudal factor in order to succeed is regeneration ofthe activated monosaccllarides . Synthesis of SLe~ and related aligosaccharides have been pe.rformed on a large scale (kilograms) using this technology. The synthesis comprises three transferase cataIysed steps. In the first, N-aceryllacrose is forme
oc z

.... . O

~

:l

O '"

'" ~

'""'" '"
"

< ()

r.¡

!-< O ~

...¡

'" '" ~

426

ANTHONSEN

CH20 H

An important !Itp 11'1

HO~ H

che production of vitamln e

I

(ucorbic acid) fro m glucase 11 me r~ose lectlve Ollidado n af o-¡Iudtol to yie ld l-$orbosa by Acetllboaer q/Inum. Thl$ bl oca~ic py ~"

EnanUopure (L)-amlno add

~,.

(lyas&)

O

~OH • Bi<x;¡¡wytk synUlesIs of am¡~oadds . ¡me.- by asym~tric. syn~ (Iower part offigurl.!) or rl.!Wlu lio~ (uP?« pare offtglJrt). Ad
PhYoCH, H NH,

+

Salt Ihal crystalllses

acids limits tbeir value. Secondly. fuere are well developed non· biocatalytic methods fur peptide syntbesis. For small quantities t he

automated Merrifield method works welL Nevertheless , one processfur synthesis oC the low calorie sweetener. Aspartame. which is a mcchyl ester ofa dipeptide (Asp-Pbe-DMe). lnvolves a biocatalytic step (the TOSQ~ process). Aspartic acid amino protected by benzyloxycarbonyl group. is reacted with two moles of racemic phenylalanine methyl ester ca talysed by the pro tease thennolysin.. Tbe exl:r.l mole of ester ma.J..."eS tbe dipeptide precipitate (Fig. 19.17).

19.7 I Further reading Bornschelll!r. U. T. and Kaz.lauskas. R.l (l999)Hyd rooues in Ofl,'anlc S,Y'Ith esl$. Wiley-Vrn. Weinheim. Faller, K. (2000 •. Biotratuformatiom In Or:;llllk Qwnil;tT)', 4th EdUlon. Springer-

Verlag. Berlin. Fenht. A. (1998). SrruCful\.' and Me,hanism ir¡ Protein Scjrnre. A Cuide taEnzyntt Gala!ysis and Prou!n t'oldlng. W. H. Freeman & Co. NewYork. Palmer, T. (1995). Undmtandlng Enzymes, 4th Edltion. Elli.5 Horwood. RDbem, S. M" Tumer. N.j., Willetll, A. and Tumer, M.. K. {199!'i).lntrodu(t!cn to Blocara!ys ls using 'vVholeI:'m:yrnes Ilnd MiCl'1).{}rgonisms. Cambridge University Press, Cambridge.

Chapter 20

Recombinant proteins of high value Georg-B. Kresse Applicatjoru ofhigh-vahle prou~ins Analytical enzymes Therapeutir proteLnS Regu1atory aspe-cts ofdH,-rapeutic protcim Olldook to tht" future ofprotein therapies Further reading

20.1 [ Applications of high-value proteins Proteins used in industrial enzyme technology. e.g. detergent proteioases orenzymes applied in me food industry. are in mus! cases rather aude preparations a.nd usually mixtures of different enzyml.'S. In con[rast, there are a number of cornmercial applications where higbly purified (and tberefore high-value) proteins are needed. Examples are : • AnaJytical enzymes aud antibodies. Por use in medical diagnostks. food anaIysis, aswelL as biochemical and m olecular biologicaJ analy¡ is (see Section 20.2).

• Enzymes used as toob in genetic engineerlng tttbnology. Gene technology has become possible through me availabilityofhighly purified euzymes sucb as resrriction endonudeases. DNAor RNA polymerases. oudeases and modifying enzymes. Similacly, glycohydcolases and glycosyl rransferases are use
Fu.rthermore, proteins with proven oc supposed biological relevance in pathomechanisms are needed as targets for the search of new ligands (agonists or alltagonists). inrnbirors and for X-r.J.y ar NMR struétur.J.I analysis in order to design nove.l interacting compounds by structureba,sed molecular modelling, Uds requires [he production ofthese proteíns on a rclatively small scalf' (10 lo 100 mg) but ofren with high purity df'pendingon tbe ¡ntended use.

430

K.RESSE

20.2 I Analytical enzymes Enzymes are highly spedfic both in the reactian catalysed as well aJi in their choice ofsubstrates. rnd\:cd. enzymes are, besidcs antibodJes . me most specific rcagents known . The use ofenzymes in analysis. thereforc. offcrs a number ofadvantages compared to cbemical reagents. The reae· tants may either bccome chemically transformcd in che presence of ,m enzyme (it they are substratesj. 0 1' (hey may modulare the enzymatic ¡¡ctivityin a manner related to their
Time

Enzyme

$ource (original)

Used forthe assay of

Cholesterol oxidase

Nocardia erythropolis or Bre\libocterium sp.

Cholesterol

Creatir-¡ase

Pseudornonos sp.

Creatine. creat inine

Creatininase j3-Galact.os,idase

Pseudomoncs sp.

Ct-eatinine

Escherichia coIt

Sodium ions: immunoassay marker en.."')'me

Glucose oxidase

Aspergillus n'ger

Glucose

Glucose-6--phosphate dehydrogenase

leucOflOSCOC mesenreroides

Glucose (indtcatorenzyme)

a-Glucos,idase

Yeast or Baúllus sp.

a -Amylase activrt.y

Glycerol-3-phosphate oxidase Hexokinase

Aerococcus viridans

Triacylglycerols

y",,,

Glucose and other hexoses

Peroxidase

Horseradish

Indic.ator enz)/me and immunoassay marker eozyme

Pyruvate oxidase

Pedtococcus sp.

Pyruvate: transaminase actMty

Sarcosine oxidase

PseudomorlOs 5p., Badllus sp.

Creatinine

Urate oxidase (uricase)

Arrhrobocrer protophormtoe

U""""

KJebslefla oemgenes

Uric acid Urea

Nffi;,s;

Al! litted I!IltY\ll
"32

I

KRESSE

HO-~CH' OOH OH

Ha OH Glucose 6-phosphate

Glucose

CH"'OH ®

®-O-vF°~H

Hbf-f1 OH

G6P-DH

~ OH

COOH

HO NADP+

Glucose 6-phosphate

NADPH

OH

6-Phosphogl LlColiate

An example of a coupled enzymatIc assay system using an indicatar enzyme: glurose assay wim hexoklnase and glucose-6-phosphate dchydrogcnase. The determinario n al glucOM! In blood or load materlals comprlses the pho~hory¡ation 01 glucose catalysed b~ yea
RECOMBINANT PROTEINS OF HIGH YALUE

Em:yme (source) Proleases

Trypsin (bovine) Chyrnotrypsin (bovine) Endoprateinase Lys-C rrom LysolxJcterc?f1zymogenes Endoproteinase Glu-C (V-S protease) from

Protein fragmentation for sequence analysis. peptide tingerprinting. limited proteolysis of enzymes or ~ceptors to 5tudy structurefun dJon reJationships

5wphylococcus QUfeuS VB

ea,ooxypepu(/a
fuund its correct conformation, the total protein concentration can be increased st:f'pwise up to economically attractive levels. Ibis process of 'pulse naturation' is commercialIy used m the production ofa plasminogen activator (see Section 20.3.5).

20.3.3 Application, delivery and rargering of rherapeutic proteins Because of their typical substance class properties, proteins genM'ally would byno means be considered 'ideal' therapeutic agents for reasons related tostability and application: 5tability Protems are polypeptides and therefore labile againstheat. extreme pH values and biological degradation. This may Icad to limited shelf-life as weU as short h •

>~

~Idl

U101

Aggregates

pH aud proteases. Therefore, oral application of protein drugs would not result in suffident bioavailability un1ess che protein is iotended to act in the oral cavity itselflas for example Iysozyme, usro ro inhibit bac[erial infectioos in m e mouchcavity) or in the gasrrointestinal tract (e.g. lípases aud amylases. used tosupport food digestion). Proteio therapeuties therefore canDor be given oraUy but have to be injected o. infused ioto the bloodstream . Immunogenicity offoreign prorel.ns Proteins mar are fereign to the human body are immunogenic. Wben injected inm the bloodstream , they may induce the formatiao afanti· bodies and cellular immune response. Furtbermore, proteinsobtained from n atural sources may contain immuoogenic contaminants. This may prevent repeated er prolonged application of the same proteio drug. (1be immunogenicity is desired when proteins are used as vaccines.)

Klrw:tlc eompetltioo between PrQl.eln foldln¡ and ;lnoelaooo. Stef»' (a) md (b) ¡¡re ptodtlctiYe fllTl-oroer 1016"8 steps whereu fleJ'$ (e) Uld (d) ue tlllproduccive Iec:ond or hlpr o.-der usoc:lation processet.. (11unn.doll eO\rte$)' of DI" R. fludolph. Halle. GermUl)t)

-«1

442

KRESSE

One way to decrease immunogenicity of proteins is cht"mical coup1ing to water-soluble polymers. especially to polyethylene glycol. Such 'pegylated ' proreins are in use as therapeutics. for example PEG-adenosine deaminase (PEG-ADA) for treatment of ADA deficiency (SClD severe combined immunodeficieney disease) by slIbstitution of tbe missing enzyme, as well as PEG-asparaginase in tumour thcrapy. It is, hOVt'eV{!r, not easy to ensure praduct homogeneity after chemical modification. and of course producrion costs are increased by the additional chemical modificaoon step. On the other hand recombinant human proteins are expected not to be immunogenic. Depending on theexpression system lIsed, however, proteins may differ from original human proteins in theirpost-tr.mslationalmodification (e.g. glycosylation, processing ofN-rerminlls. etc.).

20.3.4 First-generarion rherapeutic proteins The first-generation oC recombinant therapeutic proteins are. proteio drugs marle with the aid of gene technology. These are identical ro natural human proteins. A few typical examples are listed below.

lnsulin 'Ibis is a panereatichormonewhichhas been used fortreatrnentoftype 1 diabetes sinee 1922 because of its effect in lowering blood glucose levels. Insulin consists of rwo poIypeptide chains connected by disulphide bonds. TheA ehain has 21 amino acid residues and the Bcbain has 30 amino acid residues. Insulin biosynthesis involves proreolytic processing from me single-chain precursor moleeule proinsulin, with rclease ofa connecting (C-)peptide, as illustrated in Fig. 20.7. During me firstdecades ofinsulin ther-dpy. bovine orporci.ne inStilin had to be used. Tn these animal protcins, rhere are sorne amino acid sequence differencesfromhuman insulin thatmay lead ro formation ol' insulin antibodies dUlillg long-term applieation. In the 1970s it became possible to feplace the alanine residue, B30, ofporcine insulin witb a threonine residue by protease-catalysed semisynthesis and, mus, insulin identical to the human molecu.le couId now be produced. However, due ro me growing population of patients needing insulin (about1 in 1000). therewereconcerns thatthesupplyofpordneinsulin might become limited and the porcine or semi..synrhetic .human material bas been replaced by recombinant production of humaninsulin. Several stratf'gies have. been deve10ped to produce recombinant insulin. In the original process described by Genentech. Inc. and Eli Ully. theA and B chains are exprcssed separately inE. roli as fusion proteins with tryptopban synthease or P-gaIactosidase and, after processing by cleaVilge with cyanogen bromide, the two chains are connected by chemical reoxidation. In an alternative Qrocess, the physiological biosynthetic intennediate proinsulin (Fig. 20.7) DI: analogues with shortened connecting peptide sequences are expressed in E. rolf oryeast, and tbe connecting peptide is removed enzymaticaUy.

RECOMB1NANT PROTEJNS OF H1GH VAlUE

C-peptlda Prolnsulin

l

Proteolvtic cleavage

J'-.

-- s

S A chain

H, N -

C-peptide

@A..,""il®®@c&0OO)(D(bc~)(~)(!)@(0Q'XBJ6')~c)c~)- COOH ,

S

10

,

B chal n

H, N -

' ,I 21

Insuli n

(Ú®CN)(O)CE)(Vb®©(~{§I'®©(!)(0~~®®@X~XF)G)®
10

20

Erythropoietin Erythropoietin (Epoietin alpha and beta, EPO) is a glyt"oprotein of165 amino acid residues. It is fonned in tae foetalliveT and in tbe kidneys of adults. The EPO hormone belongs to che haematopoieticgrowth factors and induces the formarion oferythrocytes from prttursorcells (tenned BFU-E und CRJ-E) in the bone matTOW. Recombi.nanc erythropoietin has to be produced in mammalian cell sy5cems due {O the necessity ofglycosylation (Chinese. hamster ovary (CHO) cells a re used in the eom.m.erdal prCesses). and is used tberapeutieaUy mainly in renal an;)emia, but aiso in other indications. e.g. in tUIllOur anaernia. Granulocyte-colony stimulating factot(G-CSp) G-CSF belongs, as EPO, to me dass ofbaematopoietic growth fueton. GCSF stimulates proliferation and differentiation ofneutrophil precursor cells to mature granulocytes. It is therefore used as ao adjunct in chemotherapy ofcaneer to treat neutropenia caused by tbe dcstruction ofwhite blood eell5 by the cytotoxic agent. Furthermore, G-CSF is also lIsed in the rreatmenrof myelosuppt"ession afier bone marrow transplantation. chronic neutropenia, acute leukaemia. aplastic anaemia. as well aS to mobilise haematopoietic precursor cells frolD peripheral blood. G-C5F is aglycoprotein containing 174 amino acid residues, Pl'oduets bave been launched which eontam either the glyco5ylated mol&lI le produced fl"om rceombinant CHO cells (Lenograstim) or altematively an ungtycosylated, but the.rapeuticallyequallyeffcctive, form produce
Plaamlnogen

ti .,

-P"'m'n In","""

Flb rln ogen ($OIuble)

Flb ri n _ _ __

(ln&ohJbie erot)

1 Flbrlnogen

RbI'Inopeptlde5 (soluble)

peptl~

(soluble)

Tissue plasminogen activators Acute myocardiaJ ¡nfaretioo (AMI) is the principa1 cause of deaLhs in most Western hemispbere: countnes. Qne approach to improve treatment of AMI is me use ofthrombolytic enzymcs_ Plasminogen activa· tors catalyse the proteolytic processing of tbe inactive proenzyme plasminogen. which circul ates in che bl00dstream. inm the active pro-teólse plasmin. Plasmin is able ro cleave tbe insoluble fibrin of blood dors into soluble fibrin fragment peptides so that tbe dat is dissolved and [he blood vesse1 is opened. Thereactian scheme is outlilled in Fig. 20.8.

Plasminogen activators (e.g.A1rcplase andReteplase. a mutein with increasd in vivOhaLf.life) are usea increasi.ngly as thrombolytic agents in the treatment af AMI. and are aJso used in studies on relatcd disease,> such as stroke or deep veln thrombosis. ;ID

Other second generation recombinant protcin drugs In the near future, much progress is expected in me field ofrecombi· nant immunotoxin$ used in experimental trearmenr of various cancers. These arechem.ical conjugates or recombinant fusian proteins comrructed from a cell-bindingpart (mostly rh e antigen binding parts o( an aotibody), a translocation dornainmediatingtransferthrough the ceU membrane, and;¡ cytotoxic portian. e.g. pl'otein domains from bacterial toxins (such as DiphthcrUi toxin or Pseudomonnsexotoxin)or a chem¡cal cyrota; 20.4.2 Safety 1.n contrast to protl'ins isolated Í1:om buman oc animal, including tr.lD~

ge.n.ic. SOul'ces 01' from pathogenic organisms, e.g. vaccines obrained from bacteria or muses, highly purified and carefully analysed l'E'COJ1lbinant proteins do nat bcar the risk ofcontanllnaoon with allergenic subsGlIlCf:s, patbagenic viruses, e.g. HlV, or prions from Célttle or hum ans ca using newvariant Creutzfeldt-Jakab d isease. For tllls rcasoD, products such as coagulation factors (formerly produced from human blood or plasma), human growth hormane (in [he past obtained from adenohypophysis extracts), 01' hepatitis B vaccines are today manufuctured from recombinant systems.

20.5 I Outlook to che luture 01 protein therapies COllsidering tlte general advanrages and disadvantages ofpLOtein therapeutics, it can be conduded thatthey are notequally attrdctive in al! rherapeutic areas and indications when compared with competing approaches such as low-molccul;n weight chemical substaDl.:es on fue one hand , and gene therapy on the other. Proteill drugs would be especially useful in the following cases: • Inindications where no a[ternarive therapy is available, partic:uLarly forporentiaUy live-thrcatening diseases ~ut:h aS acute myocardial infarcrion, cancel' or viral infections. • For substitution therapy iCessential human proteins are rnissing or inactive, e.g. in ADA deficiency or in coagula non factor deficienc:ies.

RECOMBINANT PROTEINS OF HIGH VALUE

• Tomodulate me regulation ofbiotogicat processes such as metabolism. ce1l gcowth. wound benling, etc. OL' to influ ence the ¡mm une system byproteins acting as hormones, growtb facIors. or cyrokines (e.g. imulin. erythropoietin. c.cSF, somatorropin , interfcr ons or interleukins). ln these cases. protein- protein interac:tions have ro be modulated. This may be more effective witb therapeutic proteins as 'n a turE"s own ligands' optimised in the course ofevolu non. than with small chemical substances . • As vacOnes. especialiy against viral infectious diseases. Human proteil1s identical to [he body's own suhstances have become available through the advent of gene technology. Besides lhe 6rstgeneration biothel'apeutics, an incl'easing number of redesigned, second-gcneration pTotein muteins with improved properties are being introduced to the marketplace. Once the pl'eseut problerns oflow transfection and expression effidency have becn solved. it.maybe possible to substitute defecrgt"nes. oradd therapeutic genes. to humancells in vivo so thar (he patiem's body itselfwüJ act as (he manufacturing facility where the synthesis oftherapeutic proteins occurs . ln this sense. gene therapy may represen! the futme third-generation oftherapeutíc pr~ (eim. and may help to approach the final goal ro cure. rather roan (real, disease.

20.6 I Further reading H. u .. Grassl. M. and Bergmeyer.J. (eds.) (1983-t986). AMhods of Enzyma!lcAnuiyru, V()J.l-~1. VCH. W~¡nh~im . Bran~ , j. aoel Volunel, A (1999). rnsulin analogs witb improved pharmakoldn~tic profiles.Adv. Drug Deii~ Ro!\!. 35. 307- 335. BrulDw. A. F. (1993). Remmbinant·DNA-denvcd ins ulin analogues as potenlially us eful therapc utk ilgcnts Trrnds Bio~dmoi 11 ,301-305. B~rgmeyer.

KIegerman, M. E. and Groves. M..j. (1992). l'harmaa".lt'ical Biottdlllology: f-undum.t!lllals and ~nfillil. rnlerpbarm Press. Inc., Buffalo G~, JL. Kopctzki , E.. I.chnert, K. and Dude.!. P. (1994). EDzytues in Diagnostics: Achi{'\'('me.nts and Possibilities ofRffombinant DNA Technology. Qin. Chtm.

40.688- 704 .

Kresse. G.·B. (1995). ArIalytical uses ofcnzymes. 1n HiorMmoWgy.1nd edirion, Vol. 9 (H.:J. Rehm&G. Rred.eds.). pp. 138-163. VerJag Cb.emie. Weinbeim. Nicola. NA.(lm). Guidcbook ro Cytolincs ¡¡ndThdrR«eptors. Oxford University Press.Oxfonl. Perham. R. N. d ill. (1987). I:lUYII'Itf. In UlImoll n's fncJe/o¡miio ofllldU5hial Chemisfry. Vot 1\9. pp. 341- 5)0. AJso I~publis hed 5ep
4~

"54

VRIEZEN. VAN DIJKEN ANO I-IAGGSTRÓM

Endoplasmic reticulum

Simplitled scheme 01 mil N·&fyt.oS)'lulon parhway a/'ld

ucr.tion ola ¡fycoprnreln. A prouin Is synmeslstd 0fI ma rougt'I tndoplumlc reriwlum.l" th. tl'doplasmic relXUl.WTI me NgIyeos)'iadOl'l prtWr10r Glc)Man 9 GkNA, Is transl..-r-.d tQ th. proaoJn. Aftlrtrimmin¡ off (ha precursor, !.he: prottln Is u;uuferred to me ds·Golgl by

Cis-Golgi

vesk;u!artr.lIl1.port.. The ¡Iycortruewn: 1$ trlmm.d and eKtended al the prottlln traversas me Gol¡t tomp!tx fro m cls- f.O trans-GoIgl. Transpon: u.ku place by Ye,lcJes. When me ll'ycostr"Utw re Is tomplllU! thfI glycoproteln rnay be! I!)lCtrete d by WJy of 3 secretlon veslcle mat fuses wlm!.he: ,ell membrane. Symbob U$~ 0 , maMOse: • . GlcNAc.; +,¡ Iu cose: e, galactose; open dlamo nds. f\Kose; el, sialk aad.

Medial-Golgl

Trans-Golgl

Trans-Golgl network $ecretion Theprotein is then transponed to thecis-Golgi compartmentofthe ceH byvesicles that bud offfrom tbe ER.ln the ds-CoLgi, part ofthemannose structure is trimmed off, The proceln travel'ses the Golgi compLex via vesicular transportoDuring progression chrough the medial and trans Layen of the Golgi complex, the galacrose and GLcNAc units of !he oomplextype N-glycosylation are added. Final ly, sialicadd units maybe added in rhe trans-Golgi network. Tbe comple(ed glycoprotein is then transported to its destination. Secretory proteins are released frOIn rhe cell by fusion or the final secrerory vesicle with me cel! mem.brane. O-type glycosylation also takes place during the traflicking of the protein rhrough me GoIgi complex: [he location and reactioas for this process are, however, Jess weJl known. The specific glyoosylation panern dilJers between ce.H lines. Far instance, CHO cells do nor synthesise bisecting N-acetylglucosamine

MAMMAUAN CELL CULTURE

45

strnctuTes and mouse celllines are known lo sporadicaUy generate terminal galaetose units (Galo:l--+3Gal ) that are immunogenic in humans. Knowledge on the desired glycan strueture is therefore beneficial in se1ecting a ceU line for production, The glycan structures of gtyeoprotaos may ¡Dfluence lts key characteristics, essential fur the activity ofa phannaceutical productprotein. Forexample. cryth.ropo¡etin aetivity is total1y dependenton tbe presence and structur e ofits glyam moierles while the biological activity ofinterferons and sorne inlerleukins does llot depend on rhe presente of glyeans. The in vivo half-life of a glytoprotein 1S influenced by tbe amount of terminal sialic add which proteclS the proteio against clearanee from tbe bloodstream by hepalo'1'td or macrophages. The glycan structure also inOuenees physiC

2' .5

"'

I Media for the cultivation of mammalian cells

Mammalian cells in thebodyofan organism receive nutrients from lbe blood circulation. (en culture media for the in vlrro propagarloa of marnmalian cells must thercfore supply nutrients similar to those present in the blood strealll.lnitial attempts to grow marnmalian ceUs in vHro ¡nvolved media derived from tomple:< natural sources sllch as c1úckembryos. bIood serum ordots and lymph fluids.Since about 1950, partly defined media. consistingofa great number ofromponents. have been developed (Table 21.2). The basis for ceU culture media is a baI· anced salt solution. Thcse salt solutioos were originally used to create a physiological pH and osmolarity, required formaintainingcell viabi li ty ill \litro. To create conditions promoting proliferation. glucose, amina acids and vitamins were added ro tbe salt solut'ion, according to the requiremurs of the specific tell lineo This developmcnt resulted in various of media formulations. each des:igned for a limite
~

"'

e e e•

..

c.;

"'

f-< O

..... ..J

l

l

56

VRIEZEN, VAN DIJKEN AND HÁGGSTROM

Component Amino aods L-alanine L-arginine HCI L-asparagine H20 L-aspartic acid L-cystine L-glutamic acid L-glutamine glycine L-histidine Hel H.p l-isoleucine L-Ieucine L-Iysine l-methionine l-phenylalanine l-proline L-serine L-threonine L-tryptophan l-tyrosine l-vaJine glutathione (red) L-hydroxyproline

Vitamins D-biotin Ca D-panthothenate eholine chloride fo lie acid í-inositol nicotinamide p-aminobenzoic add pyridoxine Hel pyridoxal HO riboflavin thiamine Ha vitarnin B12

Inorganie so/ti. ea02'2H 2O CaNO]'4H 2O

Eagle's MEM

RPMI 1640

105

200 50 20 50 20 300 10 15

24 292 31 52 52 58 15 32

48 10 36 46

1 1 1 2

SO SO 40" 15 15 20 30 20

S 20 20 1 20 0.2 0.25 3.0 1.0 35 1.0 1

0. 1 1

0.2 1.0 0.005

200

Ham's FI2

IMDM

8.91 2 11 15.0 13.3 24.0 14.7 146.2 7.5 1 2 1.0 394 13. 12 36.54 a 4,48 4.96 345 10SI 1 1.91 2.042 5.43 11.7

25 84 284 30 70 75 584 30 42 104.8 104.8 146.2 30

42 95 16 84 93.6

0.007 0.26 13.96 1.32 18.02 0.037

0.013 4 4 4 7.2 4

0.062 0.Q38 0.34 1.36

4 0.4 4 0.013

44.1

218

100

CuSO~'5Hp

KCI KNO,

400

400

0.0025 0.83 223

MgSO~'7H20

220 6800

100 6000

7599

FeSO~ '7H20

NaCl

66 40

ll3

330 0.076 200 4505

MAMMALlAN CELL CULTURE

Component

Eagle's MEM

RPMI 1640

Ham's FI2

IMDM

NaHCO) Na2HPO..·7H 20 NaH¡p0.. ·2H 20

2000

2000 15 12

1176 268

3024

Other components D-glucme HEPES phenol red

150

1000

141 2000

180 1

5.0

1.2

sodium pyruvate

11 0

4500 5962 15

110

sodium selenite

0.01 7

BSA

400 1.0 100

1ransfemn soybean lipid lipoie acid linoJeie add

hypoxanthine putrescine' 2 Ha

Eagle, a pioneer in this field with many important articles published duringthe19SOs and 19605. de.tl.'rmined which amino acids were essentia! (ur mamma lian cells in culture (i.e. amiDa acid! tha[ cannot be synthesised by lhe cells themselves in amounts adequate for growth). EMEM is base
0.2 1 0.084 4.08 0.16

-iS7

458

VRIEZEN, VAN OIJKEN ANO H.AGGSTR,C M

h'3nsferrin (carnee of(ie3t ), selenium (tl'3ce element), fatty adds. dexame thason (an artificial glucocorticoid with growth_promotillg activity in certain cell types) and bovine $erum albumin (protects cells from bubble rlamage and is a carrier of lipids), Trace elements such as zinc. molybde:num and nickcl a re added to sorne media. Serurn-frec media for a variety of celllin es arE' now commerciaJly available.

21.6

1

Metabolism

Mostcu.ltured mammalian c('IIs use both glucose and glutamine as sources of energy and anabolic precursors. rus provides mammalian cells with a certaill flexibilüy. A limitt"rl supply of glucose can be compensated forby an increased consumption ofglutamine alld vice versa. As gluraminealso is a nitrogen sourre for mammalian celIs, glutamine limitation can lead to an increased cons umption of other amino acids 00 compensate for the lower nitrogen intake.

2 1.6. 1 Mecabolic routes for glucose and glutarnine Glucose is mainly metabolised via the glycolytic patbway to pyruv.ne (rig. 2 1.4) - see alsoChapter 2. Tbe main fate ofglucose-derived pyruvate is reduction to lacta te. Lactate ¡s excteted and aecumulares in the culture me tbe tricarboxylic acid cyde (TCA cyc1e). A smal) fraeríon (4-8%) of consumed glucose goes via tbe pentose phosphate pathway (PPP) which supplies ribose-5-phospbate for tbe synthesis of nudeotides. as well as reducing equivalellts (NADPH) forbí osyntbesis. ppp ¡s rhe most important partofrhe sugar metabolism for marnmalian eeJls as shown by the fo Uowingexample. Ifglucose ¡s exchanged for [ructoscovery littl e sugar is consume
-461

62

VRIEZEN. VA N DIJKEN ANO HAGGSTRÓM

coefficient forglucose {Y.•.l.1hls indicates that under glucose lim..iration a larger part orme glucose consumed is lIsed fur oxidatioll and biosyn. thesis, s.imilar to observations with micro-organisms. Limicing the amouO( ofglutamine fed to a culture likewise c\ecreases the amouots of ammoniuOl aed amino adds that are.formed.lfboth glutamine a ed g lucose are kept limiting (as with a double-limited . too-batch culture) lile production oi lactate and ammoniafammonium ions can be decreased simultaneously. Thus. Limiting one or both ofthe two major substl'ates forces the cellular metabolism ro become more efficient. Hence, understanding the interactions berween glucose: and glu tamine catabolism and amino acid metabolism is eueorja] for rationaldesign offeed and control stntegies in productioo .processes.

21.7 I large-seale eultivation 01 mammalian eells 21.7. 1 General conditions Many mammaüan celllines can be cultivated in suspension culture in the same way as rnicro-organisms. However. sorne ceH types. typically normal diploid cells, are anchorage dependent. requiring a surface to grow oo. These cells may be grown on the io's ide surfuce of plastic or glass bottles or on the sUJface ofmicl'Ocamers. Microcarners are small solid spherical particles (diamerer 100-200 jAom) whicll can be suspended in liquid cu lture medium. Porous micro(anjer beads arcan a lternative to obtaill a high surface area to volume ratio. Cellsgrowing in these carrien are prorected againsr sheal' damage. bur as they grow inm layers. diffusion limitations of nurrients and (by-)products will devclop. Mammalian cells originare n'om the body of an organism and are collsequently adapted to an environment that is kepr in homeostasis. An artificial culture environment should therefore maintain iu pH, dissolved 0 l and remperature within narrow limits. The pH optimum (pH 6.7 to 7.9) and tolerance (0.0 5 to 0.9 pH uni tsl are dependent on {he ceH lineoThe range for [he opti.mal dissolved O~ cOllcentrations is usually quite large: concentrationS between 20-80% being appropriare. In genernl. growtb maybe negative1y affected bclow 20% ofairsaruration; aboye 80% the 01 concentration becomes {oxic. As mammalian ceHs lack the rigid cell wall tha{ bacteria havc. they are sensitive to shear forces. Shear occurs nOlonly beca use ofstirring but also as a result of s.p arging. CeUs attached to ait bubbles are exposed {O enomlOUS forces when these bubblcs leave the bulle liquid at me surface and butS[due to decompression. Cells in sparged cultures can be protected from shear forces by using a medium witb a high viscosiEy. This can be achieved through high ceIl densities (> 10' cells ml - I ), addition ofextra scrum or components like Pluronic PF68. The main effecr ofsul'faceactive agents, such as PluTOnic. is via the coating of rising air bubbles. Cells do not.. atlach to bubbles coatcd with Pluronic to the same ex:tent as ro Daked bubbles and tllereby do not foUow the bubble to the surface and to the deadly bursting ZODe.

MAMMAlIAN CEU CULTURE

Cultiw.tion ml.'thodi fer mamm~IJan u lls. Open ~lTews indiQt~ a fIow of me
Fermenter

Roller bottle

Fed-Batch

Chemostat

Perfusion

In situ cell separation

Exte rnal

Hollow fibre reactor

call separatlon

Like micro-organis-m s, mammalian cclls: can be cultivated in batch, fed·batch or contiuuous mode . Cominuoos processes can be CUIl as a chemostat, or as a pcrfusion cutrure. Perfusion systcms are a specitic mode of continuolls cultivation in which the biomass i.s reuined in rhe reactor whilst ceU-free culture liquid is removed.

21.7.2 Batch Tn batch cultures, the inocuJum ofcells is added to th e total volume of medium to be-used (Fig. 21.5). During growth.cellsdeplete t he nutrients in the medium and excrete by-pl'ocIucts (Fig. 21.6). Gl'owth stops whcn a SUbstr.lfe is depleted or :J by-product b.1S re .. ched inhíbitory levels. However, in many cases it iJ¡ not obvious why gl'owth. ceases. Mammalian cells are routine1y maimained in the laboralOrybysucccssivc sub-cuItures in stationary flat·bottomed plastic Ilasks, called T· fLasks 01' Roux bottles. cODtaining 10-100 mi medium, with él largc surface·to·volume ratio. Anchornge-depcndeflt cells will attach 10 tb.e bottom of the flask so that further passages require that cells are

i6~

464

VRIEZEN, VAN DIJK.EN ANO HÁGGSTRÓM

,

~ech

hybrl~ Cll\~,

culeureof (a) locrease In

(a)

toco! "nd vl:lble cel1 conceotratioo, The lnoculum ce\\ dellsi't)' is 3bout

o

~c

0.2 x 10' cel\, mi- I an d th~ final c.~

Total ce ll co n cen tratlo nlb~.......

c

deo"t)' aboUl: \.5 x lO' c",lI$

""oc

mi - l. (b) Co ntentn.tion profíles 01

gluco,e, glutamlne, b.c;ute. '¡Ibnine

"

and ammonium lons. The Illltial

-¡;;

concentrltkm of gll.ltO$e b

ü

typially 5-25 mM and (lf gtutllmlne 2-6 mM. The f1f'\Ol1conctwltnltlon of lactat e is typlcaHy 1.7 lin\OH me

O

ilirial glurose concentratlon.

1 (b)

Ablline aOO "mlnon;u.... ions amoum: a:I 2-4 mM.. depi,lndlng 0 0 roe ili:ial gluColmioe concl!lItf1.tIon.

.; c

'E +oot ",I -z

" . c "''' ~ c

8 ~

a

'"

-¡¡;

o

~_ _- L_ __ _~_ _~_ __ _~

o

20

40 Time (h)

60

o

80

detached by using trypsin. a pcmease that 'dissolves' blidging proteins. Suspension cells will attach more loosely and can onen be removed by shaking the Oask. lt is essential that tbe inoculum size is [loe too sMall. Abou[ 2 X I0s cel1s mr ', oc mOTe. are often used togcrher with sorne spent medium which may contain secreted factors chal stimulate the censo own growth.Alternatively, the spentmedium mustberemoved by centrif\lgation to dispose ofinhibitory by-products. For large-scale cultures ofmammalian cens, 200 litres or len is often suf6cient to satisfy the demand for high-value therapeutic proteins. Even so, scaling·up requires severa! intermediate steps, the tirst being the transfer of cells from stationary culture to sbake flasks or spinner flasks . The spinner fJask. equippoo with a magnerically driven impeller hanging clown from the lid without tou ching the honom. was origí· naUy developed ro provide gentil". SriTring of microcarrier cultures, but is now used for suspensioo cultures as weU. The scale-up factor from sta· tion ary c ultures. or sbake fla sks without pH control. is Dor more than five. afien less, meaning that an inoculum volume of at least 20% must be used. In bioreacm.rs, where higber cell densities a re obtained. the scaJe-up faemc can be up ro 10 (Le. me inoculum is 10%vfv OT less). In a bioreactor.a r;ypical batch culture ofhybridomacells ¡am 3 ro 5 days and reaches a cell density of2~5 X 106 cells rnl- 1 tcorresponding to ea. l g dry wtcells 1 ~1 ). The maximum specificgrowth ratc (p.) ofhybrid·

MAMMAUAN CEll CULTURE

ama and myeloma cells i5 aboue 0.05 h- I • 111e amount of monoc1onal antibodies produced in a bacch culture ofhybridoma ceUs l'anges rrom 10 to 100 mg of protein 1- 1• A large-scale , batch production of mono-donal antibodies has been described with a 1 rn 3 stirred tank reactor. in \vhich cell densities of up to 5 X 101'> cells ml- I we.re obtained over 3.5 days. Early commercialproduction with anchorage-depe.ndent (ell! was oflen pcr:ronned in roUer bottles (Fig. 2 1.5). Rollcr bonles are kept in cODStantmotion byrotarion and anchorage-dependent cells grow on the bottIe surface. Typically a surface of750-15oo cm1 witb 200-500 mi medium will yield 1-2 x 1()A cells. A larger surface area is obtained by the use ofmicrocartiers in stirred tank reactors.

21.7.3 Fed-batch A fed-batch culture is . in the strict sense, colltrotled in [he same way as a rnemostat. i.e, the cellular growth rate is restricted by tIle dilution rate a nd the growth limiting substrate. 'lbe reasens for using the (substr:at~limited)fed·ba!ch tedmique in.microbial processes is chat01lim· jtatioD and overflow metabolism are avojded, resulting in much higher ceU densities than batch cuLtures. Although a glucose- aud glutaminelimited fed-batch culture also solves me probJem of overflow metabolism in mammalian eells, it is not enough te bring abour a substantial uH.:rease in ceU density. By feeding a balanced mixture of uutrirots. borh me cell density .md the product titre can be improved more tban lo-fuld as compared ro batch cultivation. Fed·batch cultivations can last up ro a month. Processes upto 15 m 3 have been described. Cell densities ofaround 1 rolA x lO'viablecells ml- I have been reportedforfed'batch processcs (see aIso Section 21.7.6).

21.7.4 Chemosrat Continuous. chemosrat cultivatioo is characterised by tbe continuous addidonoffresh medium and witbdrawaJ ofcuJtu re Buid. keeping the culture volume coru tant (Eig. 2 1.5), as described eJsewbere in chis volume (see also Otapter 6).10 steady-state microbial cuIHlTes tbe reja· tionship between the dilution rate (D) and the specific growth rate (~) is expressed as Il.""'D. However, in marnmalian cel] cultures, the viability ofthe culture must be talcen into aecount. Proliferating ceUs not only replace rhe viable ce11s in the effluent srream, but also the cells that die within the cul ture. TItis leads to a steady state desc:ription fur the growth rate: Il.=D(NtXN;I),

in whichN t is tbe total cel1 concentradon (viable plus deadcells) and N" is m e viable cell concentratioo. from this relaoonship it foUows that J.I. is grea{er than D wben cell death occurs in che. system. Generally, in microbial cbemostat cultures a.single nutrient is growth·Limiting and the concentration ofrhe growth·limiting s ubstrate in the feed medium dictates the maximal biomass concentradon in the culture. Furthe r. che spedfic consumption r.nes of other nutrients aTe iDdependent of the concentratioD of the limiting substrate in the feed medium ,

%

466

VRIEZEN, VAN DIJKEN AND HÁGGSTRÓM

Inmammalian cell cultures, whkh contain multiple carbQn andrritcogen sources, itis difficult ro establish steady-state growth limited by a single nuuient. Although one ofthe energy sources. glucose or glutamine. can limit the biomass yield in a steady state culture, the specifk consumption rates of other nutrients may, nevertbeless, depend on the reservoirconcentration ofthe enel·gysonrce. or on the concentration of the individual nutrient. Growth ofmammaliancells in chemostat cultures fed with complex media is therefare likely to result in multiple nlltrient limitatian. Many aspects of mammalian cell physialagy and medium optimisation, such as the intluence of p.. on product fonnation and the effects of dissolved 1 ~oncentration, pH. glucose and gluramine concentration, and amino acid and vitamin concentrations on growth and product formation. have heen investigated using chemostat cultures. Chemostat production processes with up to 2 m3 reactor volume have been described. Chernostat cul tivarion for production purposes has sorne di 5advantages. The long duration of a culture, at least five weeks. creares a marked increase in contamination risk, and tbe time needed ro re-establish a stcady-state culture aftel' contamination has occurrcd is longer than forre-startingfed-batch or batch processes.Moreover. validation of a process based on a continuous cultivation has to inelude proofthat the cellline used is stable over the cuJtivation periodo

°

21.7.5 Perfusion In perfusion cultures. biomass is accumulated as the ceUs are retalled

w:ithin the reactorvia a retention device. while fresh medium is introduced and spent medium removed. In this way, cell densities up to 3 X 10 7 cells ml- l and producttitres an arder of magnirude higher than in batch cultures can be achieved. Devices to separate cells from the culture fluid can be placed inside 01' outside the reactOl' (Fig. 21.5). The latter option.has the disadvantage that a substantial part ofthe culture. is notin the controlled environmentofthe culnlrewssel itself. Severa! perfusion systems can be distinguished , bascd on the method used to separate cells and medium. Spin-filter devices make use of a rotating cage of wire mesh with pores of 5 lo 75 ¡.lill. Spin-filters are prone to fouling. leading to a diminished flow rate t hrough the filter ¡¡nd ultimately to a total clogging of the filter mesh. Alternatively, membrane fIlters (hoUowfibres) can be used for separation oí cells from the culture fluid. Fouling of such filters can occur too but may be remediated by back-f1ushing. Settling devices, utilising the slightly higher density of cells (compared to the mediurn) to separate cells froro the culture fluid, have been developed. A specific device thaI uses grilvity te kl.'-ep cellsin the reactor is the aconstic filter. This sysIem uses static acoustie waves ro concentrare cells in tbe cHLllent stream. CeLLs accumulate in the nodes of the wave and sedimenr back into the culture. against the upo flowing ..ffiuent stream. Finally. centrifugarion as a means ofcell rctention has been applied ro large-scale processes. HoUow'fibre culture systems can be considered a spedal type of perfusion culture in which tbe cells are physically separated Erom the

MAMMAUAN CELL CULTURE

medium flow (Fig. 21.5). Ce.l1s are grown in the extra capilbry space of (he unit. while fresh medium is fed through a targc number ofhollow membrane libres. tbat pass through the unit. Ceu deruities of up to 10 8 ceUs per mi of extra-capiUary space can be acllieved and me effluent medium ftom this space contains a high product concentration.. However, concentration gradients of nutrieots and (by-)products are form ed over the fibres_ TI1ese gradients lirnit the possibiUties ofscalingup hollow tibre units to large production reactors. Nevertheless, hollow tlbre units are easy to useand have been successfuUy applied ro cornmercial production processes (see Section 21.7.6).

21.7.6 Product quality and quantity A product purifled from a mammalian ceU cuJturc may notbe 100%biologically active depending on variations in me glycosylation pattern or

on proteolytic degradatíon . Boro these parameters are i.nfluenced by the environmental conditions. The glycosylation partern changes in response ro many (actors such as the mode of cultivation. the growth pbaseo[a ba.rffi culture, whethercells are grov.rn on microcarri.ersorin suspension , me glucose concentration, the.ammonium concentration. the availability ofhomlones in tbe medium, the presence o[serum, the protein and lipid OODlent ohhe medium. pH and the 02concentration. Thus. chooring the appropriate physiological cooditioos in a production process is important fur obraining the correct glycosylation of a pharmaceutical proteio . Notoolythe qualityofmammalian teH prodllcts but also the ovcrall productivityofmammaliao ceH cultures is iofluenced by manyparameteRsuch as pH, ammonia(ammonium ion and lactate concentrations. serum concentradon. cultivatioo method, culture age, inocuJurn s[ze a nd medium campesitioD . Due lo the complexity of mammalian c~ physiology, in combination with different media and cultivadon methods that are used, itis often difficuJt to single out m e influenceof one specific factor. However, a parameter thar dearlyhas a major effect on the speci1lc productiviry ofmarnmalian ceH products is the growth rateo 80th growth-associated and non-growth associated proouction kinetics occur, Tbe specific productivily may .1150 be enhanced by compounds that are not normal campo nents ofcell culture media. Several mammalian ceHlines show a higher specific productivity io media wbere the asOlOlarity is increased from the normal 330 mOsmol to aboYe 400 mosmo!. Although no! completelyunderstood, lhis effect is, however. dependent 00 the cell line aod basal medium used. Interestingly. addition of butydc acid has been reported to enhance productivityil1 mammalian cells. Thi.s m3y depend on the ability ofbutyric.acid to arrest cells in the Gl phaseofrhe cell cyde. Conseque.ntly, for those products that exbibit non-growth associated production Jdnetics. growth arrest willlead to increased productivil)'. Thc amount ofproduct made by a culture can be expressed as me percentage of the total amollntof protein produced. With noo-growth associated production, a large fall in thls percentage occurs with

467

468

VRIEZEN, VAN DIJKEN AND HÁGGSTRÓM

increasing growth rateo For example, the specific rate afprotcin praductian in a hybridoma cellline was reported as 1.5 mg (10 9 cells)-l·h at a specific growth rate of 0.02 h - 1_The amount of product made corresponds to 28% ofthe total protein. The same celllinehad a muc:h lower specific production rate [0.2 mg (10 9 ceHsj-l.hl at a growth rate of 0.058 h- 1 • i.e. only 1% ofthe total protein production went towards the praduct dmingthese conditians. On the orber hand. in a rnyelama cell line producing a recombinant antibody with growth-associated kinetics, an increase in the percentage of product pratein from 18% to 29% was ob5erved as the growth:rate increased from 0.016h- 1 to 0.042 h- I • The protein production in marnmalian ceH cultures can be as high as in micro-organisms, as i5 cvident from the following comparison. Filamentous fungi are gen~ally regarded as very good producers of excreted proteins (see Chapter 4). Far example, an AspergiUus oryzae strain with a growth rate ofO.09 h-¡ and a.protein cantent of40% produces OA g biomass protein per hollt. The specific productivity of a-arnylase is 0.15 g (g dry biomass)-I·h. The amount of excreted product therefore amounts tú 27% ofthe total protein production. The type ofmammalian cell process thathas been mostsuccessful so far, with respect toprbduct concentration and productivity, is monoclona] anribody production witb hybridoma or myeloma ceUs. Al> shown aboye, the production potential ofrnanmlalian cens is not the lirniting factor but ratheritis the attainable biomass concentration. To meet this demand, fed-barch culture.s andhollow fibre reactors have been used to obtain high eell deruity cultures of hybridoma and rnyeloma cells. Glucose and gl utamine Iimitatíon has been combined wi(h feeding of amino acids and serum. resulting in a total eell concentration of approximately 5 X 107 eells ml-! (ofwhichless thanhalfwasviable)over 550 b. and a:final antibody concentration of2A g 1- 1 , i.e. giving a vol umetric productivity ofO.l g 1-1·day. Commercial production of monodonal antibodies in hollow:fibre reactors can yield about 700 g product per month at abont 2 g 1- 1 • Each ron Jasts foe abont (hree months but tbe first [un 15 nor productive sinee t:h.is time is required for building U]) tbe biornass in the extra capi1lary space. TIIe productivity in this system 15 0.3 g l-l.day during the harvest periodo

21.8

I Genetic engineering of mammalian cells

Geneticmodificationofmammaliancclls can be used to introduce the genetic information needed for production of a specific protein or to improve the characteristics of a production ceU lineoThere are many methods thatcan be used to introduce fore.ign DNA.into a mammalian cell. amongstothers are: electroporation, lipofeetion in whlch the DNA 15 introduced via liposomes. micro-injection of the ONA directly into the ceno fusion of the manunalian cell with a bacterial protoplast containing the DNA or viral vector systems. A transfected ceH tine will express tbeintroduced DNA stablyonly ifitis integrated in the genome. In contrast to micro-organisms. lilce S. cerevisiae and E.coH. the integra-

MAMMAlIAN CELL CULTURE

tion ofthe introduced DNA is ltlostly non·bomologous. The.gene eneod· ing a protein product m ay the.refore be integrated into regions of the genome thar are not favourable for efficient expression of the gene. Selection forthe best producing tr.msrenants is therefore always necessary. SevffaI selectablt ruarkers for marnmalian eel1lines are available. Dominan( markel'S thatean be used irrespective ofthe host ce11 line are mostly concemed with drug resisrance. Recessive markers, tbar are used in combinarion with a specmc hosr ceO genetic background, can involve enzyme5 ofme salvage patbways afthe purioe md pyrimidioe metabolismodrug resistance or amina acid merabolism. The two mos( successful systems are tbe glutamine synthetase (GS) system and tbe dibydrofulate: reductase (dhfr) system. The enzyme glutamine synthetase catalyses the furmation ofglura· mine from glutamare and ammonium iom. The GS gene can be used as a selectable marker in hybridoma and myeloma cells and other cells thatdo not possess GS. Stable transfected cells will express the GS gene and are therefure able ro growin gluramine-free media.As with the dhfr system (see below) the GS system can be used ta amplifY the product gene, a procedure aiso leading to amplification of the es gene. The me tabolicconsequence ofthis situation would be thar tbe ce.ll aculaUy overproduces glutamine. Thedhfr syS(e.m is mostlyused in combinanoo with a clhfr- CHO cell line, A dhfr- cellline is unable to synthesise tetrabydrofotate wbkh is an essential cornctor in the one-carbon metabolismoDhfr- cell Lines are only abie to grow inmedia containing thymidine. glycine and bypoxantbine, precurson and building blocks necessary to ovcrcome chis defi. ciency. Srable, trao.sfected cells that t'..'l:press tbe dhfrgene are capable of growth in unsupplemented medium. MethotTexate (MTX) can be used 10 ampli1Y rhe dhfr gene. Ibis folate anaJogue inhibits the dhfr gene producto By selecting for cells capable of growth in a medium witb increasing concentr.ttions ofMIX, cells with an increased numher of gene copies. and th~eby with enhanced expression of (he dhfr gene product. are obtained. An enhanced expression ofthe produce protein is obtained at tbe same time. A disadvantage ofilie dhfr system is rhar MTX resistance can develop that is ¡ndependent oí dhfr expression. TIte introduction of foreign genes into marnmalian eells 1S quite common, while the deletion of specific genes is notoAs mammalian cells sbow heterologous recombination, the opporrunities for sitespecific insertions and deletions are lacking as is possible in yeasts and E.. coli. Mutations ro prewnr expression of genes can be made by less specific dassical methods, like UV treatment of ceUs , combined with selection for tbe desired phenotype as has been done for the generation ofglycosylation muta.nts. A more recentapproach togene 'knock-out' is che use oí antisense oligo nudeotides that hybridise with a specific mKNA. thereby preventing its transtation Lnto roa,t un: protein. Genetic modification ofmammalian cells fal" cellline improvement is notyetwide-sprea.d but is increasing in importance. Ateas ofintetest are the prolongationofproductive ceH life,growth in serum·free media.,

46'

o

VRlEZEN, VAN DIJKEN ANO HAGGSTRÓM

the decrease ofby-product formarion and glycosylarion characteristics. Apoptosis , that oecurs in most rnammalian c:ell cultures, can be influenced by introducing the bd2 gene. an anti-apoprotic gene. TIlÍs prolongs cell life, and thereby tbe productive phase of a process. An example of decreasing by-produ ct formiltion is the introrluction ofrhe GS gene. Cells with GS produce less ammonia/ammonium as they can be cultivated in media without glutamlne. As a resultof tbis . the production ofMAbsinhybridoma celis i5 increased . CHO ceHUnes with gly-

cosylanon mutations have beeo developed with the aim of generating a less heterogeneous glycosylation ofthe product formed by tbese ceUs .

21.9

I

Fu rther reading

Butler, M. (ed.) (1991). Mn!llnwlfan Cdl BiotrdmoJogy. A Practical ApprtXlrh, Oxford Univeni.ty Press. NewYork. Spier. R. E. (ed.) (2000). Tht F.m:ydopedia ofC.ell TrdInDlo.¡;y. John Wiley. New York.

Chapter 22

Biotransformations Joaquim M. S. Cabral Introduction Biocatalyst selection BiocataIyst irnmobllisaüon aud perfurmallce lmmobilised enzyme reactors BiocataIysis in non-conventional media Conducling remarks

Furtherreading

22.1

I Introduction

Biotransformation deals with fue USe ofbiological cataIysts to convert a ~ubstrate iTIto a product in a limited llumber of enzymatic steps. The establishment of an efficieflt biotransfonnation process requires the extensive examinarían offactors affecting tbe development of oprimal biocata1ysts. reaction media and bioreactors (Fig. 22.1). There are many opportunities for industrial us~of biological cata1ysts for biotransformations. These ¡nelude not ooly the tl'aditional bydrolytk (e.g. starch and protein hydrolysisj and isomerisation (e.g. glucose conversioll to fructose) reactions bur, more recently, synthesis of chiral compounds. reversal of hydrolytic reactions. complex synthetic reactiolls suenas aromatic hydroxylations and enzymatic grOllp protection chemistry and degradation of toxic and environmentally hannful compotmds. Biological cata1ysts when compared with chemical catalysts have the advantages of their n ..gioselectivi ty and srereospecifid ty wh ich lead to single enantiomeric products witb regulatory requisites for phannaceutical, fuod and agricultural use. They are also energy effective catalysts working armoderare temperarures. presslIres and pHvalues. Hiotransfonnations have been perforrned by a variety of biological catalysts, such as isolated enzymes. cells. irnmobilised enzymes and celIs. lhe dcvelopments of recombinant DNA technology have led to improvements in the enzyme production in different host org;ulisffi.S giving the bioprocess en.g:ineer a greater choice ofbiociltalyst option. The optimal biocatalyst must be selective. active and stable under

172

CABRAL

Biotransrormatlon

Substratas

Ovarall volumatric

Product concentration

• Ouality • Purity

productivity

Product

• Scale

operational conditions in the bioreactor, which may nor be necessarily conveotional in terms of composition, concentratioo, pressure arrd temperature. lo particular ir is necessary ro evaluare rhe biocatalyst performance in non-conventional media (e.g. organic solvents arrd supercritical fluids) . A l<ey issue is the availability of suitable biocatalysts. More rarional screening and selectioD techniques are required to: (aJ isolate biocatalysts. e.g. enzymes and cells, able to cata1yse novel reactioos ofindustrial interest. and (b) select aod design catalysts suitable fur industrial use wirh improved operational stabilities and kinetic properties. This requires a much greater understanding of rhe mechanisms of protein deoaturation aod decay of catalytic activities under process conditions and an evaluatioo of methods to maiotain aod improve biocatalysr stability. e.g. chemical modification. immobilisation aod protein engineering. In the optimisation of the overall process it is also importanr to ellhance the predictability and performance oC the biocatalyst in the reaction medi9. in particular io multiphasic media fu! example iovolviog a solid phase. e.g. immobilised biocataIyst. aod one (aqueous) or two (aqueous aod organic) liquid phases. It is very imporrant to obtain reliable data and models 00 physicalfchemicaL transport and interfacial phenomena. Medium engineering plays an importantTole in the definition of the optimal biocatalyst oper.ation and to evaluare rhe effect of medium cOloposition 011 the biocatalyst. The optimal bioreactor should be simple. safe. well conttolled. easy ro design and flexible. The design ofbioreactors requires knowledge of reaction kinetics as well a1i fluid dynamics, substrate dispersion and

BIOTRANSfOflMATIONS

mass transfer.ln addition ror multiphase bioreacrions, interfac:ial pitenomena, substrate and praduct partitioning, and separatioo of two Iiquid pbasesshould also be taken into account.

22.2 I Biocatalyst selection After selecting anapPl'Opriate.startillg material to be conVH'ted into the productoit is necessal')' te select the appropriate biocatalysr with suitablc: activiry, seJeetivity and stability 10 work under the requil'ed operarional conditions (temperature, salt concentranon, pH. organjc solvents. substrate and product concencrations). Several strategies can be followed ro obtain rhe biocatalyst for the pertinenc biotr.msformation: (a) screening for novel bioca{alysts; (b) use ofexisting biocatalyru; and (e) genetic modificatioo of existing biocatalysrs.

22.2.1 Screening for novel biocatalysts Se1ection of new micrO
r-- Peptida bond ~NH ~

amine - CO-NH-

NH2

acvlhydrazlde

finkaga

HP ~

- o - N2'CI- diazonlum salt

l

- CH: -CON3 acylazlde

,1,

Peprlde bond O

- O-CH - eHl

,/

O opoxid!!

-0- CHr-CHz- SOr-CH = CH2 vinylsulphonyl

I!

-e

\ O I

acid anhydride

- e ~

O

, el

~o-t.i-

triatlny l

~R-NCS

isothiocyanate

~R - NCO

isocyanate

-

O

\ C=HN I

imldocarbonate

\ C=O I

cyclic carbonate

el F

-9-

-O

-o

NO ,

NO: m-fluo rodlnltroanalide

-

O

R' NH 11

1 NH

imidoester

~ C-OC2 H ,

-eN

1

e

cyanide

O-acyli90urea

11

NH' 1

R'

-

CH1COCI

acyl chlorlde

Schlffs base ~CHO

aldehyde

. Ex.:ampln of CQV~ll!nt dI",'U.n - - - ] -P"''';n'

Examples el bttice.

!orm¡nlon HI"llIt!gles 10r t!nlr.lflping blOCiltaljlsLli.

Di-8ldehydes Di·amlnes

-

period oftime. biocatalysts have been contined with.in semipennenbJe memhraues in !he form of hoUow libres or fiar sheet ultrafiJtration membranc reac[Ors (Fig. 22.8). lbe membratle retams the biocatalyn but ís permeable ro che products aOO sometirncs to tbe substrates. This method offers sevtT.lJ advantages relarive to other immobilisation methods. Chemkal modificanoll ofthe biocatalyst is not necessa.ry and the biocatalyst rerain its kinetic properties. TItis method is partil"ularJy suired forconversion ofhigh molecular weighl or insoluble rubstrates. slIch as starch . cellulose and protelns. as it a110m the intimare contact of the biocatalyst with (he subsu-ate achieving an cfficient conversion of!he substrates. Howcver sorne disadvantages are inherent in the metbod: the possible decrease in tbe reaction ratc as a res ul t ofthe permea bi lity resistance oftbe mem brane; and the adsorptionoftbe biocatalyst andlor substratcs and products on the me mbrane surfacc. This typt' ofimmobilisation has found applicanons on the modificatioo of fats and oils (e.g. olive oi1. palm oil) by lipa~es and dipeptide synthesis (acetylpbcnylalaninc·leucinamide) by proteases in orga nic media.

BIOTRANSFORMATIONS

(.)

lb,

substrate

Enzyme membra.nc reacton.: (a) contlnuous stirr.d t:onk reactor .....ith recin:ubtlon: (b) dcad-end cel~ and (e) tubular.

substrate

I

,

,

,

1-+ product

cb ¡

,

= (,)

product

e

Immobilisation ofmuld~nzyme systems and cells One of the strong disadvantages of single enzyme systems, free or immobiJisoo. is me;r limitation to single step tr.Insfonnations. This tiro¡(ation is particular!y acutewith thermodynamically unfavourableconversions and those requiring the regeneration of enzyrne co-factors. such as redox reactions oc phosphorylations. Among the possible soll!tioos is coupling (he intended cnzymatic reactioo ro a chemica! one or to a secolld enzymatic reactiOD_The latter alternative has bcen investigated for several NAD(P)- or ATP-dependent sysrems. using a pair of enzyrnes and two substcate5with theco-factorsbuttlingberween them. When two or more biotraruformations are c:arried out simult'aneously in me same vessel - ro regenerare co-factors. [ O shift thermodynamic equilibria. oc to favoul' process ec::onom ics - the intermediate produCLS should be rapidly convel'ted. ln this contexto co-immobilisation oft'he involved enzymes is likely te mínimise tbe diffusion paths of me intermediares between active sites. rhus acceJerating the potential ralelimiting sreps. Howevey. such systems suffer from severe problems related lO the correet relative positioning ofrhe imrnobilised enzymes and co-factor which are extremely difficult to achieve in practice. An example ofthis lypeofimmobilisation method i5 the synthesis ofL-tertiary leucine (Fig. 22.9), a chita} intennediate for chemicals. The immobiHsa.tionofthe multienzymesystemscontained in whole cell5 or cell particles can lcad to marked improvemenrs in a process wben compared to using free ceUs or immobilised single or multiple enzymes. However. a clcar distinction should be madI" between me cases in whic:h cell viability is indispensable and those employingwhole :~I:;~ II F"''"s n,on,-'; "'(e, "ude prepilrations ofsingle-activity bi(T thefirst situation, one deals with sensitive catalytic forms which very mild immobilisation and close control of operating condi""'. in o,rd,,, thatceU viability 15 preserved.1his situarian corresponds.

"ISS

CA8RAL

S¡mlhesls ofl-t.ertiary

l..cuáne dehydrogefl:!sc

leodne from trlmethylpyrIlYa[C¡¡nd

7 '\'

o 11 (CHJ), - C - C -

COOH 1l\'HJ

Trimelhylpyro~¡¡te

NADH¡

NAD

>-
-.l o,

Continuous slirred lank

""",o,

r

J

Packed be(! reactor

Fluidlsed bad raactof

el

-to{"--- - - -- -- . -"1- - ---- -- --"

Membrana reactor

Continuous membrana

*

.....

...

.. ..

r--l---.} '---.

---1>- - -

.

reaClor

Anorher altemative is ro d1311gC Che flow pattero. using a plug f]ow r:ype of reactor: the total recycle reactor ar batch rerurulation reactor, which may be a packed bed or Ouidised bed reactor, or even a coated tubular reactor. This type of reactor may be useful where a single pass gives inadc.quate conversioas. However, ie has found greatest applica· tion in me laboratory for the acquisition of kinetic data, when tbe recyde rate is adjusted so thar tbe con version in me reactor is low and it can be considered as a differential reactor. One advantageofthis cype ofreactor is that the externa! mass transfec.effects can be reduced bythe operational high fluid velocitics.

22.4.3 Continuous reactors The continuous operation of immobilised cnzymes has sorne advan·

rages when compared witb batch processes, such
22.7 I Further reading Blanch, H.W. and Clark. D. S.leds.) (1991).Applied Bi(laltalysis. Val. J, Mareel Dekker Ine .. N~w York. Cabral.J. M. S., Ikrt, D.. Boross, L. and Tramper,j. (eds.) (1994).Applied BJocatalys"is. Harwood Acadel11ic l'ress. Switzerland. JCclly, D. R. (voLed.) (1998). Blot~,ltnology Sf7ic•. (H.·J. Rehm and G. Reed. eds.). Vol. Ba, BlotrtllU"formtltions r, Znd Edldon. Wiley-VOiVerlag Gmb A, We.in)¡eim. lilIy, M. D. (1992). The design and operation ofbioU':lIlsformation processes.ln ~ccntAdwnres in BiolerlmoWgy(F. Vantar-Sukan and S.S. SUkan, eds.). pp. 4 7-(j8. XIuwer Academic. Amsteroam. Straathof, AJJ. and Adlen:reUI"Z. P.(eds.)(2000).AppUed Bioaualy$is, 2nd &litiun. Harwood Al:ademic Press, Swilzerland. Tallaka, A. Tosa, T. and KobaYilshi. T. (005., (1993).lndustriaIApplf(¿¡ tiollS 11 lmmo/7ilized Biocatalyns. Mareel De-kker.lnc.. NewYork. Tramper.J .• Vermüe, M.• Beeftink. H.H. and van Stocksar, U. (eds.111992) . ./)iocatalysis In nan·!I)/IYt!!ltiun.a1 media. Else-vier, Anuterdam. Wells.J. A. and Euell. D. A.l1988). Subtilisill: an enzyme d!!signed to be engi. !leered. TIBS13. 291-297. Wlngard.L. B.• Katchals1d·K.:lttir, "E. attd Galdstein. L (eds.) (1976).lmmabiUud Enzyml.' I'rinriplts. AcadW\1c Press, NewYork..

SO

Chapter 23

Immunochemical applications Mike Clark Gtossary focroductiOD Antibodystructml' and runc¡ions An ubody protein fra gments AnUOodyaffinity Antibody specificity lmmunisatioD and productionofpolydonaJ aumera Monoclonal antibodies

Antibodyengineering Combinatorial and phage displa.y libraries In vfrm lL~es of recombinant and monodena] antibodies In vlW uses ofrecombinant and monodonal antibodil!S

Further reading

I Glossary Adjwunts Sub.'ltances whlch when mixOO witb an antigen will make rhem more immunogenic. i.e. th ey enhance the immunl' response. AdjuVilll1:5 cause inflammation and Irrltltion ilod help to activate cens ofthe immune system. AffinityThe measured blnding mrutant ofan antibody tol' its antige n ar equilibLiulll. AlkHmmunLsudon [mrnun~tion ofan animal with ce11s al' dnues derived from another anlllw ofthe same spmes whel'e there are allellc differences in their gelles. Anlibudy Adaptive prolciru in the plasm. ofan imnlune individual with binding speci.fidty for antigensld immunoglobulin). AnNgen A moLecule orcomplex ormolecules which is recognised by an anribody (immunoglobulin) by billding to tM antibody's variable or V~ons. APC An ilntigen prcsenting ceU is a speci;tlised ce U (dendriticcells and m acrophages) which can ingest, degrade and then present o n ilS ct'1l surlace, fragmenti ofpathogens and otller antlgens, to othercdls ofthe immune sysb.'m (e.g. Ikel1s and T-cells) . .....utoimmune Immunity to molecules (antigens) wllhic 3U ilni.lllal's own body which cnn Jead to a di$e'ilse e.g. rheumatoid art htitis. 01' some forms of diabetes.

CLARK

Avldlly Anribodii'5 :frequent!y inti'l"actwith antigen using muitiple antigen

binding sites alld thu~ they have a functionaJ affinity rermed aviditywhich is a complex funcrion oftbe individual bínding affinities. Ikells A subset ofwhite ceJJs (lymphocyresJ in tbe bJood wltich produce antibodies. CDR (1,2,63) The three od.y. B-
23.2 I Antibody structure and functions Ant1bodies are proreiru made as pan afthe humoral irnmune response ro immunagenic substances and infectious agents, They serve as key adaprer molecules within the irnmune system enabling tIle bast's inherited effector functions to recognise the many unpredictable, diverse and varied antigen structures which might be encountered during an animal's lifetime. 'Ihese efIectof functioos are inh erited mechanisms ofinactivating or killing infecti aus pathogens and then ca using their breakdown and removal from the body, Howevcr, thcse effector systems do not have !he ability to easily recognise the infec· tious agcnts in all oftbeirmanydiverseforms. Th.is recognition. or targeting, of the effector syste:ms 15, in part, dependent upon the antibody's ability to intl'rfuce between antigens on the infectio us agent and also the body's effector sys[ems. Tbe effecwr syste ms aTe inherited within the germ Hne genes of an individual bU( the antibody specificitles are derived by camplex sornatic re-arrangements of the genes encoding immunoglobulins within the so-olled lk:ells (a sui}-popula· tion oftbe white bJood ce.]Js or lymphocytes). This means thateven two ¡dentical twins, or [W() mice from the same labor.:lwry strain, wiII have different irnmunoglobulin sequences expressed at any one time.

IHMUNOCHEMICALAPPUCATIONS

The basic schematic representation of an antibody is tbe familiar Y·shaped structure of an IgG with two idclltical Fab (aotigen binding) arms and a single f-c (crystaWsable) region joined by a more flexible hinge region (see Fig. 23.1). Again. mese temlS come about from the original protein chemistry in which the whole molecule was fragmente(! by cleavage with proteolytic enzymes and different properties were then assigned (O (he different isolated fragments. This basic molecular structu.re (ar subunit) is made up of two identica! heavy (H) chains and [WO identicaJ light (J,) chaios. based upon their molecular size, aod each chalo contains repeated immunoglobulin type globular domains witb a conserved structure. In protein stnlctura l terms the domains have anti-parallel strands which loop back on thernselves to fOlm ,&sheets and these sheets are then rolled up into a barrel-like struccure (see Fig. 23 .2). Ughr cbains have two of these globular domains whereas heavy chains have foul' or more (depending upon their 'dass·. see below). The heavy and lighr chains come inseveral different forms which give rise to the concept of immunoglobulin dasses and subclasses. and. for example in man (and most other mammals). we have heavy chain rypes Jl. 'Y. B and a giving. rcspectively. tbe c1as5es of antibodyIgM, IgG, IgE and 19A. Eachofthese classes can have lightchains of either the K or tbe Á type. The proportion ofimmunoglobulins in the plasma with each lighr chain type varles between spedcs ""ith man having a K:A ratio of approximately 60:40 whereas mouse has a ratio of abollr 90:10. In mano the IgG c1as5 has four sulxlasses called IgG l. 19C2. IgG3 and IgG4 using '}'1 . '}'2. y3and y4 chainswhilstthelgAclasshas rwo sub-classes 19A1 andlgA2 using al and a2 ebains. Someoftbesedasses of immunoglabulin are secreted into plasma in the forOl of more I.:omplex oligomerised StructllreS of subunits ofien assodated w:ith a molecule calledJ-chain. Thus IgM is a peotameroffive ide ntical protein subunits. and 19A is frequentlyfound asdimers and [rimen ofidentical protein subunits. again associated with } chain (see Fig. 23.3). It is the heavy cbain which is largely respoosible for the 'effector Cunetioos' (antigen destruction and removaJ) triggered lhrough ínteractions with cells of the immune system by figanon (binding ro and cross-linking) ofceJ1surface receptors {called Fcreceptors beca use they require tbe Fe fragment oftbe antl.1>ody)or. alternatively, through activatioo of the complement cascade and the binding to complement recepcors_ComplemeJlt is another family ofproteins found in the blood and whjch ate involved in immllll..e reactions. The components of complemenc are mainIy specific proteolytie enzy¡ues whose Sllbstrates are lhemselves otba complement components which are activated by proteolysis. Tbis gives rise to a classical biochemical amplification of an inidal small activation step. Once activated sorne ofthese compleroenl components also rapidly fonu covalent chemica! bonds witb antigen, thus marking tbem rOl' c1earance by complement receptors of the immune system. whilst ochers are able. ro creare pores in ceU or viral memb[anes ofinfectiou s organisms and thus kili the (elis orviruses. Each of!he different immunoglobulin (antibody) classes and also sub-elasscs exhibit a different pattern of effector functions sorne af

F, The bask; IgG invnunoglobtln smKture of 'MO heavy chalM (bla
23.4

FoFragment

FUllctional rublragrnenu 01 IgG moleru!e. The~e can b
re5ulting hybridoma cillls al"\.' 5elected fOl" growth in media which is toX!c to the parental myeloma

Cell Fusion

B-cells

Ii)(j~~

~ ~Ii) ~ (j Ii)

Drug Seleclion Cell Cloning

signals. Thus an antigen can be cambined with other substances, such as mineral oils and components derived from micro-organisms. which can act as adjuvants and activate the immune system and improve anrigen processing and 'presentation bythe APCs. Fol1owingproduction, the antisera can be used in ve!.}' many systems as a specific tool for detection of antigen. The immunoglobulin fraction ofthe antisera can be purified and then used in severa! assay and detection systems. ror example, it can be labeUed by covalent conjugation with fiuorescent dyes and used in milToscopy or flow cytometry for detecting antigen bindiog 00 or in ceUs. Equally, the antibody could be labelled with.an em:yme and used in histologyor in an enzyme-linked, immunosorbentassay(EIlSA) (see Section 23.10.2) again fur detection of the speci.fic.antigen.

23.7

I

PI'odl.lction of rnonodonal antibodies. This Involves fusio~ of sp1ilen cen~ fmm irnmun15ed rodilnt5, Wlth mYilloma cells adilpted lO cell cultul"\.'. The

Monoclonal antibodies

Conventianally, ce1llines secreting monodanal antibodies have been derived bytaking irnmune B-
23.8 I Antibody engineering Chlmaeric

Humanlsed

ChilT)3fl"lc antibodies. Through the_use ollllcombll'l:u1t DNA te.chnQlagy le Is poulble to eogioeer alltibodles with no... eI

properties. Dne "mple n.p Is to make chlrnaeric antibod\eJ. n wtllch me v,l1iable reglOfU fTc,m" rodem:

aoobody (whlu) ar. combln~ wim me c.onstan:: regiolu of a humananribody (bladt). A 1~

further In &:he technology is tO combine ¡Ust me. compl~tarlty o.te.rminlng reglan (CDR)

.ncodlng DNA lequences of lo rod.nt antibody with fnmework IIIglon (FR) encodlng DNA sequences oh human antibody. 10 ¡ive a fully numilllised l.ntibody.

lt isnow possible (O genetically engineer and express a whole r.mge of diffeling novel antibody consaucts thus :freeing biotechnologists from tbe constraints imposed by tlle naturnl biology ofthe immune system. It is the modular structure of antibody molecuJe:s which are composed ofa collection of discrete globular dornaios, eneoded by genes with a similar modular structure whereby eac.'h domain is coded in a separate exon. which makes the manipulation ofimmunoglobulin genes a [elativcly straightforward proposition (see Fig. 23.5). There are several obvious advantages of recombinant antibodies over eonventionally derived monodonal antibodies. It is technically feasible, through the use. of appropriale doning strategies, to isolatc the genes e ncoding any antibody made from any i.mmunised species a nd so furure applications need not be restricted to tbe derivation oftbe mouse. rat and human antibody classes . Often monodonal antibodies can be derived with the cotrect spedficity but they may exhibit the wrong effector functions beca use tbey are Dot of the desired SpedCli, class or su1xlass of immunoglobulin. Obviously, using recombinant DNA technology any V-regions can be expressed in combination with anyconstant regioos selected for desirable properties. TIlese antibodies are called chi.m.aeric antibodies (see Fig. 23.10), Thus variable regions from rodent antibodies speciBc for chosen antigens can be eombined with OODStant regions encoding human iromunoglobulin classes/sub-cl:.sses. the final product having potential in vivo tberapeutic uses in mano lt is abo possible lO introduee

IMMUNOCHEMICALAPPUCATIONS

furtber mutations into the Pc regíons to rnodify the properties lO suir the proposed applications of the antibody, for example to remove the ability to bind to sorne Fc receptors or to activare complement, Where in vivo therapeutic applications a re concerned, rodent a udbocHes ofien are limic.ed beca use they provoke an irnmune response in che patientto tbe antibody usuallywithin a weekof theirfirst use. This .pl'edudes any further rreaDllent beyond this time. As described aboYe. useful rodent monodona! antibodies can be partiaJly ' humanised ' by making chimenc antibodi es by combining the rodent variable-regioos with human consrant-regioos, tbus introdud ng the effecror mechanisms ofth e human whilst at the sarne time minimising me number of potential immunogenicepitopes. For irnmunotherapy, there are several leey fcatures Chal 3n antibody should h ave ifit is to be successfuUy usro. Obviously, the antibody mustpossess a de5ired spec:ificity to bind lO a relevant anrigen, 5uth
IMMUNOCHEMICALAPPUCATIONS

the conversioll ofa substrate t.o a product by che em.yme. This is stoichtometric but also ineludes an amplification of the slgnal because one molcculc of enzyme can convert many molecules of substrate over a given time. lE coloU1'cd substrates are used or coloured products are formed a simple. photomctric adsorbancy measuremeutwill qualltify the enZ)'llle reaction.It i.s also possible ro make t.ItU measuremcnt as a real time determination of the rateofthe reaction. There are many subrle variations on m e basic EiLlSA systcm. In its simplest foon, ao alltigen is adsorbed o n to a solid surface. eicher nonspecifically, OL through an affinity ligand orcovalent chemical bond, An enzyme-labelled antibody is then added in exeess to the system and sorne ofitbinds to the immobilised antigen, Excess antibody is removed by washing and then substrate is added. The amount of enzyme, and hence amouDtofantibody-antigen complex in the system. is estimated from the amount ofsubstt-ate it converts. More usually, EUSA involves a two siterecognition with two differentantibodi~ oran indirect deu~c tion (see Fig. 23.14). Por ~ample. one antibody may be immobilised on a solid matrix and used to capture ('affinjty adsorb') the antigen. The a mOllar of antigen captured. ean be determined by a Second antibody coupled toenzymewhich rffognises a differentsiteon the antigen and so does ooteompete witb rhe firse ;muoody. Indirectdetfftion sy!items can employ multiple layers of.:mti-antibodies orofbiotin·avidin layers giving even gre:01ler amplification in the systelll_ A1ternatively, tbe systems can be designed to determine the quantity ofunknown antigen in the system thcough competition with binding a known amoullt oEa labclled and pure form ofthe same :\Otigen (a method ociginaLly widely employed in radioimmunoassays). The maximum binding oflabelled antigen is seen when mere is no competitor antigen in the (est sample and the minimum binding of labelled antigen is seen when thece is a huge e.xcess ofcompetitor antigen in the test sample. Enzyme-I:lbelled antibodies are O1lso employed in immunocytochemistry. Tissue sections oc eell cytosmears are prepared on glass microscope slides. These are then incubated with antibodics specific for different fume antigens and coupled with enzymes. Meer washing away excess anribody, the enzyme substrates are added. Substrates are masen such that insohtble. ooloured prorlucts are deposited in the seetian and thesc can be visualised in light microscopy and, along with suitable counter staining. may allow fur vely detailed c1assification of the eells stained, Using appropnate counter stains and, through c.:olocalisation oftestantibodies with known markers. ir is possible to identifY which pans ofthecel1, e.g, surface. cytoplasmic or nudearstainin g. contain the antigen recognised by the antibody. Again, as described above for the EUSA system, the techniques GlD be modified ro use multipIe layers ofantibody and anti-antibodyin orderto amplify the staining, As well as c.nzyme-lahelled anrlbodies, it i.s possible to use antibodies eoupled ro fluorescent dycs (fluorophores) and to use them in fluores· cent microscopy. Fluorescently conjugated antibodies can be used in the powerful technique of confocaJ mícroscopy which allows the precise localisation ofthe Ouorescence on. orin. tbeceJl lo bevisualised

525

Stop'

J

JJ

J

J

-t,9$sZ two-site

In lbe lint wep antlbo~ ~lch islrnmob1ised OfllO 1 surfact 15 lISed U) C3ptUre th ~ ¡ntlgen from ~ olution , The excen ami therefCll't unbound amigen Is then w.uh. d away, In a second sttp an entyf!'le'bbelle dlante ,..wl!ing 'roro fonmcion of ¡ coloul'..t product is; monltored iI ¡,pecvophotomet~,

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in a timNlepcndentway.ln canfocal m icroscapy im.:¡ges coUectcd in a precise focal plome are digitised and tbcn starro in a camputer. These digitised images, whieh thus represent 'slkes' thraugh the cen. can th~n be buile up into a three-dilllensianaJ representadon ofthe inteo· sit}'oftluorescence chroughout theceU and a modeJ.can be djsplayed on a high resolution graph ics moniror.lfimages are collected atintClVa ls over a gíwn time, then thecompule rcan abo be used lO generare a tim ~ lapse movie afthe moveDlent offluorescencewithin lhe ceU. Pluorcsceot antlbody ceJI sor ting and analysis have d~oped in parallel with deve.lopment of tbe monoclonnl 3.n tibody technology. Again, the principies ofthe tedmiqueare simple. Monoc.lonal antibodies are labeJled w:itb a fluorochrome and used 10 stain cell!. lbcse cclls are then passed at high velocity thJ:Ough a nozz.le in a stream of tiquid droplels such thar me cells pass oneal a rime tbrough a laser Hght beam wruch excites the tluorophore. Detecton theo measure the fluorcscencc curput from eaeh ceU on an indjvidual oosis. At the same time, other properties ofrhe cells can be measurro through their abitities to scatrer the Iight bcam !size and g rnnularity ofthe ceUs). Abo, different fluor~ phores wirh differentemission spcctra can be used to tag different anriboches. Thus, a very sophisticated analysis of cells evcn in a complex mixture can be carried out: for example human bJood cells {AlQ ~ se~ aratcd ioto tbeir vanous types and sub-types. The wholedassification of human (ano now other a nimal) cell rurface antigens using the ·CO" which stands ror cluster designa don, nomenclalure has relied very heavily on the use of ftuores cellt celJ malysis. !he resulrs froro the ana.lysis, carried out in.many laboratories. on panels of monclonal anobad ies are used tu duS[er these antibodies in [O grou ps with similar reactivities. and provided thisdusteringseerru [O be statistically robust, an internacional cornmittee author1ses thedesignation ofa c1usterwith a new sequential numbe¡' in the CD series !e.g, COl , CD2, CD3 etc.), Although many peopLe no..... com.monJy rerer to the antigcns by the en n:une, the original designadon was ofgroups ofantibodies. Thus 'antiCOI antibodics', for examplc. is Ilonsensical in !he purest sense since t be cluster ofantibodies iseD', and the antigen ¡s thatwhich is recognised by the COl c:Iuster ofantibodies.

23.11

In vivo uses of recombinant and monoclonal

anti bodies Again, the uses of antibodies iti vÍ\'O rely heavi1y on thei}" great spedficity for antigen. lt is sometimes easy to forgerwhen dealing wich uses of monoc.Jonal antibodies in víva thatourown antibodies playa major role in our natural immunesystem in protecting us fromlnfectionby killing pathogens and by removing barmful antigens from our system. Hov.'Cve.r, despite this obvious role fur antibodies, there are. in fact, only a few therapies currendy in use whicb exploit mOlloclonal antibodies ror these properries. This is largelydue to fue commerdal. practical ano also eth ical considerations ¡nvolved in developing antibody therapies.

IMMUNOCHEHICAl APPUCAnONS

Itis an enormous ly expemive a,ud also a time-consuming undertaldng' ro get even a single antibody through clinical trials and regulatory approval for wide..pread commercial sale and use. The situation is ñn:ther coruplicated ifaccepted, cxistingtreatments are a lready in use. Thus, polyclonal human IgG is manufacture
Chapter 24

Environmental applications Philippe Vandevivere and Wi lly Verstraet e Inrroduction Treatment oí waste water Digestioil ofOJganlc slurries Trearmentofsolid W3$WS Treatment ofwaste gases Soil remediation Treatmentofgroundwale r Furtber reading

24. 1 I Int roduction Until recenUy. sanitacy engineering mOl1opolised environmental related industrial actiyities. Because sanitary engineermg gradually developed aS an offshoot of civil engineenng during the past century. emphasis has been on c:onventional engineering remnlques in which the 'bio' component ¡s Jargely ignored and dealt with.scochastically rath~ than mechanistically. S.mitary engineering ¡s weU established foro • the catchment. treatmentand distrlbution ofdrinllig water: • the treatment ofwaste water; • the treaonentand disposaJ ofsolid wastes (e.g. municipal); • the treatIDentofindustrial off-gases, Many of the conventional technologies used in sanítary engineering are, however. perfect illustrations ofMurphy's Jaw in roar tbey transform one problem ioto another ofien more intrattable one, as when water poUutanrs are nripped into the air or concentrated and dumped in tbe soil. EOv1ronmental strategies have to be conceived with respect to the <whoJe' oftbe euyironrnent in a long-rerm perspective. This integrated holistic approacb requires a detailed knowledge of environmental biology and, more particularly, of roe functioning of complex mkrobial communities .1he new focus on the environment as a whole and on [he deta.iled fu n ctioningoftbe 'bio' componenthas le
VANDEVJVERE AND VERSTRAETE

• acid rain and ozone depterion; • enrichment ofground and surface waters with nutrients and recal· citrant pesticides; • recove.ry ofreusable producrs and energy from wastes; • soil remediation ; • disposaJ ofanimal maDures. WhiJe industrial biotechnologisrs use we]l-defined rnkl'o-organisms to make products of pl'edicrable composiLioll and quality such as tactic aeid. beero!' monosodium gLutamate. enviIOnmental biotechnologists. on me other hand. stan with poody define
ENVIRONMENTAL APPUCATIONS

¡¡floxJe tank

aeratad tank

settJing tank

wa&e_--lRr1~j[~t-"i----~j:~----tl-tr==t--,J water

__• effluent

al~,:::t::::~C--J nitrare lecycling

waste sludge

sludge recycling

now

dia¡ram oIan ~ujo.... ted slud~ planr .,..im bloIogi(;l.l ~itrogen Proceu relf'l(W¡j, Since thf rol'$( tllIk lunoxle (oxyrn-fr..), mk;m.orpnlsms use nlmaw Ul 0)(1di5e die orpnlc INtur te urbon dlo)(ide and ;ammonlum, Iflveby redur.in¡ nitraUl ro dinitrogengu (denitriflaltlon). ln me subsequentHDld tank. rasldual organk mauer ¡! o)(idi$ed with oxygen a.5 fle«ron aoceptDr. sm.,.lQ,neous!y, ammonlum;1 oxidise.d Ul nlrr.ate (nltriflGltion) whlch 1, thsl recyded 10 theMlOXlc tank. 1M miuo-organisms a .... sepaDIllcl (rom Ihe clean effluenl in lhe sett9f"\11: ank.

re-injected in the aerated tank and partly w3Sred. Good performance depends on the rightchoice ofvolumetric loadingrate, which sbould He in the range 0.5-1.5 g BOO, per litre mixed ljquor per day in order to ensure proper floc formacion and obtain 90+% remova) of dissolved organic matter. As one inhabitanr equivaJent produces 30 g BOO, per day 00 the average. witb peak vaJues ofloo g per day, aer.:Jted tanks are designed to haVl! 100 litres mixed llquorforeach.inbabitantequivaJent.

533

Aerobic treatment

Residual BOD Residual N, P Sludge production

Energy Aoorarea Reliability

Anaerobi(

Activated sludge

MBR

UASB

low low

very Iow

high high

high high large

very low high verysmall

sludge bullcing

rnoosl

low

verylow low

very small granule flotation

No
ENVIRONMENTALAPPUCATIONS

flr;>c of mlO"Q_ org¡nlsms as they O-CCur In me mbced Hquor of actlvated lludgtl tanks . Note the prlilsetlce of l worm (Na& elingui5). which gr¡u;1!$ the f1ocf.Ctlvely. ThMe wonns could offer a vel")' simple and clepnl solutlon te me problam of slo.ldge disposaJ (cOlIneS)' of Prof.

Eikeli)oom).

frorn the treated effluent. In such asystem. separared sludge can be recirculated almost indefinitely in the aerated tank and, under tbese circumstances, sludge age i5 very long and exCes5 sludge production ve!}' low «0.1 kg per kg Bon removed). A second majar advantage of MBRis thata veryhigh Sllldge concentration i5 attained (up to 30 g 1- 1) which allows murn larger volumetric loading rates ro be used man in tbe activated Sllldge system.As a consequence, very compact MBR insta]· lations can be bui)t on a sma11 fraetion oftbe spa.ce required by an aeti· vated 5ludge planto This small footprint is very attractive to industries producing concentrated waste waters (BODs >2- 3 g l-I).

24.2.2 Anaerobic treatment of waste water Un til recendy. anaerobicdigestion was only appl iedfor the stabilisatiOD of concentcated organic slllrries such as animal manures and waste sewage sludge. The consensus was that anaerobic W
I'll"'ct!on A: proplooato + 2

H ~O

___ 3 H2 + ac:elale

+ C(h

reaction B: 4 H2 + CO 2

___ CH. + 2 H20

r,

A

i

8

/

pressure H2

Interval whera l>olh rsoctiOlls yiHkl > 21 I 1 1 kJ mol- ' tr.mslormed. preuure

00

lr¡nU8nl

Sdlematic diagram 01 me upnow anaerobie sJudxe bI~nkl,ll re.ct.Or ext_ i~ely for

(UASB) Ulld

the trutn'Hlnt of tontentnted ....ute watlml in lempente regions and also Ior the IreU!TM!nt 01 Ul ..... (dilUUl ....aste wUflr) In trOpical ..-t¡lOIIl.

S37

obtained. Becauseadapration ofthe association is probably a1so based on the proliferatioo of tbe right plasmids. tbere is clearly a nee;d for bctter insight in genetic evolution. plasmid transfer and species interaction in anaerohic communities dealing with xellobiotics, Another potential benefit associated with the largc-scale availabiüty of~pecial ised microbialconsortia i5 'biochemical re-routing'. Le. the inc\uction of desicable biorne.mical pathways. as fur example the dcgl'adadon ofmal· odol"Ous primary amines. anaerobic arnmonium oxidation or horno· acetogenesis. Deve10pment oC perfonnance-enhancing additives Biomass rNention through adequate granulation is of utmost importance in UASB [echnology. first in moer [O obtain a good eftIuenr quality andsecond. in order to ensure a mínimal ceH residence time of7 to 12 days wh.ich is required to avaid the wash-out of [he slov.¡est-growing anaerobic bacteria . Dile way to foster granular growtb is lO add palymees. cLay oc surfactants whkh llave aphysico-tabUise the populations of other very valuable wicro-organisms. 5Uth as bactivorous protozoa which are esscntial to obtain good qualityeffluents, orto show rile development oE detrimental micro-organisms, such as the filamcntaus bacteria which cause sludge bulking,

elean procoss water Thls

process flcw

diagram iUl,.I$lt1.tU tht state-of-me-

art technology cmplcytd In the textile industry lO c.onven la rge vol~m es 01 wastt W,ter Inte higl!-

qlJ;lWty proce.» wnerus,d for w,15l!ill&- ¡cuuri.", blnching. dyelng and prillt~. Biologkal tre.1tmenlS are cOfl,bined with physlcCKhemital treaunet1lf In orde r te achleve tlle requlnu:! purlty. Th e final biof¡ltnticm step 011

actiV;!.ted arbon. tombinlng

, physical sorptlon with in S1W

biodegracbtion.ls necen;¡,ry to remove texic compoonds produc.ed durlng the ozoni$ation uep.

539

Slope measures aClivity

e

x mg acetate

Time jmin)

me.

Respirogr.lm obalned wlth a biosensor useO to mearure on·n~e BOO and potentilll to,oclty ofWUle water befort II tllu,r$ lo treatrnent pJ.nl. TIle addltlor. of ac6elte to an aenteCe o ra toxic oompound in the wau.t waler sampJe added in B. Passlble remedial actioos are (1) me additicn cf tcXlcanl-ntutralbing additlves In lhe nuln flcwlo me pblnt, e,g, powder activate
¡''!i .;. ·'··¡

I, ..

UAS8 reactor

Completely

SOidst=

mixed reactor

""'dO>'

Effiuerrt treated

Wastewater

Organic slurry

Solid WdStes

Solid concentration in reactor(g 1- 1) Loading me (kg organics m - J'day)

Enteric v iruslls

V/brfo chO/9ra9

ki'~ng d~ys'

all P~thOl_ alter ¡ lew relention time. 1 day

Asaaris. Salmon9//8

1 week 1 momh

This extrd. nument load may cause problems in vie.w of tbe new. more stringent. standards concenling tbe nutDellt cantent of discharged efflucnts. Th1S may aJsa be. rhe case for P a.s sorne lnvt!stigators have Found tbat up [O 60% of the sludge-bound P may be release
543

-4

VANDEVlVERE ANO VERSTRAm

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.. - ~-~_:'"~:!-~~l~~~~f~~~ ···;;:~m~ ~.

':

carboo dloxldc). Blogu 11 conven:ed rnw ctcctrlcal power whlch ls sakl10 [he nctWOrk. Th e plcwre Ill.Ktr;¡tcs a p/lInt in Salzburg. Al.KtrIa, trenlng 20000 wnncs blcwastc annuaRy "";th ~ single-phue them'ophlW c so lid SQce fennen!;lltion process. T he con~yor bek In che roreground transpon! the shreder. Bioscrubben lre mo~ I~ble lO proc:en opt!n,isulon ilIld requlrt ~u floor arel than blofllters. They are h~r more cosdy an d are len effidem al rerr.ovlnl poorty ,oIl1ble VOC~ e.l. h)odroarbom. bios~ru bbers

54i

VANDEVIVERE ANO VERSTRAETE

Scr ul)ber

EHltloIint ¡II.

H>SO•• NO

".

gaseous stream to a liquid stre.un by spraying a liquid in a chamber lhough which the gas is passed.lu a bio-scrubber. the sprayed liquid i.s a suspension of micro-organisms which cyclcs back and fortb between thespray chamber and a waste water treatment unit where biodegrada· tion takes place. !b(> process parameters 5uch as adequate nutrient supply aod pH are muen more easi ly controlled (in tbe circulating Uquid) than in a biofilter, leading to fast reaction rates, While biofilters require alarge footprin t since their heignt preferably should not exceed 1 m in order to avoid clogging, bioscrubbers require muen less space because the tank where biodegradation takes place can be severa] metres high. Bioscruhbers appear best suited for large air f10ws because oftheir low bade pressure and small size. They however can be employed onIy for che removal ofgases which aresufficiendysoluble because the mass transfer rate in a spray chamber is less man thatattainable ina biofilter unit. lo case the obtained contaminant concentration in the outler gas is too bign, a second bioscnlbbcr inoculate
24.5.2 Biological removal of sulphur and n¡rrogen compounds from flue gases .. SotlO;! elemenml S

A newly develope d bloprocess Ior the t.imultancoul desulpt.....ri~don iUl
from flue gases

neps al"fl solubilisatÍOll In a scrubber, N ren'l0V31 ina tlloreactor. w lphite reducrlon to

sulphide in a UASB reilctor. sulptllde partial ox1datlon 10 eI~en~1

S'ln a submerged oltic

atLlched biofllm reacto r and Il'lcavery of solid sulphur, The l;quld phne ts contll'llJOYSly ~",,",.

Ni trogen oxides (NO.el and sulphur d ioxide fS02) are majar aiT poll utants tbrmed during tbecombustion ofroal and oil and released in flue gases. 1'here is considerable mterest in tbe development of an ef:ficienl and low-cost biotechnologyfor the simultaneous reruqval ofthese ah pollutanu. since convcntional physico-chemical technologies areeitbervery expensive or inefficie nt. Anew system is currently being propase
ENVlRONMENTALAf'PUCATIONS

reduced biologically to ~S and finally partially re-oxidized to solid ejemental sulpbur: ~SO,+3~ --+ ~S+

H2S+3 ~O

YI O~ --+ So + HzO

The rrouction ofH~SOJ takes phlce in a UASB reactor (Fig. 24.5) seeded with sulphale-reducing bacteria. Rocculant polymers are added, logether with fue necessary nuttients a nd reducing equivalenu (ethanol or 1\) to adj ust tbe (BOD ¡ HZSOJ) molar ratio at a value of one. lo the third bioreactor, ¡¡erobie bacteria oxidisesulphide back tosolid SO (end-product). TIte further oxldation of S ~ lo A!S03 and H]S04 is prevente
504

VA NDEVIVERE AND VERSTRAETE

The kinetics of biodlgndulon 01 pollu&lf\tS 111 wil S)'stems are lGually firn: order. wt1lch mfltnS th31 ¡he rate of

e

wbstnU! dJ~e Is proportion:.d to the substntll! COl1CfllltnTion (curre AJ. Wich fint order Idnetics, the h¡lf·flfe defines the time duringwhich half of ttHI! subscratt is de&r.K!ed. Flrst order kineóo:., o«ur when me substrau t:onC:Cflll" 97%) can be obtaincd for soluble paraffins «C16) and poly aromatic hydrocarbons (PAHs) after several years opeJ-ation. Bioventing is however limited to homogencou5 sub-surface formations since heterogencities would ause the. alr to move through the mos! penneable. areas causing trentmenI to occur ouly in Umited areas. Another success stot)' of In situ soil bioremediation is phytoremed.iation.. Here specific p13nts are cultivated which accumulale heavy metals in the above-ground plant msue m: sfunuJate organic breakdown in their rhizosph ere(tbe zonc immediatelyadjacent lO the I'oots). While phytoremediation is clegant. -dean' and cheap, ils main drawbacks are that oo1y (be surface layerofsoil (O-SO cm) can be. tre.ned and rbat the treatment ralces several years and leaves substantial residual levels ofrontaminants in lhe sollo Pbytoremediationis however undergoing fuU development at present. Rcmoval of oil slicks by so-called landfarnling is 3n established method based on microbial degradation (Fig. 24:14)_ Given half·livcs of the arder ofone year, it would takc abour 7years oftreannent to remove 6.4 g hydrocarbon per kg soU down to the dean-up goal of 50 mg kg' l . This low-tecll technology can be.somewhatupgraded bymixing thesoj] witb freID organic residues (compost). Elev3ted tempE'Tatures and increased microbial diversity and activity iDcrease reamoD rates. Moreover speci.fic co-substrates favour co-mctabolism. Landfarming systems can be upgraded by including anaerobic prelreaunent_ For example, anaerobic tunnels are used [O reduce rompounds such iIS trinitrotolue.ne by adding numents and co-substrates forthe indigenous bacteria. In a second aerobic srage, che reduced metabolites are eithe.r completely mineralised orpolymerised and irreve.rsibly im.mobilised in lhe soil matrix. lbis approach has also been used sllccelisfullyto decon· taminate soils polluted with chloroethene and BTX aromatics (mix· tures ofbenzene. toluene and x.ylene). Sluny-phase bioreactors may achievc the sarue dean-up levels in

IIn., CroU41!(:DoRal view

of:l solid-pkue soIl'reKU)r', or landfarming system. The sol! is eX('aYOItoo. mixed with nuuients

aOO mic.ro-orpnbms.. and evenfy sprud Out on a Uner: Wtth regular plOI.Ilhil"lg tO tmJt¡r milling UId .e
treat' r"em
uses recovery amI I"IKhar¡e weJls

tnat 'wash' the &ro..aldwater tO tIle

activated carbon

ground su/"Úce whel'! k i l treate!!

strip colurnn

filter

by ~ combmtlon of variOU\

physico-chemial ~d biological

-'+

teehnlquei.. Trealed water is reInlet:ted several time:¡ tO imFrov~ polutant recovery. Bio.1enclni. on che other hand.ls on!y a contain ~nt techniql,lll. lt ~C)fls lsu

.Ir _ _ _

InJectlon well

recovery wall

water tabla

of settlng up ¡

bloa~tive

ZOf\e, oontamin:mts are blodegraded.

groundwater

zone I t

me down-gn.dient edge 01 a conClminate d groondw.llter area via ntJU1 ent ~i«lion. As impa.«ed groundw,uer ente("l the bioaa:r..e

... Z O

~

1>-

:o

...

"

~

Bio~fencing

0

lO

nutrlents

~

O

V> a w

surface

~

InJection well

z

::>

waler la~

-

direction offlow

olean groundwater

I polluted groundwater I ¡sed. leaving a large residual fraetion in the seU. As a result of this failure. remediarlon policy and technical developments are shifting towards íncreased lIse oflTlsltl/ containment practices. e.g. biofencing (Fig. 24.15), ratber than filU treatment scenarios. In cases where full treatment is necessary. less stringent cleanup goals are seto based on risk assessment taking ioto account typeofland use. Aside frem the much-studied genericcompounds discussed a bove in mis cbapter. there is a hoS{ oftoxic compOunds usually present ar U"3ce level and whose fate remains poorly studied. One example a re !he polych10rinated dioxins and finans which are fOroled as by-products of chemical syntbesis processes. ThE1 are alsoproduced by combustion of

"-

p.

VANDEVIVERE AND VEPSTRAETE

garbage. waste oils, soils pollmed wüb oils, chemical wastes containing PCBs, and by vmous oUler high temperatl1re prcx:esses. Because of the high toxicity ofsome diorins andfurans, thesecompounds are ofmajor eco-toxicological concern. Ongo¡ng research and development has attempted to minimise theirfonnationin inr.:tner.nors and emission via tiy ashes. Yet, the biological breakdown ofUlese compounds in theenvi· ronment is of considerable importance. tndeed. they are often present in wastcs which are extreme1y difficult to t:reat properly by incineration (e.g. pollured soils and riversediments). Theyarealso ptesent in Oyashes ofincinerators which are depositcd in landfills and, notwithstanding aB precautions, can contaminare landfillleachates.

24.7.2 Natural attenuation and monitoring Several factors have recently generaroo a lot ofinterest in rrew monüor· ing techniques. Ofie such factor is the ract that remediatían tecbnolo-gies are ofien insuffident te meet stringent c1eanup targets. This limitation is making legislators reassess che target pollutant levels and making them consider the use ofrisk-based end-points in place of abso· luteend·pointvalues. 111e newconcept ofrisk·based end·points requires the development ofnew a.n..a.Iytical tools which assess the bioavailable rath~ th3.l1 the total poUulant concentration. These new rools typical1y relyon bioassays beca use the traditional analyticalmethods c;:muot dis· tinguish polJutants that are :'lva.i lable to biological systems from those thar ex:ist in ¡nerl. or complexed, unavailable ronns. Subjecting a poi· luted soil to a perlod ofinrensive microbial activity can reduce the toxicityby a factor of5 to 10. ThisecotOJCicological informatian can be easily deduced by runn.ing a simple bioassay with soil leachatcs. One type of bioassay is based on me inhibitian ofthe natural bioluminescence of tbe marine organism Phorobacreriurn pllOS"pl!orezmz, which is med , for example. in the Microtox, Lurnistox and Biotox tests . Thcse assays are, howevec, not specifk since light inhibition will occur upon exposure to any toxicant. This li.m.itation is circumvented in a new class ofbacterial biosensors which are spec:i..fI.c ro certain types oftaxicants. Forexample. biosensors able to detect bioava.ilable mecals, were conSITl1cted by pladng lu¡¡- genes ofVibrlo fischeri as reporter genes under rhe control of genes ¡nvolved in the regulation ofheavy metal resistance in the bacte-rium Alall1gC'"l'S eurrophus. Theo recombinant strains. upon mixing with metal·poIJuted soi ls orwater. emit Iight in proportion [ O the COUceD.IT"d· tion ofspecific bioavailable nletals. Light emission is easily measure
Academic Publil¡:hen, DordrechL Haug. R. T. (1993). The I'nlctfcal Hundbook uJCompost Bngülurlng. Lewis Publishe.rs.

Boca Raton, Florida . HUTSt. C_ Knudslm , G, R" Mdncercy. M.J., Stetzenbach.. L O.and Wa ller. M. V. (1997). Manual ofE1\vtrolmil'l1taf Microbiolog. Amerkan Sodety for Microbiology. Washington. oc. Grady. L C.P_ Daigger. G. T. and Um, H. C.(1999).Biological w.1St~WuterTreat·

ment, 2nd OOUtan.Mareel Dekker Inc., New York.

Sayler, G. S.. Sarueve.rino, J. and Davis, K. L (1997). Blor;:dmoJogy i1\ tn .. SU5lacnobl~ Envlrunmetll Ple:num Press. NewYork.. Versuaete. de Beer. D .. Pena. M.. Leltinga., G. a nd l..eus. P. (1996). Anaembic

w..

bioproces.sing of organic wa~tes. WnidJ. Microbio/. Biotedmol. 12 , Z21- 23B.

557

Index

Acetobacrerxylinllm. 322. 426

A,etone,34 A,mnon;um chrysogenum. 121 Acrylamide,-1S7 Adenosine tríphosphate (AIP). 20 Aerobic m icro-organisms. 29-32 ellergy production, 29 Aflinity d lJ"Omatography using

antHrodies.524 A!roligencr lall/5 PHA, 328

Aldolases. 423, 424 l -Amino acid dehydrogeuase. 421 Amino acids. see ¡¡1m ~p~,!jir names L-aspart;He,300-1

immobifued cells for 'producrion af. 301 biatin, use Dr. 287 enzyTIles use or, 426-7 flux analJ5u;. 2S5 fl1nctionrugenomiCS . 285 L-gltltarnate. 284. 285-9 prududiun plant, 288 prod lIctian proces~. 287 procluctlon straim. 286 l-lysinc. 283. 289- 93 ¡)"Sine blosynthcsis. 289 Iysine e;'(porter, 291 Iysiue synthesis, 290

production prOCess. 293 production strnins. 292 ul,ukets, 282 L-phellylalalline, 296-8 production :2roc~s, 297-1! production straJnS, 297 recombinant te
appl icuions food and agriculrure. 259-60 i:r.m5genk animals. 259 tr.Irugenic plant!o 259 medicine. 255-9 medical diagnostics. 258 othecind ustries.260 poly-bydroxylakalloall'S.260 lIantbau gu ms. 260 rompanies altitudes/c ulture, 26Z- 3 basic cowponent'l. 261 business p lan. 266-7 funding for Slart-ups. 269-75 management. 275-8 mar:ket needs. 261 patents, 278-9 plalform technology. J61 prod uClcompan.y.264 scientiliccreallvity.261 people,262 mategy. .263- 8 solulion pnwiders. 264tools company.;¿6t, drugdiscO"\'l'ry path. 257

eurobarometer 5Ul'V'f'yS. 5-6 investmenl corporale partners. 272 funding stages. 271 grants.172-3 private funding. 269- 73 pnvatein ve5tors,269 regional developmcul s upport. 273

R.eturo on Investmenl (ROL). 271

seeCl lnvestment. 269 mia U eompany su pportKhemes.

m stock mad<etand biott!Chnology. 273-4 tec:h nology transfersdlemes. 273 ~nture("",¡pitalists.

270

publieperce ption . I- 16 altitudes. 6 Borre¡iob~rftri.

61

2.3-lJ utanecUo l. 34 Ilutanol. 34 Campylobam:r jcjlmi. 61 CIIn4lrla anwrrtiCtI • •no

üllldlda WTellllla. 306 Candida Impkal!s, 306 Galldida ulilis, 43 Otrbon conllenlon coeftIciem. 43 Carboxype.ptídases.435 Catabolic palhways. 21-8 anap letotic rt3c[ion, 25 effcctOIs.24-S ¡lu("OSe degradation general eonsider¡¡tioru.1J -2 glyoxyiate by-pasli. 25 tJiearboxylic acid cyde,13-5 Catabalie reprt'5síon catabalilE actillatorprote:Jn. 40 utabolite l'eCeptor p rotein, 40 carabolite reprcssion. 38, 39 diauxiegrowth,39 CataboUsm.1 8.-46 Cataboli.sm and enelm'. 20 cONA. 95. 115 CeJ Jobiohydrolast. U5 Ge.Uulases. 409, 4t3 CentrifUg'J lÍo tl , .190-2: SI'~ abo Downstream processlnlo: Chaperone, 95. 12-4 ChloreUo pyrmoidom. 163 Cho/esterol oxidase. 431 Chromatin. 95. 99 Chromatograpby. SI't tllSO Downstream prcx:essillg adsorption., 206 affi llity. 206. 297 Oow.(h rough orped'usion. 204 bydrophobic interaction . 206 ion excttan¡.;e. 206 large-$calc.205 product fonnulation. 20'7 prote:i n $f'paration, 205 radial ftow. 2(H

size-exclw:ion.205 Chro.mosomes. 59-1>l. 95. 97 artiftdaJ. 120 bacterial.6O.61 replicatian fork. 62 bacto:!rial artillela!. 78 fungal.61 vira!. 61 Cbylllooln, 11, 125 Cit:ricacid. 306-1:S Clollil\g(DNA) lib¡·arles,76-81.H17- 15 Qoningve
filteraid . 192

Iiltr.uion.. 188-90 fiherpn.'ss. 189 membrane Il!ter presses. 190 rota.ry dnlm vacuum filt~r. 189. 190

vacuum filIen; , l89 f1occtl.la{lng agel\l~. 192

f10t.,tiOD .. 1'}2 bomogenisation oranimalfplilnt li5sue.I94- 5 membr-.me ad~oroer5. 200 membrane tlluat ion cross ..tlow or tangential flow mtratioll,200 dead-end fil tr.U.iOll. 199 Ilollow-fibre sysrem. 200

bypcrfil tration , t 99 mlcrofilo-ation,l99 mode!i of pmtein precipitation. ZO:!

perslTaction. 201 pel'V3poration, 200. 201. pred pita tion.. 201 aftInity precipital.ioll. 202 heterobi functional Hg~ nd s. :ro:! homobifunLuonalligand$,202 Te\'el'lit osmosis. 199 ultraflltrolllion, l99 melhods (o"disruption of rells. 193 deying. l 94 heat shock. 194 high-pre:uuce homog"nlsa tion, 193 mech anical disruption. 193 l11i crofluidisation, 194 non·mechanical dis rupdon, 194 orga nic solV\!Dts ;wd delergen ls. 194 o, BODs. 533 denitrifu:¡¡tion, 533 llitrílll"iltion, 533 m em brane bioreacmr. 534 wastegaK"S. 545-8 b io tlltm .547 bi ot1l 1ers and bioscrubbers. 547 biologíC"31 reman l Clfnitrogeu,

'"

volatile o rgan ic comJlQunds (VOCs). 545 Enzymesynl hcsis. tli.'gt'ildatinn. 42-) dN~pressed , 39 ccllu lase5.409 induc tion.39 modificaooll ofcnzyme a c ltvity. 40-2 ac tiun ofeffecrors, 42 1O la tion oE soluble el12ymes. 403 Iiltration, oI04 Ul trafil tratiou. 4()4 la rge-scalt' production. 396 Jegisl:lI:ive/sMt'ty, 401- 8 m anUÍilctU n:! rs of, 392

microbiaJ enzytnl'S rl'placing p lanl enzym e~. 398 micrubial soul'ccs. 395 prim'lI'ys tru cturC . 410 prod uced by Aspagíl1us"'S..... 396 p urific ~tion, 404-6 precipita tion, 104 separation by cltromatogr.lphy, 405.524 rt'COYely. 402-3 t'-" tracellular. 402 intrllcellu lar.40'2 N ductiOIlS and o xida tions. 419-:12 monlHJJo/gen ases. 4.20, 421 n ·ge ner.ltio n n t' co-lilL1urs, 4'2 \ $t'condary structure. 410 $lllLdirected mutag(!]l~ S, 4{1J sourccs of ...amylasc, 396 sou n:es of glucose homer~ e, 396 'pt!ci~lity. 393 Ll'rtiary structurc, 0110 tOlal turnO'o'ern umber (fCNI, 421 F.rythrupoieti n, 437

F,smtrlchiu ro!! c hro mosomes.59- 62 ft' rmeutltion , .ICCI/n _leTEnzymf'l¡ growt:b yield, aerobic/ana.erobic, 43 Ethanol.3r~

culture syuems,

466 microc;l rri.o: pressio ns, 129 m odl'h advillll:ed consldenttio D. 222- 8 stru ctul'e, 215-16

sUpelvision and rontrol.

228-36 Pl'oduct ronnation . 207-9 Produn finmatio n 1'lI le. 132 Promot~ (fur ONAI. 96. 99 Pro pano!. 34

SdIizosaaha~pambe,

4

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