ADVANCES IN
Applied Microbiology VOLUME 39
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ADVANCES IN
Applied Microbiology VOLUME 39
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ADVANCES IN
Applied Microbiology €Edited by SAUL NEIDLEMAN Vacaville, California
ALLEN I. LASKIN Somerset, New Jersey
VOLUME 39
Academic Press, Inc. A Division of Harcourt Brace S.Company
San Diego New York Boston London Sydney Tokyo Toronto
This book is printed on acid-free paper. @
Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.
Academic Press, Inc. 1250 Sixth Avenue, San Diego, California 92101-431 1
United Kingdom Edition published by
Academic Press Limited 2 4 2 8 Oval Road, London NW1 7DX International Standard Serial Number: 0065-21 64 International Standard Book Number: 0-12-002639-2 PRINTED IN THE UNITED STATES OF AMERICA
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CONTENTS
Asepsis in Bioreactors
M . C. SHARMA AND A . K . GURTU I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. “Invaders” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV . Sources ............................................................................... V . Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Overcautious Approaches ......................................................... VII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 3 3 4 6 24 25 25
Lipids of n-Alkane-Utilizing Microorganisms and Their Application Potential
SAMIR S . RADWANAND NASERA . SORKHOH I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. n-Alkane-Utilizing Microorganisms ....................................... 111. Total Lipid Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Acylglycerols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Sterols ................................................................................. VII . Fatty Alcohols, Ketones, and Epoxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Hydrocarbons and Waxes .......................................................... IX . Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . Glycolipids and Peptidolipids ..................................................... XI . Biolipid Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI1. Environmental Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29 30 35 42 59 61 63 66 67 73 76 78 81
Microbial Pentose Utilization
PRASHANTMISHRAAND AJAYSINGH I. 11. 111. IV . V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pentoses from Natural Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pentose-Fermenting Organisms ................................................... Pentose Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production of Solvents and Organic Acids ......................................
V
91 93 94 101 112
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CONTENTS
VI . Factors Affecting Pentose Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Product Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Strain Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX . Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
127
136 139 142 143
Medicinal and Therapeutic Value of the Shiitake Mushroom
S. C. TONG
AND J
. M . BIRMINGHAM
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Medicinal and Therapeutic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Patented Products and Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
153 154 172 175 177
Yeast Lipid Biotechnology
Z . JACOB I. I1. 111. IV . V. VI . VII .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yeasts as Potential Sources of Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Importance of Yeast Lipids in Beverages and Foods . . . . . . . . . . . . . . . . . . . . . . . . Medical Importance of Yeast Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modification of Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Commercial Significance of Yeast Lipid Biotechnology ....................... Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
185 186 187 191 192 204 207 208
Pectin. Pectinase. and Protopectinase: Production. Properties. and Applications
TAKUOSAKAI. TATSUJI SAKAMOTO. JOHAN HALLAERT. AND ERICKJ . VANDAMME I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Review of Pectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of Pectic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Pectic Enzymes in Phytopathogenesis .................................. Applications of Pectinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protopectin-Solubilizing Enzyme (Protopectinase) ............................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1. 111. IV . V. VI .
213 214 236 244 245 248 2 88
CONTENTS
vii
Physicochemical and Biological Treatments for Enzymatic/ Microbial Conversion of Lignocellulosic Biomass
PURNENDU GHOSHAND AJAYSINGH I. I1. I11. IV . V. VI . VII .
Introduction .......................................................................... Structure of Lignocellulosic Biomass ............................................. Physical Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thermal Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OF PREVIOUS VOLUMES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS
295 298 300 304 306 316 326 327
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Asepsis in Bioreactors M. C. SHARMA~ AND A. K. GURTU’ SOL Antibiotics Limited Hyderabad 500 482, India
I. 11. 111. IV.
Introduction “Invaders” Consequences Sources A. Inoculum B. Nutrient Medium C. Bioreactor System D. Air/Liquid Transfer E. The “Rogue” V. Approaches A. Sensitive Sterility Assessment Methodology B. Certified Aseptic Laboratory Inoculum C. Autoclavable Bioreactor D. Sterile Mediurn/Feed E. Aseptic Bioreactor System F. Maintenance of Asepsis during Fermentation G. Protected Fermentation H. Product Changeover I. Schedules and Procedures VI. Overcautious Approaches VII. Conclusion References
I. Introduction
The key process of biotechnology is fermentation, and the key equipment, or the heart of the process, is the bioreactor-fermentor. The fermentation process generally employs pure culture as the biocatalyst; the success of the process depends, to a large extent, on ensuring asepsis in the fermentation system and in the process. Although the terms “asepsis” and “sterility” in fermentation are microbiologically incorrect, they have been generally accepted. In fact, the term “monosepsis” would be more correct. Asepsis in biotechnology means freedom from unwanted microorganism(s), just as in clinical medicine it means freedom from pathogenic microorganism(s) (Bull et a]., 1983). Absolute sterility is a concept in probability and is an unattainable ideal in
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Present address: Biotech International Ltd., VIPPS Centre, Masjid Moth, Greater Kailash-11, New Delhi 110 048, India. * Present address: J. K. Pharmachem Ltd., Madras 600 014, India. 1 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 39 Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
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M. C. SHARMA AND A. K. GURTU
practice. A low-level, contaminated batch in a bioreactor, with normal product biosynthesis, achieves the empirical sense of “sterility,” though not the absolute one (Reisman, 1988). There are many industrial fermentations, e.g., ethanol, baker’s and fodder yeasts, and vinegar, where no serious attempt is made to maintain asepsis either in the fermentor medium or in the subsequent conduct of the process. In fact, such attempts are not warranted because of rapid culture proliferation, rapid metabolic transformations, and the resultant environment being generally nonconducive to contaminant growth (Herold and Necasek, 1959; Bailey and Ollis, 1986). It was during the first world war that Weizmann made the pioneering efforts to establish the first truly aseptic acetone-butanol fermentation (Hastings, 1978). However, the inactivation of penicillin by penicillinase-producing microbes necessitated engineering developments aimed at carrying out biotechnological processes with absolute exclusion of foreign microbes. The fermentation industry does not publish figures on the rate of nonsterile operations, yet a figure of 5-30’/0 nonsterility of bioreactors is realistic (Saudek, 1956; K. Gerlach, unpublished communications, 1986). At times the rise in figures warrants being called a “wave” of contamination. Economic considerations indicate that a contamination probability of 1in a 100 is acceptable for batch fermentations, considering the norm of 1 in 1000 as the probability of contamination commonly employed in design calculations for a sterilization process (Banks, 1979). A nonsterility rate of 1% or less is often regarded as a commendable performance (Soderberg, 1983). Artificially selected industrial microbes generally used in biotechnology endeavours are at risk of being overwhelmed by competing wild organisms. Mammalian/animal/plant cell cultures are especially prone to microbial contamination because of a long process cycle (20 or more days] and a relatively slow growth rate (doubling time as long as 100 hr). These cell cultures can be compared to an artificial organ without autoimmune protection, lacking any defense system, and therefore, extremely vulnerable to a breach of sterility (Knight, 1989).Yet the industry now operates large-scale animal cell cultures routinely with contamination rates of just 2% (Spier, 1988), even with culture lengths of several months and intermittent additions of fresh medium. The physicochemical environment generally maintained in a bioreactor is optimal for a host of microorganisms. Very rarely is the medium “protected”, i.e., selectively utilizable by a limited range of microbes (Stanbury and Whitaker, 1984) or else has an antimicrobial added to it. The intended metabolite, even if an antimicrobial, is normally produced late (in idiophase). Very often, the bioreactor in trophophase
ASEPSIS IN BIOREACTORS
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would therefore be an ideal incubator for numerous microorganisms, if provided an opportunity of entry. II. “Invaders”
The microorganisms that invade the sterile fermentation process are bacteriophages, mycoplasma, bacteria, or fungi. Phages normally infect through air and the development of phage-resistant strains has resulted in the rarity of such infections, particularly in view of the host specificity of phages. Mycoplasma and viral infections are more common in animal cell cultures through serum and can be eradicated easily (Arathoon and Birch, 1986). The microsize, omnipresence, and faculty of utilizing widely varying substrates for nutrition make the bacteria capable of causing widespread infections and maximum damage to the fermentation process. While gram-positive bacteria have air as their main source, gram-negative bacteria are transmitted through liquids, particularly water. Among the fungi, yeasts may originate mainly from insufficient sterilization of substrates. Filamentous fungi have air as their main source. Fungal infections occur rarely (Herold and Necasek, 1959). 111. Consequences
The invasion of a fermentation process in a bioreactor by a foreign microorganism can result in a variety of consequences: 1. A fast growing, wild contaminant may outcompete the normally slow growing desired strain, deplete the nutrients, and fatally interfere with the chemistry of the process and the final product. 2. The invader may not outgrow the desired strain, but may cause minor to appreciable alterations in the physicochemical characteristics of ongoing fermentation. The contaminant may also produce undesirable and possibly toxic metabolites that may lead to lower yields and productivity. 3. The contaminant may grow to a certain level and subsequently be inhibited by the metabolite(s) produced by the desired strain. The fermentation may continue to its logical end, as at times in the case of broad spectrum antibiotics. 4. Contaminants, i.e., phages, could result in the lysis of the desired microbe in a bacterial/actinomycete fermentation. 5 . Mucilage/slime produced by the contaminant may choke the filter pores to varying degrees, which in a worst-case scenario would ruin the entire batch on hand.
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6. Another consequence of nonsterility could be the degradation/ racemization of the desired metabolite, e.g., the enzymatic degradation of p-lactam antibiotics by p-lactamase-producing bacteria, leaving behind a totally unproductive batch or producting DL- or D-amino acids in an L-amino acid fermentation. 7. Undesirable moieties produced by the contaminant may lead to interference in the downstream recovery of the product, resulting in not only lower yields but a substandard product as well. The processing of the product may cause increased production costs. 8. The contaminant may render the final product unusable, e.g., single cell proteins where the cells constitute the product. 9. Not every contaminant at the fermentation stage exerts detrimental effects. The contaminant has been reported to increase fermentation yields and to better downstream processing (Reisman, 1988) due to the presence of useful enzymes like proteases and lipases.
Irrespective of the consequences of contamination, preventing the entry of contaminants is necessary. The attempt, therefore, should be to identify the sources of contamination and to conduct fermentation processes in an aseptic manner. IV. Sources
The sources of contamination in a fermentation process can be broadly ascribed to several factors. A. INOCULUM Contamination at the inoculum development stages assumes greater significance in view of the consequent nonavailability of quality “starter” material for the bioprocess and therefore the opportunity loss. The presence of a contaminant in laboratory-grown inoculum, often in concentrations low enough to escape detection up to seed bioreactor maturity, can lead to the subsequent manifestation of nonsterility in the fermentation bioreactor. In view of the relatively small volumes used for sterility testing, sensitive methodologies are obligatory. The sources of laboratory inoculum nonsterility in turn could be autoclaving deficiencies, “lumpy” medium, inadequate maintenance of sterile chambers, ineffective ultraviolet irradiation from germicidal lamps, use of inefficient germicidal solutions, wetting of plugs, and insufficient/ prolonged stocking of sterilized media/glasswares. Inadequately screened cell banks are potential sources of viral contamination in animal cell culture processes (Arathoon and Birch, 1986).
ASEPSIS IN BIOREACTORS
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B. NUTRIENT MEDIUM Nonsterility of the nutrient medium used in the seedifermentation bioreactor may culminate in a contaminated operation. An inadequate sterilization operation, “lumpy” medium, and the bioreactor itself may contribute to medium nonsterility. The choice between batch and continuous sterilization and their design depends on the scale of operations and the characteristics of the medium. In the case of separate medium sterilization, the tank/continuous sterilizer, piping, and receiving vessel could be sources of nonsterility. Animal sera used for cell culture processes may sometimes carry viral and other contaminants and render the process nonsterile (Arathoon and Birch, 1986; Maurer, 1986; Elander, 1989).
C. BIOREACTOR SYSTEM A continuous stirred tank reactor is the most commonly used bioreactor in view of its versatility and flexibility of operations therein. The sterility considerations of such a bioreactor satisfactorily cover most of the other types of bioreactors as well. The design, material, and fabrication of the bioreactor are important factors contributing to the sterility of operations. Apart from these factors, aberrations in fermentor sterilization, air supply, agitation system, sampling and monitoring ports, and uncontrolled foaming could lead to invasion of the bioreactor boundary by undesired microorganisms. The agitator shaft entry and the air exit are the most vulnerable points in a bioreactor. All types of seals, except a double mechanical seal, provide gaps as entry points for contaminants (Steel and Miller, 1970; Bull et al., 1983; Aiba et al., 1986; Reisman, 1988). Intermediate bearings on shafts, improper impeller hubs and keyways (Reisman, 1988), and pipe in pipe connections provide unhygienic places to harbor contaminants. Too many interior fittings create pockets likely to conceal microbes, are difficult to sterilize, and can result in contamination of the bioreactor. Stress corrosion/cracking in the vessel and internal coil leads to repeated contaminations. Hammering caused by steam during batch sterilization causes stress on spargerflanged joint components and creates nonuniform steam distribution-related sterility hazards. The numerous ports, pipes, and valves required in a fed-batch process are further sources of microbial entry if not properly selected or designed. Flangedhhreaded joints, pervious sealing materials, defective slopes of pipes, dead ends, pockets, indentations, crevices, solid depositions, stagnant layers, rising stem valves, leaking or “weeping” pipes/ flangeshalves, and the absence of steam seals/crosses are potential
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M. C. SHARMA AND A. K. GURTU
causes of contaminations. The continuous presence of humidity/condensed moisture and entrained medium in the air exhaust area provides an environment conducive to microbial growth at this boundary point of direct contact between sterile and nonsterile zones. Uncontrolled foam generation during fermentation causing “foaming out” through the air exhaust increases the chances of fermentor contamination (Solomons, 1967; Ghildyal et al., 1988). D. AIRILIQUID TRANSFER
Contamination through the depth filter is possible because of inadequate filter packing, channeling, free moisture, or fiber fragmentation. Individually or collectively, these factors contribute to filter inefficiency. In the case of the membrane filter, damaged 0 ringsimembrane cause losses in filter integrity. The air filter, soaked in nutrient media because of backflowloverflow, can act as an incubator for contaminating microbes. The liquid transfer systems, including those for the inoculum, feeds, and supplements, may also contaminate the process because of system deficiencies described earlier and/or inadequate sterilization. A common system for transfers to several fermentors, if having such deficiencies, would be a catastrophe.
E. THE“ROGUE” For all practical purposes even the very rare appearance of a nonproducing wild “rogue,” which occurs due to reversion mutation of the production culture during the fermentation process, leads to contamination of the bioreactor. There are no precautionary or remedial measures against such mutant appearances and early downstream processing may be warranted for salvage, if any. V. Approaches
Ever since the aseptic submerged culture technique for penicillin production was introduced, there have been attempts to perfect fermentation techniques, design, and systems in achieving asepsis. Coordinated efforts of microbiologists, technologists, engineers, and biochemists are required to evolve various bioreactor contamination control strategies. The successful commercial scale production of numerous fermentation products is a measure of the magnitude of such achievements. Exhaustive reviews on such techniques have been made by Rhodes and Fletcher (1966), Solomons (1969, 1971), Augurt (1983), Wallhausser (1985), and Bailey and Ollis (1986).
ASEPSIS IN BIOREACTORS
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Aseptic design development is based on several norms by which the intrusion of foreign microbes into a defined fermentation system can be precluded. A closer look at the behavior of a microbe at different types of boundary layers reveals the following criteria (Lundell and Laiho, 1976) relevant to asepsis: 1. Nonpenetration of homogeneous solids by a microbe. 2. No movement or growth of a microbe through holes smaller than
its own dimensions. 3. No propulsion of a microbe against the flow of carrier medium. 4. No movement of a microbe on dry surfaces without external forces. 5. No growth on a surface with temperatures exceeding a microbe’s maximum growth temperature. 6. No growth on nonmetabolizable/hydrophobic/toxicmaterials. 7. Inactivation of a microbe by high temperature, toxic chemicals, and irradiations. 8. A characteristic doubling (reproduction) period of each microbe. Most of the aseptic design considerations involve any or all of the combinations of these criteria. The sterile design of a bioreactor normally adds 15-25% to the cost toward its design, purchase, and installation (Reisman, 1988). The following approaches constitute some of the techniques essential for achieving aseptic bioprocesses:
STERILITY ASSESSMENT METHODOLOGY A. SENSITIVE The absence of a contaminant(s) in the culture inoculum, sterilized seed/fermentation media, and equipment needs to be ascertained at every stage in order to avoid nonsterility and to detect the stage at which the contaminant invaded the process. Often the results of conventional sterility checks may not be available before the culture has reached the production bioreactor or before the contaminant has reached a growth level capable of disturbing the desired fermentation. Speed of detection is germane to the salvage of a batch. A sensitive sterility assessment methodology with accelerated results would be useful for such applications. The use of a variety of nutrient media in tubes (broth, slants, and stabs) and flasks incubated at varying incubation temperatures under statidshaken conditions would be ideal in covering a range of growth conditions required by different contaminants (Soderberg, 1983). The use of thioglycolate, with a small amount of agar (0.05%), and oxidoreduction dyes like methylene bluekesazurin have been recommended for the fast detection of aerobes as well as anaerobes in a single medium (U.S. Pharmacopoeia, 1980).
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M. C. SHARMA AND A. K. GURTU
Membrane filter discs could prove useful for sterility checks of broths containing antimicrobials. Penase (a potent lactamase preparation) is recommended for the inactivation of penicillin/p-lactams during sterility testings (Difco Laboratories, 1985). Sterilized filter assemblies with membrane filter discs/glass wool pads find application in sterility checks of air/liquids passed into the fermentation equipment. Trial runs with uninoculated lean nutrient media are used to ascertain the sterility status of fermentation equipment, transfer manifolds, and air systems during validation. The use of selected radioactive carbon sources in sterility check media has been recommended under the “Bactect” system for a fast detection of contaminants (McLaughlin et a]., 1983). The mass spectrophotometric identification of 3-hydroxymyristic acid, a characteristic of gram-negative bacteria, has been applied for a rapid sterility assessment by Elmroth et al. (1990).The techniques of sampling for sterility checks were described by Elsworth (1960), and newer techniques (Charton, 1990) involving the use of thermoplastic elastomer tubings for improving sterile access to bioreactors have been described. A mobile flexible film containment cabinet attached to the bioreactor is used in sampling recombinant DNA-based fermentation processes (Hambleton et al., 1991). Elander (1989) described the use of a sterile stainless steel container with a small sterilizing filter on its vent that was attached to the fermenter, union sterilized, sample drawn, and the union finally resterilized before disconnection and further processing of the sample drawn. For animal cell cultures, the establishment and testing of suitable cell banks with screening for freedom from viruses and other adventitious agents is a major exercise (Lubiniecki and May, 1985). Further work on accelerated sterility assessment methodology in the fermentation industry is needed. B. CERTIFIED ASEPTICLABORATORY INOCULUM
The inoculum preparation room needs to have a clean room design and scheduled validation/checks of this sterile room through exposure of plates. Schedules for area fumigation and in situ integrity testing of HEPA filters by aerosols/dioctyl phthalate/dioctyl sebacate ensure sterility in these rooms. The area used for inoculum preparation needs to be isolated from that, used for in-process sterility assessment. Only validated disinfectant dilutions should be permitted in sterile rooms. The rotation of disinfectants to avoid buildup of resistant microflora is necessary and must be practiced rigidly. Principles and practices of laboratory management related to facilities, design, decontamination, access to work place, personal hygiene, apparel, sanitation, ventilation,
ASEPSIS IN BIOREACTORS
9
and safety are amply described (Rhodes and Fletcher, 1966; Soderberg, 1983; Wallhausser, 1985; Scheirer, 1987). The careful planning of premises, air circulation, and pressure differentials has recently become important, particularly in culturing animal cells and genetically engineered microbes (Scheirer, 1987; Knight, 1989). Finch (1958), Sykes (1958), Borick (l968), Benarde (1970), and Wallhauser (1985) have discussed the utility of various disinfectants and chemical sterilants for such purposes. “Biosafety in Microbiological and Biomedical Laboratories” published by the U.S. Department of Health and Human Services (1984) describes the standard and special microbiological practice guidelines and designs for aseptic laboratory operations. The proper storage of biodegradable raw materials like corn-steep liquor, soya flour, corn flour, and seed meals under hygienic conditions and preferably at reduced temperatures and humidity minimizes the further increase of microbial load. Fine powders, presoaking and preboiling, proper batching sequence, and straining help prevent the “lumpy” medium threat to aseptic processing (Soderberg, 1983). Validation and proper operation of autoclaves used for media/glassware sterilization is mandatory for pure inoculum propagation. The proper venting of air and steam ensures the absence of air pockets necessary for attainment and sensing of correct uniform temperature in the autoclave. Air, being heavier than steam, must also be expelled from the lowest level in the autoclave. Using a jacket steam ejector to remove leftover steam in the autoclave chamber after sterilization is advisable in order to minimize moisture condensation on sterilized wares (Wilkinson and Baker, 1964). Autoclave performance may be checked using “biological indicators” (Banks, 1979) or sterilizing temperature indication stickers/tapes, like those supplied by the 3M Corporation, to confirm proper autoclave operation. Such indicators have been reviewed by Augurt (1983) and Wallhausser (1985). Procedures obligatory for efficient performance of autoclaves have been described by Rhodes and Fletcher (1966), Stumbo (1976), Augurt (1983), and Wallhausser (1985). To avoid dirt accumulation, it is advisable to use rimless glassware and presterilized cotton/synthetic hydrophobic plugs. Needless to say, wrapping with waterproof papers/aluminum foil, vacuum drying through steam ejection, and hot air oven drying of autoclaved materials is very useful. Incubation of sterilized media at different temperatures for 72 hr and subsequent checks ensure their sterility before use. A need-based planning of sterile glassware and media is necessary to avoid the “emergency” use of freshly autoclaved material or prolonged stocking of autoclaved material that could be a “sterility risk,” particu-
10
M. C. SHARMA AND A. K. GURTU
larly in a humid climate. Longer necks and optimum degree of filling of containers prevent the plugs from getting wet during incubation on shaker. Attention to these details results in significant improvements in sterility at the laboratory stage. C. AUTOCLAVABLE BIOREACTOR
Smaller glass or stainless steel bioreactors (1-5 liter capacity) containing medium can be sterilized by autoclaving and later connected aseptically to various nutrients, air, pH, and pressure maintenance lines for operations. Bioreactors with capacities higher than 5 liters are difficult to sterilize in an autoclave, and the possibility of sepsis is increased if connections of larger diameters are used (Solomons, 1969). D. STERILE MEDIUMIFEED
The precautions mentioned earlier for laboratory medium preparation are necessary for bioreactor medium preparation as well. Medium mixing facilities are often neglected in the fermentation industry. Tank design for total drain out, smooth internal finish, high-pressure water jet cleaning after each operation, and chemical cleaning at regular intervals eliminate the formation of dried medium crusts and discourage microbial buildup in the tank. Sterilization of industrial media is not possible through filtration [which in fact is not recommended because suspended matter is a desirable ingredient of the medium) and is unreliable through irradiation or chemical sterilants. Steam sterilization is, therefore, the only choice and can be carried out in the bioreactor itself or in a separate pressure vesselfcontinuous sterilizer. Aseptic considerations warrant a preference for steam sterilization over other alternatives. 1. Batch Sterilization
Indirect heating through a jacket, external or internal coils, hollow baffles, and steam sparging through an air delivery system and dip pipes or any combination of the aforementioned are used for batch sterilization. Efficient mixing and circulation of the medium promote the efficiency of heat transfer and ensures the uniform heating essential for proper sterilization. In such a case the vessel also gets sterilized along with the medium. It is imperative that steam is supplied to the bioreactor through all dip pipes or ports in direct contact with the medium [Bull et a]., 1983; Stanbury and Whitaker, 1984). In fact, it is desirable to continue regulated steam supply at these points throughout the sterilization cycle since the stagnation of improperly heated medium
ASEPSIS IN BIOREACTORS
11
inside the pipe section, external to the bioreactor, is a serious sterility hazard. Relative merits of in situ sterilization and the use of separate pressure vessel have been discussed in detail by Richards (1966,1968). More often it is the in situ sterilization that is preferred. 2. External Continuous Sterilization
The external continuous sterilizer involving high temperature short time treatment (Baily and Ollis, 1986) is the method of choice when large volume bioreactors are used. The advantages of continuous sterilization of initial medium as well as nutrient feeds (Aunstrup et a]., 1979) over batch sterilization have been comprehensively described by Solomons (1969, 1971), Augurt (1983), Banks (1979), Cooney (1985), Wallhausser (1985), and Aiba et al. (1986). A shorter process time, better heat recovery, better medium quality, and cost effectiveness are ensured. The design of a continuous sterilizer is extremely important. Plate heat exchangers are suitable for media containing low levels of suspended solids. For media containing substantial solids or for viscous media, tubular heat exchangers with high flow rates and turbulent flow are used (Bull et al., 1983; Cooney, 1985). The sterilization of oils and viscous antifoams has to be planned carefully (Bader et al., 1984). Oils free of moisture are often difficult to sterilize. It is preferable to mix water in oils/antifoams before sterilization and to use a tubular continuous sterilizer. A spiral heat exchanger and an injector-flash cooler sterilizer (Banks, 1979; Stanbury and Whitaker, 1984) are useful versions of continuous sterilizers. The replacement of process water, held up inside the heat exchanger, with demineralized water prior to system sterilization with steam or superheated water (125°C) is necessary for eliminating stress corrosion cracking (Soderberg, 1983). Designing the sterilizer with near plug flow, proper retention, automatic control of the sterilization temperature, monitoring of inlet as well as outlet temperature at the holding section, and automatic switch over to the recirculation mode with a simultaneous shut off of the delivery line to the bioreactor when the sterilization temperature drops are mandatory features for aseptic performance of the system. Figure 1 represents a standard external continuous sterilization system for medium and feeds for bioreactors. Installation of a conductivity probe in the cooling water exit enables instant detection of a leak in the system. 3. Sterilization by Filtration
Sterilization through filtration by “depth” or membrane filters is a choice for clear media, especially where volumes are relatively low [Bull
M. C. SHARMA AND A. K. GURTU
12
Sterilized
* productto
bioreactor
Water detergent
Cooling Water
II Raw Product FIG.1. A typical external continuous sterilization bioreactor system. (1) Balance tank. (2) Flow control. (3) Interchanger. (4) Heater. (5) Holding tubes. (6) Cooler. (7) Controls. (8) Steam.
1 1
et al., 1983; Reisman, 1988).Membrane filters have superior operational performance because of a fixed, well-defined pore size. Sterilizable, cleanable, and reusable effective filter components have been developed and are used for plant and animal cell cultures to avoid loss of nutrients in media due to steam sterilization. Cross flow membrane cassettes, plate, disc, or cylindrical cartridge units, and even ceramic cartridges are now available for repeated sterilization operations. Their life can be prolonged by employing coarse prefilters or screens. Advance sterilization of the bioreactor and transfer lines is a prerequisite for medium sterilization through a separate pressure vessel, continuous sterilization, or filtration.
E. ASEPTIC BIOREACTOR SYSTEM In systems with strict aseptic requirements the material of construction and design requires detailed attention. The necessity of maintaining sterility has a far reaching impact on equipment design, piping, and layout (Elsworth, 1960; Soderberg, 1983; Bjurstrom, 1985; Reisman, 1988). 1. Bioreactor Internah
An aseptic bioreactor requires internals without niches and crevices to ensure steam availability to the entire surface. Internals with smooth contours ensure cleanability and complete drainage to exclude areas where condensates collect and to insulate microbes from sterilizing temperatures. Repeated sterilizations should leave no pits or a corrosive buildup in the vessel. The choice of material for such fermentation systems is restricted to borosilicate glass or stainless steel 316L (less than 0.03% carbon) with 150 grit or electro-polished internal finish
ASEPSIS IN BIOREACTORS
13
(Bull et al., 1983; Bjurstrom, 1985; Reisman, 1988). Fibreoptics and diamond sensor instruments devised by reputable bioreactor manufacturers are currently in use for inspection of welded joints and internal surface profiles. High surface qualities with grain 400 and roughness values of less than 0.06 p m are requested presently. The entries into the fermentor should be restricted to the bare minimum (Bjurstrom, 1985; Van Brunt, 1987; Reismann, 1988). The air supply pipe does not need to run through the top-head to down below the bottom impeller but instead can be introduced through the bottom cylindrical portion, This avoids “cake” formation on the pipe above the agitated broth level, particularly in the case of maintenance of the elevated inlet air temperature, thus avoiding a potential contamination pocket. Similarly, internal cooling coils should be avoided as much as possible to reduce brackets, supports, and weld joints. Internal fittings, when absolutely necessary, need to have a clear gap from the fermentor wall to facilitate proper cleaning. The use of pipes for brackets, ladders, and supports is forbidden. The ring type air sparger should have holes on its lower side for drainage of broth when the vessel is emptied. 2. Agitator Shaft and Seal
A stirrer shaft seal is the most difficult to construct in a fermentor. A side or bottom entry shaft (Richards, 1968) is undesirable since the bearings would be submerged. Eighty percent of all fermentor installations have top entry agitators (Sittig, 1982). Simple stuffing box and bush seals are potential sterility hazards because of frictional damage to packing, uncertain sterilizations, and oozings. Stellite hardening of the agitator shaft in the stuffing box region helps in maintaining the shape of the shaft, which normally gets worn and leads to increased leakages, Chemicals are sometimes used to disinfect the area. A double mechanical stainless steel 316 seal that can be sterilized with live steam and lubricated with cooled steam condensate is always preferred (K. Gerlach, unpublished communications, 1986; Liberman et al., 1986; Reisman, 1988). Seals able to withstand live steam/hot steam condensate lubrication have a decided edge over others. Polyethylene tetrafluoroethylene (PTFE) secondary seal, ethylene propylene dimer (EPDM), fluorocarbon and viton “0” rings, resin impregnated carbon/ tungsten carbide faces with a ceramic seat are the state of the art components of such seals (Fig. 2) like those from A.W. Chesterton Co. (Stoneham, MA) and John Crane, U.K. Ltd. (Slough, Berks, U.K.). Use of a back pressure indicator and regulator in the seal lubricant exit line helps ensure sterility and also acts as a forewarning device for loss of seal integrity. A low seal pressure/low level alarm system with an
14
M. C. SHARMA AND A. K. GURTU
Tungsten-Catbide stationary ring Cahn
Condensate out FIG.2. A state of the art double mechanical seal for a bioreactor agitator.
automatic stirrer switch off are features of recent versions (K. Gerlach, unpublished communications, 1986). Another attempt in solving the problems related to the sealing of the impeller shaft involved the use of a high-torque magnetic drive system (Cameron and Godfrey, 1969; Bull et aI., 1983). The driven magnet is on one end of the impeller shaft and the driving magnet is outside the vessel. Such magnetic drives, without piercing for an agitator shaft, can only be used for small-sized fermentors (Knight, 1989). Using neodymium-iron-boron supermagnets, up to 50 horsepower can be delivered to the agitator. The air lift bioreactor does not involve any moving parts within the bioreactor and obviates the need for piercing the shell for agitation. Such bioreactors are useful in animal cell cultures and in single cell protein fermentations (Smith, 1980; Arathoon and Birch, 1986). 3. Bioreactor Ports
Every port of entry and exit on the fermentor is a potential source of contamination and therefore has to be properly designed to avoid
15
ASEPSIS IN BIOREACTORS
stagnancy of the medium therein. It should have a positive sealing penetration capable of maintaining its integrity when subjected to repeated sterilizations and chemical exposure. The bottom discharge valve in a batch sterilization fermentor has to have a flush bottom valve with steam seal 0 rings to avoid the risk of becoming a repeated source of sepsis. A totally bottom closed bioreactor is preferable except for operational complications (K. Gerlach, unpublished communications, 1986). Headplates of small bioreactors and the manhole lid of large industrial fermentors need to be steam sealed. Entry ports, when not permanently piped to addition vessels, seed tanks, or feed manifolds, need to be sterilized initially by heat conducted from the shell of the fermentor or heated by a double valve and steam bleed arrangement. The entry ports may have needle-penetrable self-sealing diaphragms or a special connection with quick connecting valves (Bull et al., 1983). These connections have to be made sterile by standard aseptic measures (steam, flaming, and/or chemical disinfection). The ports for probes-pH, redox, and dissolved oxygen-should be slanted, with 0 rings at a point closest to the inner surface of the fermentor wall (K. Gerlach, unpublished communications, 1986; Reisman, 1988). Figure 3 shows a resterilizable side-mounted process port with a steam lock for aseptic operations. Application of hydrophobic toxic grease between two 0 rings has been recommended by Lundell and Laiho (1976).Double 0 ring seals with steam tracings between the seals have been recommended by Hambleton et al. (1991) for lid fittings and filter housings
Steam in
0
Out
FIG.3. A typical resterilizable side-mounted process port with steam lock.
16
M.C. SHARMA AND A. K . GURTU
of containment requiring bioreactors. Triple elastomer seals are used where steam tracing is not practical. The inspection glasses located at the dome of the bioreactor have to be cleaned quite often with a jet of steam to wash down broth splashings. It is important that only live steam is delivered and not the steam condensate. Steam sealing of sight and light glasses has been suggested by Lundell and Laiho (1976). The circular shape of sight glass offers improved seal characteristics as compared to other shapes (Hambleton et al., 1991). Asepsis in samples drawn from bioreactors is usually accomplished by providing a fixed sample line through a permanent penetration. Two valves in tandem on the pipe with regulated steam flow help maintain sterility between sampling operations. A sterilizable sampling device with piston valves having an 0 ring seal and flush with the inside vertical wall of the fermentor in the closed position eliminates the dead space that may cause sterility problems. Encasing the sampling pipe with a removable screw cap with a pinhole ensures that external as well as internal pipe surfaces are continually exposed to live steam between samplings. The importance of aseptic sampling systems can be judged from the variety of systems proposed for bioreactors (Heatley, 1950; Chain et al., 1954; Heden, 1958),including automated, computercontrolled sterilizable sampling systems (Ghoul et al., 1986; Seifert and Mattaeu, 1988). The air exhaust region has been the focus of attention recently because of its vulnerability to back-in infection. Protection of the exit point by an absolute membrane filter is recommended, although it has practical difficulties of membrane clogging because of “entrainment” of the medium. Mechanical foam separators like “Turbosep” [Anonymous, 1990; Hambleton et al.,1991) offer some relief by ensuring effective separation of foams, aerosols, and liquids from the air stream. Liquid drops into the bioreactor and the exit gas heated by 10-15°C passes through the exit filter. The incineration of effluent gas is a recommended step (Melling and Allner, 1981; Bull et al., 1983) in containing organisms as well. Systems of double inlet and outlet air filters permitting isolation, replacement, and sterilization without undue interference in the ongoing fermentation are now available (Hambleton et al., 1991). The positioning of an air exit at the highest elevation on the top dome, an inverted “U” configuration of the air exhaust line, and an exit valve positioned on the downward leg of the loop are sometimes adopted. This creates a sufficient distance between the fermentor and the outside nonsterile environment. It also prevents the “fall back” of collapsed foam or overflown broth from the exhaust valve into the bioreactor,
ASEPSIS IN BIOREACTORS
17
thus avoiding direct physical contact between the sterile and nonsterile environments. To discourage microbial growth in the air exhaust, the pipe immediately after the valve, as well as the air filter, if used, is kept sufficiently heated through a steam jacket. Disinfectants sometimes become trapped in the air exhaust line. The air exhaust line after the valve should be easy to dismantle for the upkeep of internal hygiene. 4. Piping and Valves In a typical fed batch fermentation process requiring the addition of four nutrient feeds, pH maintenance, and partial withdrawals, the bioreactor needs about 10 ports, 60 valves, and a considerable length of pipe connections. The design of this ancillary pipeware is crucial for asepsis. Sterile piping is usually of welded construction with internally and externally polished welds and has the minimum possible of flanged connections (Perlman, 1950; Smith, 1980; Stanbury and Whitaker, 1984; Bjurstrom, 1985; Bailey and Ollis, 1986; Van Brunt, 1987; Reisman, 1988). Threaded fittings are not acceptable in aseptic services. Sharp turns, indentations, and dead legs in lines should be avoided (Reisman, 1988). Any upturn in a sterile line should have a drain valve and steam sealing on the downstream side. The lines should also be sloped slightly for free flow and complete drainage (Stanbury and Whitaker, 1984).Of special significance is the worksmanship during erection. Misaligned pipe sections bolted under tension invariably have flanges as a perpetual source of leakage (Soderberg, 1983).Cutting stainless steel pipe with a hacksaw and making a V-notch prior to argon arc welding are necessary. Sterile sections of pipe should be separated from nonsterile sections by a barrier such as a sterile filter or a live steamheated section of pipe (Bailey and Ollis, 1986; Reisman, 1988). Gaskets and 0rings made from elastomers, e.g., EPDM, viton, silicone rubber, or high temperature-resistant Teflon, are ideal for sterile operations since they are impervious, long lasting, and easily cleanable (Bull et a]., 1983; Reisman, 1988). The selection and placement of the valves used for a sterile system need special care. Microorganisms have been known to grow through closed valves under conditions suited to them (Bjurstrom, 1985). Valves must meet cleanliness, maintenance, and sterility requirements. Diaphragm and pinch valves with Teflon sealing are ideal for aseptic operations (Stanbury and Whitaker, 1984; Bjurstorm, 1985; Threfall and Garland, 1985; Reisman, 1988). Diaphragms made out of high temperature butyl rubber are advantageous because of their long life expectancy under most operating conditions, including liquids containing solids and abrasives. Ball valves are also used while butterfly valves are rarely used. Globe and gate valves are
18
M. C. SHARMA AND A. K. GURTU
considered unsuitable because of internal crevices and the inherent lack of cleanability. Valves should be designed and positioned to permit the total drainage of materials. Most of the valves in a bioreactor system must be operated during sterilization and any mistake may lead to contamination. The microprocessor controlled operation of valves in the right sequence, activating all individual valves, is desirable for aseptic processing (K. Gerlach, unpublished communications, 1986). 5. Air System
The production of sterile air is currently based on “depth” or membrane types of filters. With depth filters, the design is very crucial (Aiba et al., 1986). Long staple glass fibers packed in pressure vessels were initially used, and prepacked cartridges (Perkowski, 1983) are now available in a wide range of sizes for this purpose. Generally, glass fibers with a diameter less than 10 p m are packed to a minimum density of 180 kg/m3. A packing density of 300 kg/m3 is achieved by high compaction under wetted conditions. Incorporation of a built-in mechanical bed compaction device in the filter should take care of the sagging packed bed without opening the lid. The filter bed should be perfectly dried out before being used as an air supply to a sterilized bioreactor. Excessive heating with a steam jacket in packed regions should be avoided to save glass fibers from fragmentation and the consequent reduction of filter efficiency. To maintain an elevated air temperature, air should be passed through a heater prior to entry into the filter in order to inactivate the microorganisms trapped in the filter bed while effectively drying the packing. However, this requires increased energy consumption for heating and the subsequent cooling of air to make it acceptable for the bioprocess. The relevance of high air temperature may be judged from the fact that for every cubic meter per hour of influent air flow, approximately 66 million microbes can be expected to challenge the filter annually, even with rather clean air (1700 organisms/m3 of air). Membrane filters have increased acceptance, and membranes made out of PTFE and polyvinyl difluoride, incorporating 0.1 pm pore size, repeated sterilizability, and high void volumes (80%), permit air flow with low pressure drops (Smith, 1981; Stanbury and Whitaker, 1984). Polyhexamethylene adipamide, polyamide, cellulose nitrate, cellulose acetate, and regenerated cellulose membranes are also used. The superiority of a membrane filter system and criteria for selecting fermentation air filters have been reviewed extensively by Leahy and Gabler (1984), Conway (1984, 1985), and Hambleton et al. (1991). The advantages described also include in situ validation of the integrity of filters by a
ASEPSIS IN BIOREACTORS
19
forward flow system. Such a validation is impossible in the case of depth filters, and hence a nagging doubt persists about the depth filter being the cause of nonsterility. Provision of moisture and oil-removing prefilters enhances the performance of membrane filters. Special attention needs to be paid to mechanical damages to the 0 rings and the membranes during cleaning/assembling. Strict adherence to the prescribed norms and limits of thermal and pressure differentials is helpful in maintaining integrity and a long operational life of sterile membrane filters. Even the steam used for their sterilization should be filtered. It is essential that each bioreactor has an individual sterile filter on its air supply line. A common storage tank for a sterile feed to many bioreactors should preferably have two membrane air filters in series as a precautionary measure. 6. Liquid Transfer System
When transferring liquids from one bioreactor to the other, sterilization of intervening piping, prior to passage of liquid, is invariably done by steam. Short flexible pipes with sanitary quick connecting ends/ valves may be used after autoclaving or in situ sterilization. The application of stericonnectors for the sampling and transfer of liquids has been emphasized by Heden (1958) and Steel and Miller (1970). Tolbart and Feder (1982) described an air-shielded quick connecting system for the sampling and transfer of liquids for aseptic processes. In fixed piping systems, steam supply and condensate trappings are a must on every section of sterile operation lines. Separate sections of the plant, with a double block protection, should be sterilizable without interfering with other ongoing operations. Lines with double/triple valves and steam bleeding are necessary for the maintenance of sterile zones at desired places (Bull et a]., 1983). Sterilizable diaphragm metering pumps are recommended for feed transfers instead of glass tube rotameters. Various devices have been suggestedhsed for the aseptic transfer of inoculum from the laboratory to the seed culture reactor as well as from one bioreactor to another. These transfers have to be made while maintaining a differential in positive pressure in the donor and recipient vessels. The inoculation port should be equipped with a steam supply. Some of these systems have been described by Parker (1950), Jackson (1958), Steel and Miller (1970), and Meyrath and Suchanek (1972). For laboratory inoculum transfer, the system could have a “pressure flask” with a side nozzle fitted with silicone tubing with a needle fixed at the distal end. The transfer of inoculum is aided by a peristaltic pump, after the sterile needle pierces the presterilized silicone/rubber diaphragm on the inoculation port of the seed bioreactor. Another possibility could
20
M. C. SHARMA AND A. K. GURTU
be to use a metallic pot, filled with inoculum in sterile room, clamped onto a vertical steam cross system on the bioreactor, and sterilized at the junction by steam with transfer effected through a pressure differential manipulated in the pot and bioreactor. Any inoculation procedure requiring total depressurization of the bioreactor is not advisable. The pooling of inoculum out of many containers at any stage should be avoided as much as possible. For continuously fed industrial bioreactors, the considerations of investment, space, energy, and process economics determine the setup of feed systems and the choice between a dedicated/common feed tank or a dedicated point of use continuous sterilizer for each feed. Because of feeds, the dedicated feed tank is ideal for containing large-scale outbreaks of nonsterility. When in use, concentrated acid and alkali solutions and anhydrous ammonia gas would normally be considered self-sterilizing. However, the air supply to the acid and alkali tanks must be filtered via a membrane filter, the feed line to the bioreactor must hold the acidlalkali for a certain amount of time prior to the addition, and the stream should remain unbroken during a fermentation run (Hambleton et al., 1991). Many of the aforementioned concepts necessary for aseptic operations of bioreactors have been incorporated in Fig. 4 . F. MAINTENANCE OF ASEPSIS DURING FERMENTATION The maintenance of asepsis is necessary throughout the complete process cycle. During the cool down from the sterilization temperature, the systems should be pressurized with sterile air to avoid pulling a vacuum and drawing in contaminating organisms. Positive pressures have to be maintained throughout all processes (Stanbury and Whitaker, 1984; Bjurstrom, 1985;Reisman, 1988).Air pressure fluctuations should be minimized to avoid the back flow of nutrients into the air filters. The availability of a standby automatic changeover, captive power generation unit, compressed air buffer reservoir, and the automatic closure of air inlet and outlet valves at a preset air flow/pressure fall, could help in avoiding the depressurization of bioreactors. The use of a nonreturn valve on the air line is also helpful. Special attention needs to be paid in eliminating thermal and pressure differential shocks to membrane filters through check valves and proper operations. Effective foam control through foam breakers (Hall et al., 1973; Viesturs et al., 1982), foam probes, and antifoam additions help in aseptic fermentations. A new breed of foam sensors for accurate foam level detection and control has been developed with corrosion-resistant construction material without ridges or crevices to prevent the growth of organisms on the probe
ASEPSIS IN BIOREACTORS 20
8
21
8
17
t:
9
11
24
FIG.4. A state of the art bioreactor system with asepsis concepts. (1)Bioreactor with SS 316 L material, electropolished internal finish, internal fittings with gaps, and total drain concept. (2) Manhole with steam seal. (3) Viewglass with steam seal. (4) Double mechanical agitator seal, steam lubricated, with level indicator. (5) Steam filter. (6) Air filter. (7) Inoculum vessel. (8) Steam inlet. (9) Steam/condensate outlet. (10)Steam trap. (11) Air from prefilter. (12) Inverted “U” loop on air supply line. (13) Inverted “U” loop on air exhaust line. (14) Steam heated jacket. (15) Exhaust air filter bypass. (16) Vessel pressure controller. (17) Air exhaust. (18) Pressure gauge. (19) Quick connecting aseptic coupling. (20) Inlets for medium, feed, and antifoam. (21) To cleanable, selfdraining sparger. (22) Flush bottom valve. (23) To harvest. (24) Drain. (25) Sampling point. (26) Filter cover for sampling point. (27) Limpet coil/jacket. (28) Exhaust cooler condenser.
(Russell and O’Hare, 1991). The choice of antifoams is a subject in itself (Solomons, 1967; Ghildyal et a]., 1988). The maintenance of aseptic transfer lines is a process needing care and attention. The sequencing of valve operations to ensure the bleeding off of the condensate and the heating up each portion to sterilization
22
M. C. SHARMA AND A. K. GURTU
temperature is necessary. The use of “Tempilistiks” (Soderberg, 1983) or thermocouples, color-changing adhesive tapes or Browne’s tubes (Threfall and Garland, 1985) is recommended to ensure proper sterilization. The use of a dedicated steam trap on every sterile process line is necessary to build up pressure. A common steam trap serving several pipe lines reduces sterilizing efficiency (Bull et a]., 1983) and results in incapacitating a number of lines, even if only a single component fails. Once the transfer from a donor vessel is finished, the pipe line must be steam sterilized prior to receiving the contents of another donor. G. PROTECTED FERMENTATION
The application of antimicrobial substances for the protection of fermentations has been in practice since 1923, particularly in manufacturing alcohol, yeast, and antifungal antibiotics (Hayduck, 1923). Herold and Necasek (1959) have written a comprehensive review on “Protected Fermentation.” Bisulfites, formic acid/formaldehyde, boric acid, picric acid, pentachlorophenol (Underkoffler and Hickey, 1954), and antibiotics like polymyxin, penicillin, and chlorotetracycline (Strandskov and Bockelmann, 1953; Day et a]., 1954; Borzani, 1956; Borzani and Aquarone, 1957; Herold and Necasek, 1959) have been used as antimicrobials in maintaining asepsis in beer/alcohol fermentations. The addition of neomycin for prophylaxis in fermentations of nonantibacterial products was suggested by Bull et aI. (1983). The present day animal cell cultures are usually protected by “antimicrobials’’ (Arathoon and Birch, 1986). Perlman (1979) reviewed the use of antibiotics in cell culture media and recommended using penicillin/ streptomycin, chloramphenicol, or tetracycline against bacterial contaminants; gentamicin or tylosin against mycoplasma; and amphotericin B against yeasts. Lambert and Birch (1985) advocated the use of penicillin, gentamicin, amphotericin B, or nystatin for contamination control in cell growth media. Despite the threat of development of antimicrobial-resistant contaminants, the protected fermentations are currently in use. As it may be unethical for surgeons to disregard aseptic techniques in surgery, it would be unprofessional for biotechnologists to cover the inefficiencies of techniques and equipments by protecting the fermentation process with antimicrobials (Herold and Necasek, 1959). However, the use of antimicrobials in lowering the cost of production, simplifying equipment and maintenance, or salvaging a contaminated batch may at times become necessary. Bull et al. (1983) consider such prophylactic measures as a last resort and regard them as poor substitutes for proper equipment maintenance and operating practices.
ASEPSIS IN BIOREACTORS
23
H. PRODUCT CHANGEOVER In the event of a “wave” or siege of contamination in the production unit, the advisable recourse, pending investigational findings, is to temporarily switch over to the production of any other fermentation product, if available, or to substitute the strain with an immunehesistant strain, especially in case of a phage spread over (Soderberg, 1983).There have been cases of stoppages of production in the case of single product plants whenever such contamination “waves” appeared.
I. SCHEDULES AND PROCEDURES Routine and preventive maintenance of vessels and systems plays a crucial role in asepsis. Checklists for equipment elements to be inspected after every harvest should be maintained and adhered to (Reisman, 1988). Separate checklists for weekly/monthly inspections also should be made. A third checklist should include a thorough internal inspection of the bioreactor during downtime for preventive maintenance (once or twice a year). A special checklist has to be made for a postcontamination check. A record of the checks made and adjustmentirectification carried out helps in correcting measures in the future. Leak tests are generally performed during preventive maintenance or if the contamination frequency is high. A fixed schedule for the hydraulic pressure testing of the vessel, jacket, and coil should be adhered to irrespective of the sterility status in the past. The use of an iodine solution with a starch indicator for detecting leaks in cooling coils may prove useful. Perkowski et al. (1984) recommend the use of a popping sound generated by a patented liquid leak amplifying chemical in conjunction with ultrasonic waves in detecting microscopic leaks in cooling coils that could not be detected even with a halogen leak detector after Freon-12 pressurization in coils. Radiographic detections have been described as very reliable in testing leaks. Biological tracers (Bacillus subtilis var. niger spores) have been recommended by Hambleton et al. (1991) for the microbiological assessment of the integrity of fermentors and their components. Nutrient feed lines should be checked for hermeticity prior to each sterilization. Control instruments validation and calibration for pressure and temperature should also be included in the maintenance schedule. The inspection of a bioreactor should cover the shell, dome, agitator seal, air exhaust line, nozzles, sight glasses, 0 rings, valves (inputs and effluents), air filters, probes, shaft, keyways, impellers, hubs, hangers, brackets, coils, sparger, ladder, thermowells, dip pipes, inoculum header, feed lines, and sampling line (Reisman, 1988).
24
M. C. SHARMA AND A. K. GURTU
The use of a high pressure water jet during regular postharvest cleaning operations should ensure a thorough cleaning and removal of undesired materials (Reisman, 1988). Vessels should be boiled with a 5% alkali solution and the feedlines and air exhaust system should be cleaned with a hot alkali solution at regular intervals. In case of severe contamination, checks and cleaning should be rigorous. Leakages, however minor, should not be overlooked. No transfer line should be in use for repeated aseptic transfers for prolonged periods without steaming in between for sterilization. The good housekeeping of fermentation areas and proper disinfection of contaminated batches, prior to discharge, helps in the maintenance of asepsis in bioreactors. Initiating and adhering to standard operating procedures, training personnel (Soderberg, 1983),rigorous checking, following proper procedures, obtaining feedback on observations, ideas, and errors, and communicating at regular/informal technical discussions are essential for good operating practices. The logging of data, deviations, and infrequent observations do forewarn and indicate the areas for corrective/remedial measures. VI. Overcautious Approaches
Driven by the fear of adverse economic consequences of nonsterility in the bioreactor, the fermentation industry often tends to adopt rather overcautious approaches. Some such practices include: 1. Sterilization of emptying vessel prior to batch sterilization 2. Incorporating chemical disinfectants like formaldehyde during
empty vessel sterilization by steam 3. Prolonged maintenance of medium at around 100°Cprior to raising to sterilization temperatures 4. In situ batch sterilization of insoluble medium ingredients coupled with external continuous sterilization of soluble components 5 . Exceeding the classical “121°C for 30 min” sterilization regarding temperature and/or time in case of equipment, piping, air filter, and thermostable feeds for the process 6. Providing a standby fiber-packed filter and switch over before the designed filter span 7. Prolonging drying of the fiber-packed air filter 8. Maintaining a high air temperature even when using a validated membrane air filter 9. Using an air sampler or bubbler on the sterile air system for detecting contaminants, despite knowing that the sample tested is a very minute part of the total air supplied to the bioreactor (Soderberg, 1983)
ASEPSIS IN BIOREACTORS
25
10. Using laminar air flow benches inside sterile rooms/chambers for microbiological handlings.
It is a matter of debate whether the practice of such techniques is necessary, desirable, or avoidable. VII. Conclusion
Asepsis in a bioreactor can be achieved through integrated efforts on system design, materials and layout, effective validation and operating procedures, scheduled checks and maintenance, and trained motivated personnel. The effectiveness of these efforts depends on the stringent adherence to schedules and procedures and on the limits of sensitivity of sterility assessment methodology. Every effort made on the design, operation, and maintenance of a bioreactor system for minimizing opportunities of invasion by unwanted microbes is worth the trouble. REFERENCES Aiba, S., Humphrey, A. E., and Millis, N. F. (1986). “Biochemical Engineering.” Academic Press, New York. Anonymous (1990). “Process Filtration News,” Biopharm Edition. Domnick Hunter Filters Ltd., Durham, England. Arathoon, W. R., and Birch, J. R. (1986). Science 232, 1390-1395. Augurt, T. A. (1983). Kirk-Othmer Encycl. Chem. Technol. 3rd Ed. 21,626-644. Aunstrup, K., Andresen, O., Falch, E. A., and Nielsen, T. K. (1979). In “Microbial Technology” (H. J. Pepler and D. Perlman, eds.), 2nd ed., Vol. 1, pp. 282-309. Academic Press, New York. Bader, F. G., Boekeloo, M. K., Graham, H. E., and Cagle, J. W. (1984). Biotechnol. Bioeng. 26, 848-856. Bailey, J. E., and Ollis, P. R. (1986). “Biochemical Engineering Fundamentals.” McGrawHill International Edition, Singapore. Banks, G. T. (1979). Top. Enzyme Ferment. Biotechnol. 3, 170-266. Benarde, M. A. (1970). “Disinfection.” Dekker, New York. Bjurstrom, E. E. (1985). Chem. Eng., (N.Y.) Feb. 18, pp. 126-158. Borick, P. M. (1968). Adv. Appl. Microbiol. 10,291-312. Borzani, W.(1956). Bol. Dep. Quim. Esc. Politec., Univ. Sao Paulo 2, 1-4. and Aquarone, E. (1957). J. Agric. Food Chem 5, 612-616. Borzani, W., Bull, D. N., Thoma, R. W., and Stinner, T. E. (1983). Adv. Biotechnol. Proc. 1, 1-30. Cameron, J., and Godfrey, E. I. (1969). Biotechnol. Bioeng. 11, 957-985. Chain, E. B., Paladino, S., Ugolini, F., Callow, D. S., and Van der Sluis, J. (1954). Rend. 1st. Super. Sanita (Engl. Ed.) 17, 61-86. Charton, R. (1990). Am. Biotechnol. Lab. Dec., p. 60. Conway, R. S. (1984). Biotechnol. Bioeng. 26, 844-847. Conway, R. S. (1985). In “Comprehensive Biotechnology” (M. Moo-Young, ed.), Val. 2, pp. 279-286. Pergamon, Oxford and New York. Cooney, C. L. (1985). In “Comprehensive Biotechnology” (M. Moo-Young, ed.), Vol. 2, pp. 287-298. Pergamon, Oxford and New York.
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Day, W. H., Serjak, W. C., Stratton, J. R., and Stone, L. (1954).J. Agric. Food Chern. 2, 252-258.
Difco Laboratories (1985). “Difco Manual,” lo th ed. Difco Laboratories Inc., Detroit, MI. Elander, R. P. (1989).In “Genetic Engineering Technology in Industrial Pharmacy: Principles and Application” (J. M. Tabor, ed.). pp. 115-129. Dekker, New York. Elmroth, I., Valeur, A., Odham, G., and Larsson, L. (1990). Biotechnol. Bioeng. 35, 787-792.
Elsworth, R. (1960). Prog. Ind. Microbiol. 2, 103-130. Finch, W. I. (1958). “Disinfectants, Their Values and Uses.” Chapman & Hall, London. Ghildyal, N. P., Lonsane, 8 . K., and Karanth, N. G. (1988). Adv. Appl. Microbiol. 33, 173-222.
Ghoul, M., Ronet, E., and Engasser, 3. (1986). Biotechnol. Bioeng. 28, 119-121. Hall, M. J., Dickinson, S. D., Pritchard, R., and Evans, J. I. (1973). Prog. Ind. Microbiol. 12,169-234.
Hambleton, P., Griffiths, J. B., Cameroon, D. R., and Melling, J. (1991). J. Chern. Tech. Biotechnol. 50, 167-180. Hastings, J. J. H. (1978).In “Economic Microbiology” (A. H. Rose, ed.), Vol. 2, pp. 31-45. Academic Press, London. Hayduck, F. (1923). U.S. Pat. 1,449,112. Heatley, N. G. (1950).J. Gen. Microbiol. 4, 410-412. Heden, C. G. (1958). Nord. Med. 7, 1090. Herold, M., and Necasek, J. (1959). Adv. Appl. Microbiol. 1, 1-21. Jackson, T. (1958). In “Biochemical Engineering” (R. Steel, ed.), pp. 183-222. Heywood, London. Knight, P. (1989). Bio/Technology 7(5), 459-461. Lambert, K. J., and Birch, J. R. (1985).In “Animal Cell Biotechnology” (R. E. Spier and J. B. Griffiths, eds.), Vol. 1, pp. 85-122. Academic Press, London. Leahy, T. J., and Gabler, R. (1984). Biotechnol. Bioeng. 26, 836-843. Liberman, D.I., Fink, R., and Shalfer, F. (1986). In “Manual of Industrial Microbiology and Biotechnology” (A. L. Demain and N. A. Solomons, eds.), pp. 402-409. Am. SOC.Microbiol., Washington DC. Lubiniecki, A. S., and May, L. H. (1985). Dev. Biol. Stand. 60, 141-146. Lundell, R., and Laiho, P. (1976). Process Biochem. 11, 13-17. Maurer, H. R. (1986). In “Animal Cell Culture: A Practical Approach” (R. I. Freshner, ed.), pp. 13-31. I.R.L. Press, Oxford and Washington, DC. McLaughin, J., Bruno, C. F., and Forrest, T. (1983). Biotechnol. Bioeng. 25, 1229-1236. Melling, J . , and Allner, V. (1981). In “Essays in Applied Microbiology” ( J . R. Norris and M. H. Richmond, eds.), Chapter 11, p. 1. Wiley, New York. Meyrath, J. and Suchanek, G. (1972). Methods Microbiol. 7 , 159-209. Parker, A. (1950).“Recent Advances in Fermentation Industry.” Royal Institute of Chemistry, London. Perkowski, C. A. (1983). Biotechnol. Bioeng. 25, 1215-1222. Perkowski, C. A., Daransky, G. R., and Williams, J. (1984). Biotechnol. Bioeng. 26, 857-859.
Perlman, D. (1950). Bot. Rev. 16, 449-523. Perlman, D. (1979). In “Methods in Enzymology” (W. B. Jakoby and I. H. Pastan, eds.), Vol. 58, pp. 110-116. Academic Press, New York. Reisrnan, H. B. (1988). “Economic Analysis of Fermentation Processes.” CRC Press, Boca Raton, FL. Rhodes, A., and Fletcher, D. L. (3966).“Principles of Industrial Microbiology.” Pergamon, Oxford and New York.
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Richards, J. W. (1966). Process Biochem. 1(1), 41-46. Richards, J. W. (1968).“Introduction to Industrial Sterilization.” Academic Press, London. Russell, M., and O’Hare, D. (1991). Am. Biotechnol. Lab. July, p. 30. Saudek, E. C. (1956). Bacteriof Rev. 20, 279-281. Scheirer, W. (1987). Trends Biotechnol. 5(9), 261-265. Seifert, G. K. E., and Mattaeu, P. (1988). Biotechnof. Bioeng. 32, 923-926. Sittig, W. (1982).J. Chem. Tech. Biotechnol. 32, 50. Smith, S. R. L. (1980). Philos./Trans. A. SOC.London, Ser. B 290, 341-354. Smith, S. R. L. (1981). In “Microbial Growth in C1 Compounds” (H. Dalton, ed.), pp. 342-348. Heyden, London. Soderberg, A. C. (1983). In “Fermentation and Biochemical Engineering Hand Book” (H. C. Vogel, ed.), pp. 111-117. Noyes, Data Corp., Park Ridge, NJ. Solomons, G. L. (1967). Process Biochem. 2, 47-48. Solomons, G. L. (1969). “Materials and Methods in Fermentation.” Academic Press, New York. Solomons, G. L. (1971). Adv. Appl. Microbiol. 14, 231-248. Spier, R. (1988). Trends Biotechnol. 6, 2-6. Stanbury, P. F., and Whitaker, A. (1984).“Principles of Fermentation Technology.” Pergamon, New York. Steel, R., and Miller, T. L. (1970). Adv. Appl. Microbiol. 12, 153-188. Strandskov, F. B., and Bockelmann, J. B. (1953). J, Agric./Food Chem. 1, 1219-1223. Stumbo, R. (1976). In “Industrial Microbiology” (B. M. Miller and W. Litsky, eds.), pp. 412-450. McGraw-Hill, New York. Sykes, G. (1958). “Disinfection and Sterilization.” Spon, London. Threfall, G., and Garland, S. G. (1985). In “Animal Cell Biotechnology” (R. E. Spier and J. B. Griffiths, eds.), Vol. 1, pp. 123-140. Academic Press, London. Tolbart, W. R., and Feder, J. (1982). Biotechnol. Bioeng. 24, 1885-1887. Underkoffler L. A., and Hickey, R. J. (1954). “Industrial Fermentation,” Vols. 1 and 2. Chemical Publications, New York. U.S. Department of Health and Human Services (1984). “Biosafety in Microbiological and Biomedical Laboratories.” HHS Publication, Washington, DC. U.S. Pharmacopoeia (1980). XX Revision, NF XV. USP Convention Inc., Rockville, MD. Van Brunt, J. (1987). Bio/Technology 5(11), 1133-1138. Viesturs, U. E., Kristapsons, M. Z., and Levitans, E. S. (1982). Adv. Biochem. Eng. 21, 169-224.
Wallhauser, K. H. (1985). In “Biotechnology” (H. J. Rehm and G. Reed, eds.), Vol. 2, pp. 699-724. Verlag Chemie, Weinheim. Wilkinson, G. R., and Baker, C. L. (1964). Prog. Ind. Microbiol. 5, 237-283.
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Lipids of n-Al kane-Utilizing Microorganisms and Their Application Potential
s.RADWAN’” AND NASERA. SORKHOHt * Institut fur Mikrobiologie, Universitat Munster,
SAMIR
D-4400Munster, Germany f
Department of Botany and Microbiology, Faculty of Science, University of Kuwait, safat 13060, Kuwait
I. Introduction 11. n-Alkane-Utilizing Microorganisms
111. Total Lipid Contents A. Lipids and Fats B. Basic Studies C. Biotechnological Considerations IV. Fatty Acids A. Basic Studies B. Biotechnological Considerations V. Acylglycerols VI. Sterols VII. Fatty Alcohols, Ketones, and Epoxides VIII. Hydrocarbons and Waxes IX. Phospholipids X. Glycolipids and Peptidolipids XI. Biolipid Extract XII. Environmental Considerations References
I. Introduction
Until the beginning of the 1970s the results of many studies on hydrocarbon-utilizing microorganisms were covered by patents. Such studies comprised the isolation of microorganisms, oxidation and hydroxylation of hydrocarbons, biomass production, biosynthesis of proteins, carbohydrates, “fats,” vitamins, enzymes, nucleotides, antibiotics, and organic acids, as well as recovery procedures for these products. During the past two decades the number of original studies published in scientific journals continuously increased, and it now exceeds by far
’
Present address: Department of Botany and Microbiology, Faculty of Science, University of Kuwait, Safat 13060,Kuwait. 29 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 39 Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
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SAMIR S. RADWAN AND NASER A. SORKHOH
the number of patents. As far as the history of studies on lipids of hydrocarbon-utilizing microorganisms is concerned, a parallel development occurred. In the beginning of the 1970s there were numerous patents covering results on “fat” production by oil-utilizing yeasts. Within the past 25 years much original research work has been published on the lipids of microorganisms that utilize hydrocarbons, especially n-alkanes, as substrates; a review of these studies appears to be timely. Earlier reviews on the microbial degradation of hydrocarbons (Ratledge, 1978, 1980; Rehm and Reiff, 1981; Fukui and Tanaka, 1981; Biihler and Schindler, 1984) and on lipids of oleaginous microorganisms (Boulton and Ratledge, 1984; Ratledge, 1986) devoted parts of their discussion to lipids and/or fatty acids of n-alkane-utilizing microorganisms. This article presents a comprehensive review of this subject, referring to the potential commercial values of various lipid classes and fatty acids. In addition, reference is made to environmental considerations associated with the proposed application of microorganisms in controlling oil pollution and in enhanced oil recovery. During many of such processes microorganisms liberate surfactive lipids, whose impact on the environment is not known, so far. That lipids of n-alkaline-utilizing microorganisms should be expected to differ from lipids of the same organisms grown on conventional substrates is apparent from the following arguments. 1. n-Alkanes, being water insoluble, expectedly induce in cell membranes alterations that allow for their enhanced active transport. Such alterations may involve the membrane lipids which contribute to about 50% of the membrane weight. 2. Alkanes, themselves lipids, are taken up, chemically unchanged, and thus directly contribute to the total cell lipids. 3. Initial phases of n-alkane metabolism involve oxidation of these substrates to fatty alcohols and fatty acids which become, in part, incorporated into complex cell lipid compounds. 4. Several n-alkane-utilizing microorganisms reveal cytological entities that are associated with their growth on n-alkanes as substrates. Thus, certain bacteria produce intracytoplasmic membranes, and yeasts produce peroxisomes. Like other biological membranes and organelles, these cytological entities are expected to be rich in lipids. I I. n-Alkane-Uti lizi ng Microorganisms
This subject has been repeatedly reviewed (Klug and Markovetz, 1971; Levi et a ] . , 1979; Einsele, 1983) along with the physiology of
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n-alkane-utilizing microorganisms and alkane metabolism (Rehm and Reiff, 1981; Fukui and Tanaka, 1981; Boulton and Ratledge, 1984). The most interesting microbial genera capable of utilizing n-alkanes as sole sources of carbon and energy are listed in Table I. This list, which may grow in the future, includes microorganisms reported in earlier reviews together with additional members reported in original research publications. It is apparent that the utilization of n-alkanes as substrates is a widely distributed activity among microorganisms. This activity is achieved by both prokaryotes and eukaryotes, including mainly organotrophs and a few photoautotrophs. Although Table I comprises a relatively large number of genera, most studies in the literature have been done on TABLE I MICROBIAL GENERACONTAINING HYDROCARBON-UTILIZING SPECIES OR STRAINS' Prokaryotes Photoautotrophic: Rhodospirillum, Rhodopseudomonas, Oscillatoria [Cerniglia et al. (1980a)l Organotrophic cocci: Acinetobacter, Micrococcus, Sarcina Curved rods: Vibrio, Azospirillum [Roy et al. (1988)] Gram-negative rods: Aeromonas, Alcaligenes, Chromobacterium, Flavobacterium, Klebsiella, Pseudomonas [Klug and Markovetz (1971)l Gram-positive rods: Bacillus [Loginova et al. (1981)], Bacillus stearothermophilus [Sorkhoh et al. (1993)j Actinomycetes and related organisms: Arthrobacter, Brevibacterium, Corynebacterium, Rhodococcus [Egorov et 01. (1986)], Mycobacterium, Actinomyces, Nocardia, Streptomyces Eukaryotes Photoautotrophic: Chlorella [Schroeder and Rehm (198l)], Scenedesmus [Schroeder and Rehm (1981)l Organotrophic: Yeasts: Candida, Debaryomyces, Endomyces, Leucosporidium, Lodderornyces, Metschnikowia, Pichia, Rhodosporidium, Rhodotorula, Saccharomycopsis, Schwannio-myces, Selenotila, Sporidiobalus, Sporobolomyces, Torulopsis, Trichosporon, Wingea Filamentous fungi: Absidia [Hoffmann and Rehm (1978)], Aspergillus, Aureobasidium, Beauveria [Davies and Westlake (1979)], Botrytis, Cephalosporium, Cladosporium, Corellospora [Kirk and Gordon (1988)], Cunninghamella, Dendyphiella [Kirk and Gordon (1988)], Fusarium, Hormodendrum [Lin et al. (1971a,b)] Lulworthia [Kirk and Gordon (1988)], Mortierella, Mucor, Penicillium, Phialophora, Phoma [Davies and Westlake (1979)], Scedosporium [Ornodera et al. (1989)], Scoleobasidium [Davies and Westlake (1979)], Sporotrichum. Varicosporino [Kirk and Gordon (1988)], Verticillium a
Unless otherwise specified, the information is according to Levi et al. (1979)
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SAMIR S. RADWAN AND NASER A. SORKHOH
much fewer ones. Thus the prokaryotic genera most frequently studied include Acinetobacter, Pseudomonas, and the actinomycetes Arthrobacter, Corynebacterium, Rhodococcus, Mycobacterium, and Nocardia. In this context, a problem related to the nomenclature should be mentioned. Thus, for example, Acinetobacter used to be called Micrococcus cerificans in earlier studies (see Makula and Finnerty, 1970). Similarly, many of the Nocardia species in the Eighth Edition of “Bergey’s Manual of Determinative Bacteriology” are classified in the more recent first edition of “Bergey’s Manual of Systematic Bacteriology,” as Rhodococcus species (see Nakajima and Sato, 1983). Koronelli (1988) used the term “saprophytic mycobacteria” for Rhodococcus, Corynebacterium, and related genera. In view of the striking morphological similarity among such actinomycetes (Sorkhoh et al., 1990a),misleading identities of these organisms should be expected, especially in very early publications. In the genus Bacillus, only B. stearothermophilus has been reported to utilize n-alkanes (Loginova et al., 1981); other reports (Kachholz and Rehm, 1978) were not confirmatory. It appears, however, that the ability to utilize n-alkanes as sole sources of carbon and energy is lacking among mesophilic Bacillus species (Kvasnikov et al., 1973; Kachholz and Rehm, 1977). The yeast genus most frequently studied is Candida; fewer studies have been done using Lodderomyces, Rhodotorula, and Torulopsis. Among the filamentous fungi, Aspergillus, Cladosporium, Cunninghamella, Fusarium, and Penicillium received most of the researcher interest. In this context, it is noted that Lindley and Heydeman (1985) emphasized the importance of an extended lag phase when assessing substrate optima for alkane utilization by filamentous fungi. Reportedly, failure to take the progressively longer lag phase (in respect to carbonchain length) into consideration may have led workers, who used single point biomass measurements as an indicator of growth, to underestimate the potential of fungi to grow on alkanes such as octadecane. Of course, none of the microorganisms are capable of utilizing all nalkanes; each organism can utilize only a certain range of compounds. But collectively, all compounds from the gaseous low molecular weight (van Ginkel et al., 1987; Ornodera et al., 1989) up to the medium and high molecular weight constituents (Demanova et al., 1980b) can be attacked by microorganisms. This activity is maintained during immobilization of both unicellular (El-Aassar et al., 1988) and filamentous (Heinrich and Rehm, 1981) microorganisms. Among the interesting alkane-utilizing microorganisms are the thermophiles Thermus ruber (Loginova et al., 1981), Thermoleophilum album (Zarilla and Perry, 1984) and Bacillus stearothermophilus (Sorkhoh et al., 1993), and the nitrogen-fixing Azospirillum sp. (Roy et al., 1988).
n-ALKANE-UTILIZING MICROORGANISMS
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Mixtures of n-alkane are much more efficiently oxidized via the activities of microbial associations than in pure cultures. This has been demonstrated experimentally using an association of Pseudomonas and Mycobacterium (Koronelli et al., 1984). Particularly interesting is the ability of the cyanobacterium Oscillatoria sp. and the green algae Chlorella vulgaris and Scenedesmus obliquus to utilize n-alkanes as sole sources of carbon and energy (Cerniglia et al., 1980a,b; Schroeder and Rehm, 1981; Zawdzki and Langowska, 1983). Oscillatoria sp., diatoms, as well as green, red, and brown algae can also oxidize naphthalene to at least six metabolites including 1-naphthol. The degradation of hydrocarbons by associations of cyanobacteria and organotrophic bacteria has been documented (Gusev et al., 1981, 1982; Sorkhoh et al., 1992).
Microorganisms may exhibit, during n-alkane utilization, characteristic morphological and cytological features (see Table 11). One of these features is hydrocarbon inclusions. The production of intracytoplasmic inclusions by oil-utilizing microorganisms has been documented by early investigators working on bacteria (Scott and Finnerty, 1966; Atlas and Heintz, 1973; Kennedy and Finnerty, 1975a) and filamentous fungi (Cundell et al., 1976; Koval and Redchitz, 1978: Redchitz, 1980). These inclusions may occupy up to 40% of the total cell volume (Griffin and Traxier, 1983). On conventional carbon sources these inclusions are absent in most cases or, in a few cases, have only a very minute size. Another characteristic is intraplasmic membranes. Such membrane systems have been observed in alkane-utilizing bacteria, viz. Acinetobacter sp. (Kennedy and Finnerty, 1975b) and Rhodococcus rhodochrous (Ivshina et al., 1982).Volutin inclusions have been observed in the hyphae of Aspergillus spp. (Redchitz and Koval, 1979) grown in the presence of hydrocarbons and Rhodococcus rhodochrous incubated in an atmosphere of propane (Ivshina et al., 1982). In addition, Penicillium sp. grows in shaken cultures in media containing n-hexadecane as hollow mycelial balls enclosing hydrocarbon droplets, whereas the mycelial balls were solid in media containing peptone as substrate (Cundell et al., 1976). Alkane-grown cells, but not glucose-grown cells of Candida tropicalis possess a mannan-fatty acid complex on their surfaces (Kappeli et al., 1978). Also, alkane-grown cells show a radial arrangement of the wall polymers, with protruding parts, in contrast to the smooth surfaces of glucose-grown cells. The authors believe that the mannan-fatty acid complex may be involved in the cell binding to alkanes. Omar and Rehm (1980) noticed that n-tetradecane- and npentadecane-grown cells of Candida parapsilosis produce much more pseudomycelia and show much higher catalase activity than glucosegrown cells. According to Fukui and Tanaka (1981) the conspicuous
SAMIR S. RADWAN AND NASER A. SORKHOH
34
TABLE I1 CYTOLOGICAL CHANGES INDUCEDDURING MICROBIAL GROWTHON HYDROCARBONS Microorganism Acinetobacter sp.
Acinetobacter calcoaceticus Arthrobacter sp.
Substrate Paraffinic hydrocarbons Hexadecane or hexadecene Hexadecane Hexadecane
Flavobacteriurn, Brevi bacterium
Crude oil
Rhodococcus rhodochrous
Propane
Candida lipolytica
Hexadecane
Candida tropicalis
n-Alkanes
Aspergillus oryzae, Aliphatic A. effusis, A. hydrocarbons ochraceus, A. sydowi, A. niger Cladosporium Hydrocarbons resin ae
Penicillium sp.
n-Hexane
Cytological changes
Reference
Intracytoplasmic hydrocarbon inclusions Intracytoplasmic membrane Thin fimbriae
Kennedy and Finnerty (1975a); Scott and Finnerty (1966) Kennedy and Finnerty (197 5b) Rosenberget al. (1982)
Intracytoplasmic hydrocarbon inclusions Intracytoplasmic hydrocarbon inclusions Volutin, hydrocarbon inclusions, intracellular membrane system Complex membrane and vesicles on the outer cell surface, peroxisomes Protruding parts on the outer cell surface, peroxisomes Fatty inclusions, volutin inclusions
Griffin and Traxier (1983)
Thinner walls, vacuoles, microbodies, increased catalase Grows as hollow balls enclosing hydrocarbon, hydrocarbon inclusions
Atlas and Heintz (1973) Ivshina et al. (1982)
Gutevskaya and Shishkanova (1982)
Kappeli et al. (1978); Fukui and Tanaka (1981) Koval and Redchitz (1978); Redchitz and Koval (1979); Redchitz (1980) Smucker and Cooney (1981)
Cundell et aI. (1976)
appearance of peroxisomes in the cells is one of the specific features of alkane-utilizing yeasts. Smucker and Cooney (1981),investigating the cytological changes in Cladosporium resinae, shifted from a glucose to a hydrocarbon medium and found that the cell walls become thinner (in hyphae and spores), large vacuoles appear, enrichment with micro-
n-ALKANE-UTILIZING MICROORGANISMS
35
bodies occurs and catalase activity increases. Rosenberg et al. (1982) presented evidence that special thin fimbriae on the cell surfaces of Acinetobacter calcoaceticus are the agents that mediate the adherence of this bacterium to hydrocarbon droplets. Reportedly, only strains capable of adhering to hydrocarbon droplets, and consequently of utilizing these compounds, possess such thin fimbriae. Neufeld et al. (1983) observed that cells of Acinetobacter sp. lose their structural integrity when cultivated on hydrocarbon substrates, probably because of extraction of lipophilic surface components by the hydrocarbon. The plasma membrane of hydrocarbon-grown Candida lipolytica becomes thicker and contains deep projections (Ludvik et al., 1968). Ill. Total Lipid Contents
A. LIPIDSAND FATS Sometimes, especially in early publications, the terms “lipids” and “fats” have been used synonymously. It is, however, well known that the term “fats” is the common name of the chemical lipid class of triacylglycerols. In contrast, “lipids” is a collective term which comprises several chemical classes that possess the common property of being soluble in lipophilic solvents. The most common lipid classes in biological materials, including microorganisms, are triacylglycerols, sterols and steryl derivatives, phospholipids, and glycolipids. Triacylglycerols are storage products in the cell, whereas the other classes are constituents of cell membranes and organelles, where they have both structural and physiological functions.
B. BASICSTUDIES Although research on lipids of alkane-utilizing microorganisms dates back to the 1950s and 1960s, most of our current information about this subject has been published in the past two decades. However, many of these publications unfortunately were not concerned with a “classical” analysis of lipids, but were devoted primarily to the fatty acid composition of the total lipids. This is understandable in view of the early recognized fact that n-alkanes are oxidized to fatty acids during their assimilation. Nevertheless, this fatty acid-oriented interest was at the expense of the interest in elementary information about the total lipid contents and lipid composition, which were not as extensively investigated. Table 111 presents the total lipid contents of some alkane-utilizing microorganisms. Whenever available, the lipid contents of the same
36
SAMIR S. RADWAN AND NASER A. SORKHOH TABLE 111 TOTALLIPIDCONTENTS OF ALKANE-UTILIZING MICROORGANISMS" Microorganism
Substrate
n-Hexadecane Ahodococcus rubropertinctus Micrococcus n-Alkanes freu denreich i i Mycobacterium Acetate convolutum M. convolutum M. convolutum M. convolutum M. convolutum M. convolutum M. convolutum M. convolutum C28 Nocardia sp. n-Alkanes Pseudomonas Glucose aeruginosa P. aeruginosa "-Cm Candida 107 n-Alkanes Candida tropicalis Glucose C. tropicalis n-C16 C. tropicalis n-Alkanes C. tropicalis n-Alkanes C. tropicalis n-Alkanes Candida lipolytica Glucose C. lipolytica n-Alkanes C. lipolytica n-Alkanes C. lipolytica n-Alkanes C. lipolytica n-Alkanes Candida rugosa Glucose C. rugosa n-Alkanes Candida Glucose parapsilosis C. parapsilosis CI, Candida maltosa n-Alkanes Mycotorula japonica n-Alkanes Pichia vonriji n-Alkanes Rhodotorula glutinis n-Alkanes Rhodotorula gracilis n-Alkanes Absidia spinosa Glucose A. spinosa c 1 2 A. spinosa c13 A. spinosa n-Paraffin
Total lipids 14.0-22.0
Reference Egorov et al. (1986)
5 .O-12.4
Kvasnikov et al. (1977a)
0.19
Hallas and Vestal (1978)
0.55 0.55 0.49 0.60 0.53
Hallas and Vestal (1978) Hallas and Vestal (1978) Hallas and Vestal (1978) Hallas and Vestal (1978) Hallas and Vestal (1978) Hallas and Vestal (1978) Hallas and Vestal (1978) Raymond and Davies (1960) Koronelli et al. (1982a)
0.49
0.56 56.0 6.7 7.0 26.0 2.8-5.0 6.5-12.5 17.0 6.0 10.0
6.0-8.5 12.0-17.0 27.0 17.0 47.0 5.5-6.5 16.0-19.0 9.4 12.3 11.0-16.5 15.0
Koronelli et al. (1982a) Thorpe and Ratledge (1972) Hug et al. (1974) Hug et 01. (1974) Mishina et al. (1977) Thorpe and Ratledge (1972) Hug and Feichter (1973) Mishina et al. (1977) Mishina et al. (1977) Pelechova et al. (1971) Nyns et al. (1968) Jwanny (1975) Iida et 01. (1980) Iida et al. (1980) Omar and Rehm (1980)
35.0 32.0 25.8 15.6 14.9 48.6
Omar and Rehm (1980) Blasig et al. (1989) Yamaguchi and Kurosawa (1976) Pelochova et al. (1971) Pelochova et al. (1971) Pelochova et al. (1971) Hoffmann and Rehm (1978) Hoffmann and Rehm (1978) Hoffmann and Rehm (1978) Hoffmann and Rehm (1978)
38.4
Hoffmann and Rehm (1978)
20.0
(c13-c17)
Cunninghamella echinulata
Glucose
n-ALKANE-UTILIZING MICROORGANISMS
37
TABLE 111 (Continued) Microorganism C. echinulata C. echinulata C. echinulata
Substrate
Total lipids
Reference
29.9 49.4 52.5
Hoffmann and Rehm (1978) Hoffmann and Rehm (1978) Hoffmann and Rehm (1978)
27.2
Hoffmann and Rehm (1978)
c 1 2
17.1
Cl, n-Paraffin
10.9 22.7
Hoffmann and Rehm (1978) Hoffmann and Rehm (1978) Hoffmann and Rehm (1978)
c 1 2
CI, n-Paraffin (c13-c17)
Mortierella isa bellina M. isabellina /M. isabellina M. isabellina
Glucose
(C13-C17)
Data are expressed in percentage of dry biomass except for Mycobacterium convolutum whose data are given in mg lipidlmg protein.
organisms growing on conventional substrates such as glucose, acetate, or peptone are also given, for the purpose of comparison. Alkane-utilizing bacteria have a lower lipid content than yeasts and filamentous fungi. Oleaginous microorganisms are much more frequent among eukaryotes than prokaryotes. As a rule, the growth on alkanes as sole sources of carbon and energy is associated with increased lipid content of the microorganisms, as compared with the values recorded when conventional carbon sources are used. This fact was realized by investigators in the 1960s (Johnson, 1964; Mizuno et al., 1966; Dunlap and Perry, 1967; Nyns et al., 1968; Koronelli, 1968). Microorganisms grown on alkanes and on conventional carbon sources have similar lipid compositions, with only quantitative differences (see Table IV). However, sometimes there may be qualitative differences. Dodecanegrown cells, but not glucose-grown cells of Rhodococcus rhodochrous contain sterols, diacylglycerophosphocholines, and an unidentified glycolipid (Sorkhoh et a]., 1990b). The lipid classes responsible for increased total lipid content during the shift to alkane utilization are listed in Table V. Makula and Finnerty (1970), through phosphorus analysis of the total lipids, concluded that Acinetobacter HO1-N grown on hexadecane contain 50% more phospholipids than when the cells are grown on glucose. Also, Mycobacterium vacca grown on propane contain 66% more phospholipids than on acetate (Vestal and Perry, 1971). Kvasnikov et al. (1974, 1977b] found that lipid synthesis by Micrococcus freudenreichii grown on n-alkanes is much more active than on glucose; it was twice as active during slow growth and six times more active during rapid growth. Reportedly, free
38
SAMIR S. RADWAN AND NASER A. SORKHOH TABLE IV LIPIDCOMPOSITION OF Acinetobacter
SP.
HO1-N GROWNON n-HEXADECANE'
Intracellular lipids (pmol/g dry cells) Nutrient broth- yeast extract
Lipid classes Phospholipids Triacylglycerols Monoacylglycerols diacylgl ycerols Free fatty acids Free fatty alcohols Wax esters
+
Extracellular lipids (Fmol/liter)
Hexadecane
46.0 1.8
0.4
Nutrient broth-yeast extract
Hexadecane
129.0 2.5 6.8
0.0
0.0
2.4
25.6 410.0
8.2
4.0 0.0 0.0
7.5 Trace 11.5
2.6
18.0
0.0
60.0 0.5
280.0
"Results from Makula et al. (1975)
TABLE V LIPIDCLASSESREPORTEDTO ACCUMULATE IN MICROORGANISMS SHIFTED TO n-ALKANE UTILIZATION Microorganisms Acinetobacter sp.
Lipid classes
Phospholipids, mono- and diacylglycerols A. lwoffi Phospholipids Mycobacterium vacca Phospholipids Micrococcus Phospholipids, free fatty acids, waxes freudenreichii Arthrobacter ceroformans Wax esters Phospholipids Mycobac terium convolutum Sterols, Rhodococcus monoacylglycerols, rhodochrous unknown glycolipids Candida tropicalis Phospholipids C. tropicalis Phospholipids, fatty acids C. Iipolytica Phospholipids C. guilliermondii Triacylglycerols, wax C. rugosa Ergosterol C. albicans Total sterols Cladosporium resinae Hydrocarbons Paecilomyces persicinus Triacylglycerols, free fatty acids
Reference Makula and Finnerty (1970) Makula et al. (1975) Vachon et al. (1982) Vestal and Perry (1971) Kvasnikov et al. (1974, 1977b) Koronelli et al. (1978) Hallas and Vestal (1978) Sorkhoh et al. (1990b)
Mishina et al. (1977) Kvasnikov et al. (1977b) Mishina et al. (1977) Demanova et al. (1980a) Iida et al. (1980) Sorkhoh et al. (1991) Walker and Cooney (1973) Boyer and Pisano (1974)
39
n-ALKANE-UTILIZING MICROORGANISMS
fatty acids make up about 60% and phospholipids about 20% of the total lipids from cells grown on glucose. On alkanes, the phospholipid content increased to 46% and the cells accumulated wax esters (20-47% of the total lipids). The total lipid content of Mycobacterium convolutum grown on n-alkanes is two to five times higher than on acetate (Hallas and Vestal, 1978). The newly synthesized lipids have their origin in the alkane substrate as shown by results of [14C]acetate incorporation into cellular lipids and proteins (Table VI). Less than 10% of the labeled acetate is incorporated into lipids of n-alkane-grown cells compared to acetate-grown cells, whereas incorporation into proteins is not lower. This result indicates that in n-alkane-grown cells the lipids should have been synthesized mainly from the alkane substrates. In contrast, a few authors failed to find any substantial difference in the total lipid content between alkane-grown cells and cells utilizing conventional carbon sources. Koronelli et al. (1982a), working with Pseudomonas aeruginosa, analyzed “free” and “bound” lipids and found that hexane- and glucose-grown cells contain 7.0 and 6.7% free lipids, respectively. The values for bound lipids are 6.7 and 5.6%, respectively, of the dry biomass. There are comparatively more reports on total lipids of yeasts than of bacteria and filamentous fungi. This is apparently because only yeasts have been successfully used for biomass production as fodder, with hydrocarbons as substrates. In the majority of these studies Candida yeasts were used. Shigyo and Takeuchi (1972) developed a technique for the complete extraction and separation of lipids from hydrocarbongrown yeast. Hug et al. (1974) observed that lipid synthesis by Candida TABLE VI INCORPORATION OF [14C]ACETATEINTO TOTALCELL LIPIDSAND PROTEINS OF Mycobacterium convolutuma Substrate
Lipid incorporation (pmol acetatdpg lipid)
Acetate Nonadecane (C1J Eicosane (Czo) Decosane (C,) Tricosane (C,,) Tetracosane (Cz4) Hexacosane (Cz6) Octacosane (C2J From Hallas and Vestal (19781.
Protein incorporation (pmol acetatdpg protein)
315
86
24 10
15
84 80 80
11
97
29
76
18
65 82
15
40
SAMIR S. RADWAN AND NASER A. SORKHOH
tropicalis is activated by the presence of the hydrocarbon, and that little substrate uptake is possible until the lipid concentration in the cells is sufficiently high. These authors confirmed an earlier viewpoint (Dunlap and Perry, 1967; Vestal and Perry, 1971) that high lipid content is necessary for hydrocarbon uptake, and is not merely a reflection of the lipophilic nature of the substrate. Mishina et a]. (1977) reported that the total lipid content of Candida tropicalis almost doubled when shifted from glucose to alkane utilization. The chain length of the nalkane substrate did not exert any obvious effect on the total lipid content. However, these authors found in the same study that the lipid content of another species, C. lipolytica, comprised about 4% of the dry biomass, irrespective of whether the substrate was glucose or an nalkane. Kvasnikov et al. (1977b) reported that the activated lipid synthesis by C. tropicalis on hydrocarbons occurs primarily at the expense of the substrate. Davidova et al. (1978), working on C. tropicalis and using I4C substrates, compared the kinetics of label incorporation from noctadecane and glucose into the main groups of organic substances, viz. proteins, nucleic acids, polysaccharides, lipids, free amino acids, organic acids, free carbohydrates, and nucleotides. When the cells utilize n-octadecane, the radioactivity in the lipid fraction is higher than when they are grown on glucose. A few investigators working on yeasts failed to observe any striking effect of the alkane substrate on the total lipid content. Reference has already been made to C. lipolytica, which had a similar lipid content when grown on glucose or on n-alkane. Only small quantitative differences in the total lipid content of C. tropicalis were observed when the cells were grown on n-alkanes, glucose, or glycerol (Giewicz et al., 1983). Moreover, there is some contradiction in the literature regarding the effect of the alkane-chain length on the total lipid content. While Mishina et al. (1977) did not find any effect of the alkane-chain length on the total lipids of C. tropicalis, a result which was later confirmed on Mycobacterium convolutum (Hallas and Vestal, 1978), Demanova et al. (1980a) found that the lipid content of C. guilliermondii grown on n-octadecane (C18)is three times higher than that of cells grown on . et al. (1980) showed that the lipid content of C. docosane ( C Z 2 )Iida rugosa significantly increases from 16 to 19% of the dry biomass when the n-alkane chain length increases from C,, to Cz0. The effect of the alkane substrate concentration also has been investigated. Zalashko et al. (1983) reported that the optimum concentration of n-hexadecane for growth and lipid production by C. tropicalis is 1-2%; higher concentrations are inhibitory. The optimum paraffin concentration for growth and lipid synthesis by C. maltosa is 1.5-2.5% (Maksimova eta]., 1988).
n-ALKANE-UTILIZING MICROORGANISMS
41
Andreevskaya and Zalashko (1984) studied the effect of temperature on biomass and lipid production by C. tropicalis grown on C, to C,, nalkanes. The highest lipid content was obtained at 10°C, although this temperature was not optimal for growth. A few studies on total lipid contents and lipid composition of nalkane-utilizing filamentous fungi have been published. Walker and Cooney (1973) showed that the total lipid content of Cladosporium resinae grown on n-alkanes as substrates is higher than that on glucose or glutamic acid. Similarly, Paecilomyces persicinus grown on nhexadecane contains more total lipids than when glucose is utilized as substrate (Boyer and Pisano, 1974). The increase was particularly noticeable in the triacylglycerol and free fatty acid fractions, which were, respectively, three and five times higher in alkane- than in glucosegrown mycelia. Three oleaginous fungi belonging to the mucorales behaved differently on n-paraffin with C,,-C,, alkanes (Hoffmann and Rehm, 1978). Absidia spinosa and Cunninghamella echinulata grown on n-paraffin contain more total lipids than on glucose, whereas Mortierella isabellina contain less total lipids. The lipid content of the three fungi grown on n-dodecane is obviously lower than on glucose. C. BIOTECHNOLOGICAL CONSIDERATIONS
Although there are numerous patents covering the production of useful compounds by n-alkane-utilizing microorganisms, there are so far no industrial fermentation processes based on hydrocarbon substrates. According to Shenman (1984), even gas oil and n-paraffin single cell protein projects are changing to methanol or carbohydrate feedstocks. The prediction, made two decades ago, that some of the world's supplies of oil and fats could be produced microbiologically using n-alkanes as a starting material (Ratledge, 1970) remains too optimistic. n-Alkane-utilizing microorganisms appear to be the least suitable as fat sources, particularly for food and feed purposes, for two main reasons: (1)Fats, i.e., triacylglycerols, as demonstrated in this article, frequently are not a major lipid class in such microorganisms. The increased total lipids during shift to n-alkane utilization is primarily due to increased biosynthesis of phospholipids, fatty acids, monoacylglycerols, and wax esters, but rarely of triacylglycerols (see Table V). (2) Even if it is possible to enrich such microorganisms with triacylglycerols, for example, by nitrogen and other element starvation (for review, see Ratledge, 1986) and/or by genetic manipulation, a problem regarding the suitability of the fat product to be returned into the food chain remains unsolved. Because of the relatively high content of odd-chain
42
SAMIR S. RADWAN AND NASER A. SORKHOH
fatty acids (see Section XI) associated with microbial growth on nalkane mixtures, the fat product cannot be recommended as a nutrient. However, the chance for lipids from n-alkane-utilizing microorganisms to find application in the diverse field of oleochemical industry (for reviews, see Richter and Knaut, 1984; Leonard and Kopald, 1984) is rather good. Some aspects of these applications are discussed in the following sections dealing with individual lipid classes of n-alkaneutilizing microorganisms. IV. Fatty Acids
Fatty acids occur in all biological systems, mainly in complex lipids such as triacylglycerols, wax esters, steryl esters, phospholipids, and glycolipids. Sometimes they occur, although only in small amounts, in the free form. Fatty acids perform important physiological functions in the living cell. Acyl moieties, stored, for example, in triacylglycerols, may be degraded to acetyl-CoA, and via the citric acid and glyoxylate cycles can provide the cell with energy and cell material. The acyl moieties of phospholipids and glycolipids in cell membranes and organelles exhibit different degrees of unsaturation and thus determine the degree of fluidity and consequently the active transport properties of these membranes and organelles. The usual fatty acids in lipids of biological origin are predominantly those with 16 and 18 (in marine eukaryotes also 20) carbon chains. They may be saturated or unsaturated, with one or more double bonds per molecule. As far as the microbial fatty acids are concerned, differences exist between prokaryotes and eukaryotes. Unlike yeasts and filamentous fungi, bacteria are usually low in polyunsaturated fatty acids with two or more double bonds. Unusual fatty acids among microorganisms include the hydroxy acids, branched acids, cyclic acids, di- and tricarboxylic acids, and mycolic acids. In this section major emphasis is put on the usual fatty acids. Some of the unusual fatty acids, e.g., dicarboxylic acids and mycolic acids, will be considered elsewhere in this chapter. A. BASIC STUDIES Most studies have been done on the constituent fatty acids of total lipids from n-alkane-utilizing microorganisms. Only rarely were the constituent fatty acids of individual lipid classes investigated. Various studies had two different, yet not contradictory, objectives. The first is rather practical; n-alkane-utilizing microorganisms were investigated as potential sources of fatty acids and fats. The second objective is
n-ALKANE-UTILIZING MICROORGANISMS
43
academic; fatty acids were studied as a means for elucidating the biochemical mechanism(s) by which n-alkanes are microbiologically oxidized. Oxidation mechanisms leading to fatty acids are particularly relevant to the present section. However, for more detailed information the reader may refer to specific reviews (Klug and Markovetz, 1971; Einsele and Fiechter, 1971; Rehm and Reiff, 1981; Fukui and Tanaka, 1981; Buhler and Schindler, 1984). There are three different mechanisms known so far for the initial attack on n-alkanes by microorganisms. (1)Hydroxylation by a monooxygenase system in which cytochrome P450 may or may not be involved; (2) dehydrogenation leading to the n-alkene, which is subsequently hydrated; and (3) hydroperoxidation through a free-radical mechanism followed by reduction to the corresponding alcohol. The alcohol produced by any of the three mechanisms is then oxidized to the correspondingfatty acid. Here too are different pathways: (1)The monoterminal oxidation pathway in which one of the terminal methyl groups is oxidized leading successively to the corresponding 1-alkanol, 1-alkanal, and monocarboxylic fatty acid; this pathway is prevailing in many bacteria, yeasts, and fungi; (2) the diterminal oxidation pathway involves the oxidation of both methyl groups leading to the corresponding a,w-dicarboxylic (dioic) acid; (3) the subterminal oxidation pathway involves the oxidation of methyl groups leading successively to the corresponding secondary alcohol, ketone, and fatty acid. The composition of fatty acids and oxidation intermediates in the biomass and medium are indicative of the oxidation pathway prevailing in the culture. Cooney (1979) and Rehm and Reiff (1981) tabulated results of total fatty acid analysis for numerous microorganisms growing on n-alkanes [and n-alkenes) as substrates. In reviewing this subject, only representative data are tabulated, and readers interested in more details can refer to the reviews cited previously. Studies on bacteria date back to the sixties. Dunlap and Perry (1967) cultivated Mycobacterium sp. OFS on n-alkanes with 13, 14, 15, 16, or 1 7 carbon chains as sole sources of carbon, and analyzed the total cell fatty acids after 3 days. These authors showed that the cells accumulated in their total lipids fatty acids with chains equivalent in length to those of the substrates (Table VII). Cells grown on odd-chain alkanes tridecane [C13),pentadecane (CJ, and heptadecane (C17)have larger proportions of fatty acids with 1 7 carbon chains than cells grown on even-chain nalkanes. The same authors extended their work a year later (Dunlap and Perry, 1968) to cover the additional n-alkanes C,,, C,,, C,,, C,,, C,,, and C,,, and also studied Mycobacterium sp. 7EIC grown on C,,, Corynobacterium sp. grown on C17,and Brevibacterium sp. JOB5 grown
TABLE VII CONSTITUENT FATTYACIDSOF TOTALLIPIDSFROM Mycobacterium AND Micrococcus GROWNON II-ALKANES WITH DIFFERENT CHAIN LENGTHS' Mycobocterium sp. OFS Fatty acids 1o:o 11:0 12 : O 13:O 14:O 15:O 15:l 16:O 16:l 17:O 17:l 18:O 18:l
GIb
C,2b
-
-
2.1 5.1 0.4 4.1 1.1 Trace 3.3 7.0 2.9 1.4 0.4 0.1 30.6 27.5 18.1 13.8 1.2 0.5 2.0 0.6 0.8 20.7 17.5
C13c C,,'
C,,"
Micrococcus cereficons
GGC C,,c
CIab
-
-
-
-
-
-
1.4 0.9 21.4 1.8 6.6 1.7 15.3 14.3 3.6 9.8
1.0 2.9 0.9 39.0
0.5 1.0 0.2 5.9 1.5
-
7.2 4.0
-
50.0 28.2
-
0.8 5.7
-
-
-
9.6
19.4
-
2.5 0.3 2.3 0.5 18.1 2.7 3.8 2.5 19.4 27.2
-
-
0.8 0.5 6.9 0.9 74.0 8.9
-
5.0
-
19.5
2.9 1.3 0.4 28.3 15.5 0.4 0.5 1.9 26.3
Data expressed in relative percentage of the total fatty acids Data from Dunlap and Perry (1968). Data from Dunlap and Perry (1967). Data from Makula and Finnerty (1968).
C,,b
C2,b -
-
3.0
Trace Trace 22.0 18.0
-
0.1 0.3 3.0 10.4 2.5 21.8 13.8
Trace Trace
-
30.0
21.6
C2ab Clod -
3.8
0.4 0.8 3.1 6.0 21.1 3.7 15.8 10.6 3.0 8.5
-
13.8
C,,d
C,,d
0.5
7.6
0.7 0.9 2.6
-
Trace
-
4.4
1.7 1.9
-
1.6 1.7 7.2 7.7 2.2 1.2
Trace
-
Trace
18.7 14.2 11.2 15.6 6.3 26.1
23.3 14.3
17.3 11.6 7.7 7.7 6.7 27.4
39.5 14.5
6.7 23.6
-
6.6 0.8
-
6.6 47.5
C,,d 1.7
7.3
28.4
10.9 16.9
11.6 23.2
CIjd 1.1 1.2 1.3 1.1 0.2 32.6 36.2
Trace 4.5 3.3 3.8 11.3 3.3
CIGd C,,d 0.2
Trace
-
2.0 1.4 1.8 0.4 6.6 3.6 2.7 1.6 21.0 49.9 1.6 7.5
7.1
2.0 -
29.6 43.3
-
7.1 2.0
C,,d Trace
-
3.9
-
1.9 -
16.3 20.8 -
10.4 41.6
n-ALKANE-UTILIZING MICROORGANISMS
45
on C,, and C,,. The results (Table VII) also confirmed that cells grown on odd-chain alkanes accumulate larger proportions of fatty acids with 17 carbon chains and to a lesser extent 15 carbon chains in their lipids. Interestingly, cells grown on the very long chain alkanes, C,,, C,,, and C,,, do not accumulate any fatty acids with the same chain length and contained instead the usual fatty acids with 16 and 18 carbon chains, in addition to considerable concentrations of fatty acids with 15 and 17 carbon chains. The accumulation of C,, and C,, fatty acids indicates that very long even-chain alkanes are also attacked by mid-chain oxidation. Makula and Finnerty (1968), working on Micrococcus cerificans (Acinetobacter sp.) grown on C,,, C,,, C,,, C,,, C,,, C,,, C,,, C,,, and C,, n-alkanes, confirmed these results (Table VII). The data in Table VII indicate that the tendency to accumulate fatty acids with chains equivalent in length to those of the n-alkane substrates in cell lipids is valid within the alkane chain range C,,-C,,, even if the alkane is odd or even chained. Total lipids from Nocardia salmonsicolor PSU-N-18 grown on nhexadecane contain hexadecanoic (46%),hexadecenoic (4.5%),and octadecenoic (14.5°/0)acids as predominant fatty acids (Abbott and Casida, 1968). Killinger (1970) showed that total lipids from Pseudomonas sp. grown on acetate, propionate, or n-alkanes with different chain lengths contain fatty acids predominantly with 15 to 18 carbon atoms, and that fatty acids with odd chains accumulate in cell lipids after growth on propionate or odd-chain n-alkanes (Table VIII). Reportedly, the carbon chains of the fatty acids are synthesized mostly de novo, but can also be taken unshortened from the substrate. Confirming these results on Mycobacterium, Micrococcus (Acinetobacter), Pseudomonas, and other genera, Yano et al. (1971) found that lipids of Arthrobacter simplex contain significant amounts of odd-chain fatty acids only when grown on odd-chain n-alkanes (Table VIII). On the other hand, Edmonds and Cooney (1969) showed that lipids of Pseudomonas aeruginosa grown on n-tridecane contain stearic, palmitic, and palmitoleic acids as predominant fatty acids but only small proportions of pentadecanoic and heptadecanoic acids and no tridecanoic acid at all (Table VIII). bassilnikov et al. (1972)found that various strains of Mycobacterium lacticolum var. aliphaticum grown on n-hexadecane accumulate in their total lipids only hexadecanoic, tetradecanoic, and dodecanoic acids. Similarly, Koronelli et al. (1981) reported that total lipids of two Arctic, pigmented Mycobacterium strains grown on n-hexadecane contained palmitic acid (16 : 0)as a predominant usual fatty acid. Mycobacterium vaccae JOB5 grown on n-pentadecane accumulates large pro-
TABLE VIII CONSTITUENT FAITY ACIDSOF TOTALLIPIDSFROM Pseudomonos AND Arthrobacter GROWNON n-ALKANES WITH DIFFERENT CHAIN LENGTHS' Arthrobacter simplex
Pseudomonas sp. Fatty acids Acetateb Propionateb
1o:o
4.0
1l:O 12:o 13:O 14:O 15:O 15:l 16:O
-
16:l 17:O 17:l 18:O 18:l >I8 a
5.6
4.0
29.4 23.5
-
1.8 1.9 3.5 0.4 0.5 6.6 1.2 7.0 6.3 28.4 22.4
Glob
Cllb
18.3
0.7 2.6 2.4 1.6 Trace 1.4 2.6 2.4 6.6 3.6 15.8 32.6 15.3 1.5 16.5 2.3 13.8 44.0 18.4 7.4
1.5
-
2.0
-
23.4 29.8
-
-
-
34.5
19.6
25.0
-
-
-
-
C13'
-
Data expressed in relative percentage of the total fatty acids. Data from Killinger (1970). Data from Edmonds and Cooney (1969). Data from Yano et al. (1971).
Cl,b
CISb Cl,b
1.4
1.0 2.8 1.8 3.3 1.6 2.4 1.3 1.1 0.4 0.9 15.8 6.2 3.1 9.1 5.1 7.8 18.3 12.2 12.0 17.6 15.4 21.6
-
4.8 -
8.3
37.7 19.8
Cl,d
Cl,d
C,,d
2.0
-
-
5.5
-
-
-
-
5.1
7.0
1.1
0.5 6.9 1.9
1.5 33.4
1.5 2.5
-
-
C,,b
-
-
5.8
-
-
-
35.6 15.9
9.9 1.2 0.4 2.9 1.7 54.5
-
-
_
-
28.0
18.1
20.8
7.5 26.5
20.4 7.7 2.9 17.4 2.0 37.7
-
-
-
-
-
-
-
C,,d
1.1 Trace
10.0 38.9 53.0 2.3 2.3 Trace 1.1 29.1 1.4 Trace 18.8 3.0
-
-
CITd
Cl,d
C,,d
-
-
-
Trace Trace 1.8 Trace
-
Trace Trace
9.7
0.9
9.6
-
-
-
1.8 0.2 22.6 46.4
30.3 6.8 0.9
Trace
Trace
Trace
19.2
5.0 54.3
-
-
2.0 21.2 3.4 0.7 13.1 39.9
n-ALKANE-UTILIZING MICROORGANISMS
47
portions of pentadecanoic (51%) and pentadecenoic (14.4%) acids in its total lipids; cells grown on n-heptadecane contain high concentrations of heptadecanoic (22.6%), heptadecenoic (19.1%),and pentadecanoic (17.9%) acids in their total lipids (King and Perry, 1975). Reportedly, cells grown on n-tetradecane, n-hexadecane, and n-octadecane contain only minute amounts of odd-chain fatty acids, if at all. Makula et al. (1975) found that Acinetobacter sp. grown on n-pentadecane and n-heptadecane contain relatively large proportions of fatty acids with 15 and 1 7 carbon chains. On the other hand, cells cultivated on nhexadecane do not contain any odd-chain fatty acids. In the total lipids of glucose-grown cells of Corynebacterium cyclohexanicum, methyl tetradecanoic (35%) and methyl pentadecanoic (35%) acids are the major constituent fatty acids, whereas in the lipids of cells grown on cyclohexanecarboxylic, m-hydroxybenzoic, butyric, and acetic acids, methyltetradecanoic acid is the major fatty acid, making up 65-81Yo of the total fatty acids (Kaneda, 1983). In addition to n-alkanes, bacteria can also terminally oxidize 1alkenes (Dunlap and Perry, 1968; Makula and Finnerty, 1968), cyclohexylalkanes (Beam and Perry, 1974),and chlorinated alkanes (Murphy and Perry, 1983). Table IX indicates that Mycobacterium convolutum can oxidize 1-chlorohexadecane to 1-chlorohexadecanoic acid, which it can then desaturate, shorten, elongate, and incorporate into different lipid classes, including diacylglycerophosphoinositolmannosides,the predominant phospholipid (Murphy and Perry, 1987). Koronelli et al. (1988) suggested the use of tritium-labeled n-alkanes in estimating the activity of alkane-oxidizing bacteria. Oxidation of octadecane in the unlabeled CH, group leads to labeled stearic acid, whereas its oxidation in the labeled CH, group results in the loss of radioactivity. In their comparative study they concluded that Rhodococcus erythropolis is 70 times more active in alkane oxidation than Pseudomonas aeruginosa. Yeasts and filamentous fungi are eukaryotes; they differ from bacteria in possessing organelles. Therefore, early studies on n-alkane-utilizing yeasts have debated about whether the substrate oxidation occurs in mitochondria, cytoplasmic membranes, or elsewhere in the cell (Van der Linden and Huybregste, 1967; Lebeault et al., 1970;Liu and Johnson, 1971). The whole subject has been reviewed by Fukui and Tanaka (1981). In Candida yeasts, n-alkanes are first hydroxylated to fatty alcohols in microsomes (see also Blasig et al., 1988). These alcohols are subsequently oxidized to fatty acids via aldehydes in microsomes, mitochondria, and peroxisomes. Further P-oxidation of the fatty acids occurs exclusively in peroxisomes, whereas fatty acids produced in microsomes and mitochondria are incorporated into various complex lipids.
48
SAMIR S. RADWAN AND NASER A. SORKHOH TABLE IX CONSTITUENT FATTY ACIDS OF TOTAL LIPIDS AND
DIACYLGLYCEROPHOSPHOINOSITOLMANNOSIDESOF Mycobocterium C O n V O l U t U m GROWN ON n-HEXANE AND 1-CHLOROHEXADECANE"
Total lipids Fatty acids 14:O 15:O 16:O 1 7 : Obr 16:l 9 1 6 : l 11 18:O 1 9 : Obr 18:l CI 1 2 : o c1 13:O C1 14:O C1 15:O Cl 1 5 : l Cl 16:O C1 17:Obr c1 1 6 : l c1 1 8 : O C1 1 9 : Obr Cl 1 8 : l
Hexane
Chlorohexane
Trace Trace
Hexane
Chlorhexane
2.0
6.2
Trace
Trace 48.6 3.8 35.8 3.1
Diacylglycerophosphoinositolmannosides
3.9
Trace 1.1
Trace Trace Trace Trace 1.6
Trace
10.6 1.2
52.1 30.4 6.2 5.8
Trace Trace
-
-
3.0
1.0
Trace Trace Trace
26.2 4.2
19.7
Trace
-
38.1 4.4 14.2
29.7 17.3 4.2
-
-
1.6 4.5
7.2 1.1
8.0
Data from Murphy and Perry (1987). Values are expressed in relative percentage of total fatty acids.
In this context, Ermakova and Lozinov (1976) isolated 13 species belonging to seven genera of yeasts, which could neither grow on liquid paraffin nor oxidize n-octadecane, but grew well on tridecanol, and oxidized various aliphatic alcohols and fatty acids. These authors concluded that the only reaction that can be considered typical of n-alkane-utilizing yeasts is the oxygenase reaction leading to the fatty alcohols. Schunck et al. (1983) isolated and reconstituted the alkane monooxygenase systems of the yeast Lodderomyces elongisporus. On the cytological level, the possession of large numbers of peroxisomes is considered to be one of the specific features of alkane-utilizing yeasts (Fukui and Tanaka, 1981). As has been demonstrated in bacteria, yeasts also tend to accumulate fatty acids with chains equivalent in length to those of the alkane sub-
n-ALKANE-UTILIZING MICROORGANISMS
49
strates in their total lipids (e.g., Klug and Markovetz, 1967a,b). This is, however, particularly valid for n-alkanes with C,, to C,, chains. Before reviewing these studies, attention should be directed to some contradictions in the literature probably because of strain and/or cultural condition variations. Such contradictions are obvious in studies concerned with the proportions of saturated and unsaturated fatty acids in Candida yeasts. A group of earlier workers reported that lipids from yeast cells grown on n-alkanes contain larger proportions of unsaturated fatty acids at the beginning of the incubation period than at the end (Dyatloviskaya et a]., 1965; Pelechova et al., 1971).Another group of authors conversely showed that the proportion of unsaturated fatty acids is low in the beginning and increases at the end (Hug and Fiechter, 1973; Volvova and Pecka, 1973). A third group demonstrated that the proportion of unsaturated fatty acids remains rather constant throughout the incubation period (Mishina et al., 1973, 1977). Rattray et al. (1975) realized that yeast cells grown on odd-chain nalkanes contain large proportions of odd-chain fatty acids in their total lipids. Mizuno et al. (1966) showed that Candida petrophilum grown on n-hexadecane contains in its total lipids almost exclusively C,, and C,, fatty acids. In contrast, n-tridecane-grown cells contain, in addition to these acids, considerable proportions of heptadecenoic (38.8%)and pentadecanoic (9.3%) acids. The results of Klug and Markovetz (l967b), Mishina et al. (1973), and Jwanny (1975) on Candida Iipolytica grown on different n-alkanes are summarized in Table X and the results of Hug and Fiechter (1973) and Mishina et al. (1973) on C. tropicalis are presented in Table XI. Despite obvious differences in the values given by various authors, it may be concluded that yeast cells grown on n-alkanes with chains between C,, and C,, accumulate fatty acids with equivalent chain lengths in their total lipids. Moreover, the largest proportions of oddchain fatty acids are present in cells grown on odd-chain n-alkanes. Similar results have been recorded for Candida rugosa grown on nalkanes with C,, up to C,, chains (Iida et al., 1980) and Mycotorula japonica grown on C,,, C,,, C,,, C,,, C,,, and C,, n-alkanes (Yamaguchi and Kurosawa, 1976). Souw et al. (1976) showed that tetradecanoic acid is produced by Candida sp. through monoterminal oxidation of n-tetradecane with ntetradecanol as an intermediate, and demonstrated the desaturation of the fatty acid to its monoenoic homolog. No odd-chain fatty acids are present in the cell lipids. Lipids from n-tetradecane-grown cells of C. parapsilosis have similar fatty acid patterns to lipids from glucosegrown cells (Omar and Rehm, 1980).Similarly, it has been demonstrated
TABLE X CONSTITUENT FATTYACIDSOF TOTALLIPIDSFROM Candida lioolvtica GROWNON ALKANES WITH DIFFERENT CHAINLENGTHS' Fatty acids
Cllb
CIzb
C13b
C,,b
C,,"
Cl,b
CISc
Ul
0
11:o 12:o 13 : O 14:O 14:l 15:O 15:l 16:O 16:l 17:O 17:l 18:O 18:l 18:2
1.1
-
-
11.7
0.3
-
Trace -
5.0 0.6
2.5
Trace
-
-
13.4 14.6
10.4 17.4
Trace
-
5.7 2.4 47.9 12.1
0.9 3.1 43.1 7.9
Trace 8.5 0.3
16.6 1.8 2.0 6.1 1.5 31.6 0.6 20.6 10.5
C,,b
C,,'
-
Trace Trace
-
Trace
Trace Trace Trace
19.5 2.6
3.0
0.3
0.8
0.1
5.0 5.0
-
-
-
-
-
Trace Trace
4.5
-
8.5 24.3
13.5 7.5
Trace
Trace Trace
20.0 2.9 0.3 1.4 1.5 56.5
20.8 1.7 2.6 4.3 0.8 25.4 12.3 8.6 19.7
1.0 0.8 29.6 13.5
44.8 10.1 14.9
Data expressed in relative percentage of the total fatty acids. From Mishina et al. (1973). From Klug and Markovetz (1967b). From Jwanny (1975).
-
14.3 7.2
-
0.6
5.0
-
-
30.2 27.7
30.0 20.0
-
Trace Trace
1.0 2.1 30.0 8.3
Cl,C
CIBb
C1,C
-
0.8
-
Trace Trace -
16.8 12.3
21.4 6.7
-
Trace
3.7 7.9 41.2 16.7
5.3 6.7 16.7 34.8
~
Trace Trace
-
Cl,b
C,,d
~~~~
~~
10.0 25.0 -
-
-
1.3
1.7 2.9 2.0 3.0 10.0 3.7 26.7 9.0 2.9
-
-
0.2 0.4
2.0
-
Trace -
-
5.6 17.0 6.3
-
2.1 0.8 0.7 0.6 4.6 77.9
-
0.5
3.9 3.9 5.2 2.6 14.3 36.6 19.6 3.9 5.2
-
2.6 4.0
TABLE XI CONSTITUENT FATTY ACIDSOF TOTALLIPIDSFROM Candida t r o p i c a h GROWNON ALKANES WITH DIFFERENT CHAINLENGTHSO ~
Data from Hug and Fiechter (1973) Fatty acids Glucose
L ul
ll:o 12:o 13:O 14:O 15:O 15:l 16:O 16:l 17:O 17:l ia:o ia:i ia:z i8:3 19:o
0.5 Trace 0.6
1.3 0.4 13.4 12.0 0.8
2.2 14.6 23.0 30.5
Acetate
CI2
0.5 Trace 1.5 1.3 0.7 16.0 11.4 1.3 2.o 14.3 22.2 28.5
24.4 Trace 1.4 Trace
C,,
C,,
0.5 2.4 0.4 28.4 Trace 0.8 33.3 7.2 Trace 0.4 10.4 9.6 7.3 6.8 5.0 11.8 Trace 1.3 Trace 2.0 5.5 5.3 12.5 14.1 5.3 33.5 24.7 19.1 8.4 7.0 4.5 Trace Trace
Data expressed in relative percentage of the total fatty acids.
~~
~
Data from Mishina et al. (1973)
C15
C,,
1.2 0.9 0.5 0.5 22.8 4.5 2.6 2.6 2.0 15.6 18.6 16.9 4.6 5.4
-
0.1 Trace 0.4 0.1 Trace 28.9 27.7 Trace 5.5 8.3 21.2 7.6 -
-
C17
Cll
C12
1.0 1.3 1.2 1.4 1.9
-
0.5
-
1.4 Trace _ _ _ 2.7 3.8 10.6 0.2 0.8 Trace 0.4 11.5 Trace 18.3 0.5 0.4 0.9 17.2 2.0 15.1 0.8 33.8 0.5 12.5 2.3 24.9 1.4 33.7 0.3 3.4 0.5 6.0 Trace 17.8 2.4 49.2 2.1 50.9 1.1 65.6 0.6 0.6 2.9 Trace 40.5 17.9 28.1 18.3 23.0 12.5 16.2 7.1 14.0 4.5 6.0 1.8 1.8 0.8 3.9 1.2 1.0 0.8 -
1.9
8.1 5.8 5.1 2.1 17.1 8.6 25.3 30.0 24.8 1.3 3.4 27.0 0.8 20.3 - 2.9 5.5 -
C13
C14
CIS
C16
C,,
C18
7.2 1.3 0.3 5.6 67.0 15.8 2.7
52
SAMIR S. RADWAN AND NASER A. SORKHOH
that n-pentadecane is monoterminally oxidized to pentadecanoic acid via pentadecanol by Candida sp. (Souw et al., 1977). Moreover, evidence for diterminal and subterminal oxidation, as well as for the desaturation of pentadecanoic acid, is presented. Blasig et al. (1984) incubated glycerol-grown cells of Lodderomyces elongisporus for a few hours with n-hexadecane and n-heptadecane as sole carbon sources, and demonstrated that the levels of hexadecanoic and heptadecanoic acids in the cell lipids increase dramatically (Table XII). Table XI1 indicates that these fatty acids are actively desaturated by the yeast cells to the corresponding monoenoic acids (16 : 1 and 17 : 1).These findings were confirmed by studying the distribution of radioactivity of [l -l4C] hexadecane, as a sole carbon source, among the fatty acids of total lipids from this yeast. The results (Table XIII) indicate the occurrence of both fatty acid elongation and @-oxidation.The latter pathway is substantiated by the appearance of [14C]myristic(14 : 0)and -1auric (12: 0 ) acids. There is little information about the oxidation of very long n-alkanes with C,, and longer chains by yeasts. The only available study is that of Blasig et al. (1989) who demonstrated that Candida maltosa could TABLE XI1 CONSTITUENT FAITY ACIDSOF TOTALLIPIDSOF Lodderomyces elongisporus BEFORE AND AFTER INCUBATION WITH n-ALKANES' ~
4
Fatty acids 12:o 14:O 15:O 16:O 16:l 16:2 17:O 17:l
Before incubation 0.57 0.94 18.84 9.35 0.52
hr after incubation with C,,
C,,
0.05
0.05 0.22 1.36 7.72
0.99
0.35 68.28 41.31 2.18
-
-
18:l 18:Z 18:3
1.73 24.97 1.16 0.36
20:o
-
0.15 29.20 20.31 5.30
18:O
Total
58.41
?
6.02
167.91 ? 24.06
-
58.52 27.65 0.35 8.25 0.45 1.09 105.65 ? 22.96
Values are expressed in f i g fatty acid per mg protein. Cells were grown on glycerol as a carbon source before they were incubated with alkanes. Results from Blasig et of. (1984).
53
n-ALKANE-UTILIZING M I C R O O R G A N I S M S T A B L E XI11
DISTRIBUTION OF RADIOACTIVITY AMONG FATTY ACIDS OF Lodderomyces e l o n g i s p o r u s AFTER INCUBATION WITH [1-'4C]HEXADECANEa Fatty acids
0.2
hr
-b
12:o 14:O 16:O 16 : 1 16:2
10.1 1.97 ? 0.22 10.1
18:O
wine yeasts > distillers yeast > brewers yeast (Casey and Ingledew, 1986; Rose, 1987). There are two basic hypotheses for the mechanism of alcohol inhibition of fermentation: (1)damage to cell membrane and (2) end product inhibition of the glycolytic enzymes. It is apparent from in vitro studies that enzymes of the glycolytic pathway are resistant to the ethanol concentration produced during fermentation (Miller et aI., 1982). In addition, for many ethanologenic organisms the potency of alcohol as an inhibitor has been correlated with lipid solubility (Ingram and Buttke, 1984), implying that the hydrophobic site of the membrane is a prime target of ethanol inhibition. Results obtained so far also accredit the cell membrane as the major cause of ethanol inhibition in ethanologenic mesophilic organisms, e.g., Saccharomyces and Zymomonas. However, this does not appear to be the case for some ethanologenic thermophilic bacteria. Among various functions of the membrane, alcohol inhibits the uptake of various nutrients viz. glucose, ammonium ions, and amino acids; changes the physicochemical properties of membrane; and also causes leakage of various essential cofactors (Ingram, 1986; D’Amore and Stewart, 1987). So far, only limited information is available on the ethanol tolerance of xylose-utilizing yeasts. An ethanol concentration of 20 g/liter begins to affect specific ethanol productivity and xylose consumption in the pentose-utilizing yeast P. tannophilus (Silinger et al., 1982; Watson et al., 1984b). The concentration of ethanol that stops ethanol production is much higher than the concentration that is growth inhibitory (42 g/liter). Ethanol added at a concentration of 80 g/liter results in specific productivity around 0.03 g/g/hr which is about half that observed in the absence of added ethanol. Results indicate that although P. tannophilus can tolerate ethanol up to 100 g/liter, a maximum of 38 g/liter ethanol accumulates in cultures even with an excess of xylose (Silinger et al., 1982). Thus it is apparent that ethanol toxicity is not the factor limiting ethanol accumulation when xylose is the substrate.
138
PRASHANT MISHRA AND AJAY SINGH
This is in accordance with the observation by Jeffries (1985) that P. tannophilus is able to produce more than 50 g/liter ethanol when glucose rather than xylose is the substrate. Candida shehatae is found to tolerate 80 g/liter ethanol when a mixture of hexose and pentose sugars is used as the substrate (Wayman and Parekh, 1985). Ethanol above 15 g/liter inhibits the growth of Fusarium oxysporum ATCC 10960; however, no growth has been reported at concentrations above 42 g l liter (Rosenberg et a]., 1981). Further, ethanol has no inhibitory effect on xylose fermentation by F. oxysporum VTT D-80134 at 3.5 to 4% (w/v) (Suihko, 1983). Butanol is the most hydrophobic fermentation product of acetonebutanol and ethanol-producing bacteria, and it is generally thought to be the most toxic major product in limiting fermentation. The growth of Clostridia is inhibited by butanol concentrations of less than 1%. The half-maximum growth inhibitory concentration of butanol is seen at a concentration of 0.15-0.18 M in C. acetobutylicum (Leung and Wang, 1981; Costa and Moreira, 1983). Butanol toxicity in mesophilic C. acetobutylicum appears to be related to membrane damage, while ethanol inhibition in thermophilic clostridia, e.g., C. therrnocellurn and C. thermohydrosulfuricum, is due to the direct inhibition of glycolysis. In C. acetobutylicum, growth inhibitory concentrations of butanol dissipate the pH gradient across the plasma membrane and partially inhibit glucose transport and ATPase activity (Bowles and Ellefson, 1985). Efforts have been made to isolate mutants resistant to butanol (Lin and Blaschek, 1983; Hermann et al., 1985). Butanol-resistant mutants lose their sporulation ability which may be due to the pleotropic nature of mutations (Hermann et al., 1985). Butanol-tolerant mutants of C. acetobutylicum do not show increased tolerance to either acetone or ethanol, suggesting that although these products may damage the membrane, their specific action must be somewhat different. In addition to alcohols, weak organic acids are also produced during the fermentation of pentoses. The effect of weak acids, such as acetate and butyrate, on C. acetobutylicum has been studied (Herrero et a]., 1985; Bowles and Ellefson, 1985). These weak acids act as uncouplers of proton transport across the cell membrane. Thus high levels of accumulated weak acids increase ATPase activity, leading to the loss of cellular ATP. Herrero et al. (1985) observed depleted cellular ATP contents in cells of C. thermocellum incubated with 0.8 M acetate. In another study, the total acid produced (0.1 M ) by C. acetobutylicum under normal growth conditions had no effect on cellular physiology but butyric acid added at a concentration of 0.17 M acted as an uncoupler (Bowles and Ellefson, 1985). The half-maximum growth inhibition by
MICROBIAL PENTOSE UTILIZATION
139
butyric acid is 0.07-0.16 M in C. acetobutylicum (Leung and Wang, 1981; Costa and Moreira, 1983). It is apparent from these studies that weak acids are toxic products and lead to end product inhibition of the C. acetobutylicum fermentation. In fact, a switch from acid production to solvent production has been regarded as a detoxification mechanism (Rogers, 1986). Cultures of C. acetobutylicum that fail to switch from acidogenesis to solventogenesis die because of acid accumulation (Gottwald and Gottschalk, 1985). Similarly, cultures of acidogenic bacteria, e.g., C. thermoaceticum (which produces only acetic acids), also do not survive once acid is accumulated. Murray et al. (1983) isolated mutants of C. saccharolyticum with less acetic acid production which showed an increased ethanol resistance. Alcohol-tolerant mutants of CIostridium have also been reported by exposure to increasing concentrations of alcohols (Herrero and Gomez, 1980; Lovitt et al., 1984; Bowles and Ellefson, 1985).These mutants may prove to be useful for higher alcohol production in Clostridia. VIII. Strain Improvement
From the aforementioned material, it is apparent that the available yeasts or bacteria are not completely satisfactory for the bioconversion of D-xylose to ethanol. There are various limiting factors like conversion rate, product yield, and low ethanol tolerance. Thus these strains are amenable to strain selection and genetic improvement using recombinant DNA technology. One approach in improving a strain for xylose utilization involves the construction of yeasts that convert D-xylose into ethanol using genetic engineering methods. Brewers yeast, Saccharomyces cerevisiae, which is one of the most ethanol-tolerant yeasts, is unable to produce ethanol from D-xylose; however, a ketoisomer of xylose, xylulose, can be utilized by many Saccharomyces species for ethanol production (Wang et aI., 1980a,b; Gong et al., 1983). Thus these species have the potential to produce ethanol, if they are transformed with a gene coding for the enzyme that can convert xylose to xylulose and is expressed in the yeast. However, most yeasts do not efficiently utilize D-xylose because of cofactor (NADPH/NADH)regulation (Batt et a]., 1986). Hence practical strategies in developing yeast strains to utilize xylose for ethanol production involve attempts to circumvent the xylose reductase-xylitol dehydrogenase pathway. The cloning and introduction of genes from a pentose-metabolizing yeast (e.g., P. tannophilus, C. shehatae, or P. stiptis) in S. cerevisiae would not alleviate the problem of cofactor limitation. Alternatively, transformation with a xylose isomerase gene
140
PRASHANT MISHRA AND AJAY SINGH
from another source for the direct cofactor free conversion of D-xylose to xylulose is the most useful approach. This involves isolation and characterization of the E. coli gene for D-xylose isomerase. Hence the xylose isomerase gene from E. coli is purified and characterized (Ho and Chang, 1989). Several hybrid plasmids bearing this gene have already been isolated bearing different sizes of the insert. Using this approach the E. coli gene has been expressed both in S. cerevisiae and Schizosaccharomyces pombe (Sarthy et al., 1987; Chan et al., 1989). Schizosaccharomyces pombe has been transformed with hybrid plasmid pDB 248 XI which contains the xylose isomerase gene from E. coli. In transformed yeasts two enzymes are involved in the conversion of D-XylOSe to ~-xylulose-5-phosphate(Chan et a]., 1986). The xylose isomerase converts D-xylose to D-xylulose without NADH or NADPH as a cofactor and xylulokinase converts D-xylulose to ~-xylulose-5phosphate with ATP as a cofactor. Although cloned strains grow on xylose as the sole carbon source, ethanol production is slow (3%, w/ v) and xylitol production is very active. A later study indicated that the low isomerization of xylose in the transformed yeast is the limiting step for D-xylose fermentation. Although yeast proteases decrease xylose isomerase activity in vitro, this finding needs to be confirmed using protease-negative mutants. Xylitol, a by-product of D-xylose fermentation, has no effect on the activity of xylose isomerase activity (Chan et ai., 1989).A low activity of xylose isomerase in transformed yeast might also be due to a low expression of the xylose isomerase gene. Hence construction of an isomerase gene under control of a highly active yeast promoter is likely to improve the expression of the xylose isomerase gene (Chan et al., 1989). Attempts have also been made with xylose isomerase genes from Bacillus subtilis and Actinoplanes missourienis (Amore et al., 1989). In order to increase the production of ethanol from xylose, another approach employed cloning and the expression of the xylose uptake gene from E. coli (Kurose et ai., 1987) and the xylulokinase gene from P. tannophilus (Stevis et al., 1987) and S. cerevisiae (Ho and Chang, 1989).Some of the yeasts like Candida and Rhodotorula which are thought to contain the D-xylose isomerase enzyme are potential organisms in conversion. However, the disadvantage lies in xylitol production. This trait can be altered through conventional mutagenesis. Attempts have also been made to increase ethanol production using genetic methods. For example, xylose consumption and ethanol production by E. coli are increased by a coordinate expression of Zymomonas mobilis pyruvate decarboxylase (pdc) and adh I1 genes (Ingram and Conway, 1988; Neale et al., 1988). Similarly, Klebsiella planticola, which produces acetate, formate, lactate, ethanol, and CO, as end prod-
MICROBIAL PENTOSE UTILIZATION
141
ucts of hexose and pentose metabolism, has been used to improve ethanol production. In this organism, pentoses and hexoses are degraded to pyruvate which is dissimilated by the enzyme pyruvate formate lyase to yield acetate, ethanol, and formate (1: 1:2) (Tolan and Finn, 1987). A mutant of Klebsiella lacking pyruvate formate lyase was selected, which accumulated 70% lactate with residual acetate and 2,3butanediol and traces of ethanol. These mutants were further transformed using a plasmid carrying the pyruvate decarboxylase gene from 2. mobilis (which has a highly active pdc system but is incapable of fermenting pentose sugars). These transformed Klebsiella mutants show efficient ethanol production (Feldmann et al., 1989). Various approaches using mutants have been developed to improve the performance of microbes for pentose utilization. Using ultraviolet irradiation mutagenesis, mutants are selected for their ability to utilize xylose for the production of various products (Gong et al., 1981a; McCracken and Gong, 1983). One of the mutants of Candida, Candida SP XF217, which produces 31 g/liter of ethanol from 100 g/liter Dxylose, shows the maximum increase in ethanol production (McCracken and Gong, 1983). In these mutants the specific activity of xylitol dehydrogenase and xylulokinase is increased; however, D-xylose reductase activity remains the same. The increased xylitol dehydrogenase and xylulokinase activity of the mutant enabled them to shift from xylitol to ethanol production. Thus, instead of excreting xylitol as the final product, these mutants convert more xylitol to D-xylulose and ultimately to ethanol. Mutants of P. tannophilus were selected on plates containing nitrate and xylitol as the sole source of nitrogen and carbon (Bolen and Detroy, 1985). These mutants were able to use nitrate for rapid growth. Mutants of P. tannophilus, which can not grow on ethanol as the sole carbon source, also accumulate more ethanol and less xylitol (Lee et al., 1986). These mutants lack enzyme activity for the further degradation of ethanol. For example, mutant eth-2-1 is deficient in the enzyme malate dehydrogenase required for the metabolism of a two carbon compound either by the TCA or by glyoxalate cycles (Lee et aI., 1986).Further hybridization of the eth 2-1 mutant with another mutant, a rapid grower in nitrate-xylitol, improves ethanol production from Dxylose (Clark et a]., 1986). Another approach in obtaining improved strains of P. tannophilus is through the construction of polyploids (Jamesand Zahab, 1982,1983). Pachysolen tannophilus is homothallic in nature and is usually diploid for only a brief period between mating and meiosis. However, auxotrophic haploids of this strain are produced by ultraviolet irradiation of vegetative cells. The diploids are induced to undergo mitosis rather than meiosis. Using this approach a series of
142
PRASHANT MISHRA AND AJAY SINGH
triploids, tetraploids, and aneuploids were developed (James and Zahab, 1982,1983). A correlation between ploidy and production of ethanol and xylitol is observed in P. tannophilus (Maleszka et al., 1983b). In general, an increase in the chromosome number above the haploid level shows an increase in ethanol production and a decrease in xylitol production, but the level of by-products, e.g., acetic acid and arabitol, follows no pattern (Maleszka et a]., 1983b). An increase in the ploidy of C. shehatae leads to a small increase in ethanol production from Dxylose. The reason for the increase-in-ploidy-enhanced ethanol formation from xylose seems to be due to complex physiological changes that are still not clear. Efforts using a combination of both classical genetics and modern genetic engineering techniques are required to further improve strains for increasing pentose utilization and ethanol production. IX. Future Prospects
The current and projected scarcity of liquid and gaseous fossil feedstocks has promoted renewable (biomass) resources for the production of substitutes. These substitutes include sugar, starch, and lignocellulosic residues. Traditional starch and sucrose-based fermentations can be expanded to increase the supplies of liquid fuels. However, utilization of these agricultural commodities competes with food production because of their use in human and animal food, resulting in a less favorable energy balance. In contrast, lignocellulosic residues are cheap raw materials, containing glucose and pentose sugars. Sugars derived from cellulose and hemicellulose components of biomass are more attractive substrates for the microbial production of solvents. However, the expense involved in converting hemicellulose components has been responsible for the limited success in developing industrial processes. Thus the economic importance of lignocellulose utilization depends primarily on the bioconversion of both hexose and pentose sugars. If these pentoses could be converted to ethanol, some 4 billion additional gallons of ethanol could be obtained in addition to that derived from D-glucose. In developing a simple industrial process for solvent productivity, a biological system that could ferment both types of sugars simultaneously, with high product yields and higher bioconversion rate, is essential. Fermentation of the pentoses is generally slower than that of the hexoses. These rates could be improved through process optimization, but it is likely that the slow rates of pentose fermentation result from the biochemical pathway employed. Yeasts, in general, are the best choice in fermenting pentoses to ethanol. Basic studies related to the regulation of pentose metabolism using
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molecular biological techniques will help in developing new organisms devoid of limitations of ethanol production from pentoses. The cloning of the n-xylose isomerase gene in yeast to by-pass the oxidoreduction steps; the cloning of transhydrogenase (EC 1.6.1.1)to carry out regeneration of NADPH through the interconversion of “NADP+ + NADH NADPH + NAD+;” and the cloning of n-xylulokinase to shift the equilibrium of conversion of D-xylose to ~-xylulose-5-phosphate are among the possible approaches (Gong, 1983). Some pentose-fermenting bacterial strains produce organic acids in addition to the neutral products such as acetone, butanol, isopropanol, butanediol, and ethanol. These acids are usually not desirable products. Knowledge of the regulation of these pathways should suggest biochemical strategies for minimizing acid production in the fermentation. Thermophilic bacteria like C. thermoaceticurn have the potential for homoacetate fermentation of pentose sugars present in the hydrolysate of natural substrates (Brownell and Nakas, 1991).However, the acetic acid fermentation process is hampered by the low end product tolerance. Acetic acid levels above 10 g/liter are reported to stop cell growth and product formation (Wang and Wang, 1984; Sugaya et a]., 1986; Brownell and Nakas, 1991). Thus improvement in pentose-fermenting organisms in terms of yield, productivity, and end product tolerance is likely to be involved in the overall process. These improvements could be achieved by searching for new isolates with desirable properties, understanding the physiology of the strains, and making them accessible to genetic manipulation using advanced recombinant DNA techniques in manipulating carbon flow to desirable products. Therefore, the overall emphasis of future genetic engineering research in this area would be to produce better fermentation biocatalysts with wider substrate ranges and improved kinetics of product synthesis. In addition, improved methods for separating these products from fermentation broths should be sought. The large-scale production of solvents and chemicals from pentose sugars in addition to products derived from hexoses found in agricultural wood and industrial residues should be realized in the near future. Last, but not the least, careful consideration should be given to the idea that the organisms that have been described in the past are not the only candidates of particular interest, new organisms exhibiting desirable new fermentation pattern remain to be discovered.
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Medicinal and Therapeutic Value of the Shiitake Mushroom
s.c.JONG AND J. M. BIRMINGHAM Mycology and Botany Department American Type Culture Collection Rockville, Maryland 20852
I. Introduction 11. Medicinal and Therapeutic Properties
A. B. C. D. E.
Hypolipidemic Activity Anti-thrombotic Activity Antibiotic Activity Antiviral Activity Anti-cancer/Anti-tumor Effects F. Lentinan, a Biological Response Modifier 111. Patented Products and Processes A. Anti-hypertensive and Anti-cholesteremic Compositions B. Antibiotics C. Viricides Including Anti-AIDS Agents D. Neoplasm Inhibitors E. Immunoregulatory Substances F. Anti-ulcer Composition G. Anti-clotting Composition H. Anti-asthma Composition I. Bone Formation Accelerator J. Dermatological Compositions K. Postoperative Treatment L. Assay Processes IV. Discussion References
I. Introduction
The shiitake mushroom, Lentinula edodes (Berkeley) Pegler [Lentinus edodes (Berkeley) Singer], is the second most popular edible mushroom in the global market. According to ancient Chinese medical theory, consumption of the shiitake was recommended for long life and good health. Wu Juei, a Chinese physician of the Ming dynasty (1368-1644), claimed that it preserved health, improved stamina and circulation, cured colds and, in modern terms, lowered blood cholesterol (Mori, 1974). Many Japanese people believe that the shiitake is an elixir. In order to explore and possibly exploit the shiitake myth, many scientists have attempted to document its traditional therapeutic value. 153 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 39 Copyright 0 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.
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The shiitake mushroom contains proteins, fats, carbohydrates, vitamins, and minerals (Breene, 1990). Hypolipidemic and anti-thrombotic substances have been identified; the nucleic acids induce interferon production (Vo, 1987; Eder and Weig, 1988; Chang and Miles, 1989). Reputed antitumor components vary in chemical nature, but the most important may prove to be a polysaccharide that acts as a host defense potentiator (Jong et al., 1991). II. Medicinal and Therapeutic Properties
A. HYPOLIPIDEMIC ACTIVITY
The ability of the shiitake to lower blood cholesterol was first reported by Kaneda and Tokuda (1966), who found that a diet supplemented with the dried ground sporophores of L. edodes lowered average plasma cholesterol when fed to rats. The active principle was identified as an amino acid and named lentinacin by Chibata and co-workers (1969), and lentysine by Kamiya and co-workers (1969).Tokita and co-workers (1972) isolated two closely related compounds from the dried mushroom. The main and active component, 2(R),3(R)-dihydroxy-4-(9adenylj-butyric acid, was called eritadenine, the name currently in use. The minor component, 2(R)-hydroxy-4-(9-adenyl)-butyric acid, had no effect. Eritadenine lowers all lipid components of serum lipoproteins in both animals and humans (Takashima et a]., 1973; Tokuda et a]., 1976; Tokuda and Kaneda, 1979; Suhadolnik, 1979). It has very low toxicity in rats and is effective when administered orally, although only 10°/o is absorbed from the intestinal tract. The effect continues even when it is removed from the diet (Yamamura and Cochran, 1976a). Intravenously administered eritadenine is ineffective; it is rapidly cleared from circulation and excreted through the kidneys. Of the 1 2 4 derivatives of eritadenine that have been synthesized and tested, the most active are carboxylic acid esters with short-chain monohydroxy alcohols. It appears that a carboxyl group and one hydroxyl group, along with an intact adenine ring, are necessary for biological activity. Kabir and co-workers (1987) examined the effect of dried shiitake on the blood pressure and plasma lipids of spontaneously hypertensive rats (SHRs), and found that it decreased both the VLDL- and HDLcholesterol levels. In human testing S. Suzuki and Ohshima (1976) reported that serum cholesterol was decreased in groups of women fed fresh, dried, or UV-irradiated shiitake. A similar experiment, conducted on people 60 years or older, showed that serum cholesterol decreased after 1 week.
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Because shiitake mushrooms are a rich source of dietary fiber, Kurasawa and co-workers (1982) fed rats a standard control diet, and one containing cholesterol, along with whole shiitake or neutral detergent fiber (NDF) extract. The eritadenine-free NDF had a cholesterollowering effect distinct from that of eritadenine, which was attributed to its ability to bind to cholic acid salts. B. ANTI-THROMBOTIC ACTIVITY
Hokama and Hokama (1981) discovered that low molecular weight compounds extracted from some mushrooms, believed to be nucleosides and/or other nucleic acid derivatives, were capable of inhibiting aggregation of blood platelets. The highest yield of the inhibitors was obtained from L. edodes with an IC,, (inhibition concentration) of 80 pglml.
C. ANTIBIOTIC ACTIVITY Bianco (1981) reported that L. edodes was active against Candida albicans, Staphylococcus aureus, and Bacillus subtilis.
D. ANTIVIRAL ACTIVITY Goulet and co-workers (1960) were the first to show that antiviral substances were present in mushrooms. Tsunoda and Ishida (1969) found that an aqueous extract of the fruiting body and spores of the Donko variety of L. edodes was effective against influenza A/SW15 virus infection in mice. The active principle, identified as doublestranded RNA (ds-RNA), originated from attached virus-like particles (Ushiyama et al., 1971; Takehara et al., 1979) and induced interferon production (Kleinschmid, 1972). F. Suzuki and co-workers (1976) extracted ds-RNA from spores; a single dose produced a survival rate of 60% in rabbits infected with influenza virus. Virus-like particles in three basic shapes, spherical (S), filamentous (F),and rod-shaped (R), were detected in normal mycelia and fruiting bodies by Mori and Mori (1976). The S and F particles induced interferon production in sera of rabbits after intravenous (i.v.) injection. Takehara and co-workers, (1979, 1981, 1984) and Toyomasu and coworkers (1986) purified S and F particles and extracted ds-RNA from the S particles. In vitro tests with rabbit kidney cells (RK-13) showed that all induced interferon production. S-derived RNA was the most effective and F particles the least effective. In vivo studies indicated that a single intraperitoneal (i.p.) administration of S particles, prior to
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virus challenge, significantly reduced the mortality of mice infected with western equine encephalitis virus; F particles were considerably less effective. RNA with the same molecular weight as that extracted from the S particles of the spores could be obtained from fruiting bodies. Viral growth was inhibited in vitro, but less effectively than the spore RNA; the same was true for in vitro induction of interferon. When administrated i.p., purified S and F particles and the extracted ds-RNA demonstrated antitumor activity against Ehrlich ascites carcinoma in mice. Antiviral and antitumor activity appears to overlap. A peptidomannan (KS-2) extracted from cultured mycelia grown on stillage from whiskey manufacture (T. Fujii et a]., 1978) also exhibited antiviral activity (F. Suzuki et al., 1979). KS-2, composed of a-linked mannose and a small amount of peptide, has a molecular weight of -6 x lo4-9.5 x lo4.When administrated orally or i.p. to mice infected intranasally with influenza virus, KS-2 afforded therapeutic as well as prophylactic protection through its interferon-inducing activity. Yamamura and Cochran (1976b) determined that compound Ac2P isolated from the aqueous extract of dried shiitake was effective against the viral disease scrapie. Ac2P is a high molecular weight polysaccharide composed mainly of pentose sugars. IR vitro and in vivo tests in mice showed it to be a selective inhibitor of orthomyxoviruses, such as influenza viruses.
E. ANTI-CANCER/ANTI-TUMOR EFFECTS Ikekawa and co-workers (1968, 1969) found that an i.p. injection of an aqueous extract of L. edodes greatly inhibited growth of tumors (81%) arising from sarcoma 180 ascites cells implanted in Swiss albino mice. The active principle, a polysaccharide, was isolated and named lentinan by Chihara and co-workers (1969,1970),who observed complete regression with no toxicity. Additional data (Maeda and Chihara, 1971) showed that lentinan strongly inhibited the growth of transplanted tumors, but it had no effect on spontaneous mammary adenocarcinoma in mice when applied after reimplantation of autologous tumor tissue (Tokuzen and Nakahara, 1971). Because of its clinical and commercial importance, lentinan is considered separately in the following sections. T. Fujii and co-workers (1978) found that when given either orally or i.p. the polysaccharide KS-2 suppressed the growth of Ehrlich ascites tumors as well as sarcoma 180 tumors in mice. Sugano and co-workers (1982) obtained a water-soluble fraction (LEM) and two alcohol-insoluble fractions (LAP and LAP1) from the culture medium of mycelia with activity against Ehrlich ascites carci-
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noma in mice. LAP and LAP1 contained 58 and 65% sugar and 25 and 34% protein, respectively. The major sugar was xylose (>30%) with appreciable amounts of glucose, galactose, and arabinose (13-20%), about 9% mannose, and 1-2% each of fructose and rhamnose. Until the 1980s most of the research on the anti-tumor activity of mushrooms involved administration by injection to test animals. Lentinan and various other polysaccharides were shown to be ineffective when administered orally. More recent work (Mori et a]., l983,1987a,b; Nanba and Kuroda, 1987; Nanba et al., 1987) involved oral administration of powdered, dried mushroom fruiting bodies and powders from which the carbohydrate fraction (P-glucan) and/or lipid fraction was removed. Results of tests with L. edodes indicated that tumor growth could be inhibited 67% by the whole powder, 57% by defatted powder, 39% by polysaccharide-free powder, and 0% by powder free of both lipid and carbohydrate. Addition of extracted lipid elevated inhibition by 25%. The inhibition rate increased when mushrooms were fed over a longer period of time.
F. LENTINAN,A BIOLOGICALRESPONSEMODIFIER 1. Chemical Structure
Lentinan is a P-glucan (Hamuro et al., 1976) with a backbone of P-~-(l+3)-glucanand side chains of both p-~-(143)and P - ~ - ( l + 6 ) linked D-glucose residues, together with a few internal p-~-(1+6)-linkages (T. Sasaki and Takasuka, 1976). There are two P-D-(1+6)-glucopyranoside branches for every five linear /3-~-(1+3)-glucopyranoside linkages (Chihara, 1990). Based on crystalline structure studies (Bluhm and Sarko, 1977a,b), the probable structure of lentinan was determined to be a right-handed triple helix. Saito and co-workers (1977, 1979) concluded that the ordered conformation of both the p-n-(1+3)-linked main chain and side chains is a single-helix conformation which tends to form multiple helices as junction zones for gel structure. High resolution solid-state 13C NMR studies of the secondary structure (Saito et al., 1987; Saito, 1988) indicated that lentinan takes the curdlane-type single-helix conformation and is converted to the triple-helix form by lyophilization after dissolution in a 8 M urea solution and dialysis against distilled water. N. Suzuki and co-workers (1982) studied the hydrodynamic behavior of lentinan and established the relationship between the molecular weight and the diffusion coefficient. The anti-tumor fraction of lentinan is of high molecular weight (T. Sasaki et al., 1976);the average weights
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of two samples were 6.9 x lo4 and 5.2 x lo4 (R. Sakamoto, 1982). Maeda and co-workers (1988) investigated the correlation between higher structure and biological functions. Denaturation and renaturation of lentinan using urea and DMSO were associated with the decrease and recovery of anti-tumor activity against P-815 mastocytoma and vascular dilation and hemorrhage-inducing activity, which are Tcell-mediated responses. The change of the higher structure did not affect the increase of serum acute phase proteins, a non-T-cell-mediated response. 2. General Mode of Action
Lentinan appears to be a potent host defense potentiator which improves homeostasis of the host against cancer or infection (Chihara et al., 1989). It has no direct cytotoxicity to target cells; its action is host mediated. It activates the classical and alternative pathways of the complement system and augments the responsiveness of the host through maturation, differentiation, and proliferation of lymphoid and other physiologically important cells. The fact that lentinan is a T-celloriented adjuvant, in which macrophages play some part, distinguishes it from other well-known immunopotentiators (Hamuro et al., 1976). Although it does not specifically accelerate the production of interleukin-2 (IL-2) from helper T cells, it potentiates the induction of different types of anti-tumor effector cells, such as killer T cells, NK cells, and cytotoxic macrophages (Chihara, 1983; Chihara et aI., 1987). The effector cells may act either selectively or nonselectively on target cells. Various kinds of bioactive serum factors appear immediately after the administration of lentinan, most induced by macrophages. They act on lymphocytes, hepatocytes, vascular endothelial cells, or synovial fibroblasts, causing the many host defense reactions associated with inflammation and immunity. 3. Role of the Thymus and Cell Response
The action of lentinan is part of a thymus-derived immune mechanism (Maeda and Chihara, 1971,1973b; Maeda et a]., 1971,1973). Haba and co-workers (1976) showed that the selective suppression of T cell activity in both sarcoma 180 tumor-bearing mice and cell-free Ehrlich ascitic fluid-treated mice can be prevented by treatment with lentinan. In contrast to strong anti-tumor activity in vivo, lentinan showed no inhibition of sarcoma 180 cell cultures and failed to inhibit tumor growth in thymectomized mice bearing subcutaneously transplanted sarcoma 180 cells, or to stimulate conventional immune responses, such as antibody formation or phagocytosis. Shiio and co-workers ( 1 9 8 7 ~ ) demonstrated that the administration of Thy 1.2 antibody prevents lenti-
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nan suppression of tumor growth in C3H/He mice with sarcoma 180 solid tumor, and diminished its effect on tumor growth suppression in BALB/c nu/nu mice with grafted sarcoma 180 solid tumor. Coadministration of thymus homogenate or extract greatly enhanced its effect. Arai and co-workers (1971) found the anti-tumor activity of lentinan in mice with transplanted sarcoma 180 was reduced if the animals were x-irradiated, given benzylthioguanosine, an immunosuppressive agent, or blocked by injection of anti-lymphocyte serum after tumor transplantation. Dennert and Tucker (1973) showed that lentinan has only minor effects on the plaque-forming cell response to sheep red blood cells, but significantly stimulated antibody dependent, cell-mediated immunity. It did not increase sensitization of T killer cells in an allogeneic system. A simple assay system for ascertaining the cellular orientation of adjuvants and the action of lentinan on T cells using mice was described by Dresser and Phillips (1973, 1974). Maeda and Chihara (1973a) found that lentinan activated the antitumor effect of peritoneal-exudate cells in rats against sarcoma 180 in vivo. Hamuro and co-workers (1979, 1980) showed that it may potentiate cellular-immune responses by reducing synthesis of immunesuppressive prostaglandins from peritoneal-exudate cells. Injection of lentinan (i.p.) rendered murine peritoneal-exudate cells highly cytotoxic which may be related to their ability to activate the alternative path of the complement system. Zakany and co-workers (1980a,b) studied the effect of lentinan on the retardation and regression of transplanted tumors in murine allogeneic- and syngeneic-tumor hosts and concluded that a tumor-induced immunosuppression can be overcome, most likely through enhancement of the migration inhibitory factor (MIF) production. Izawa and co-workers (1982) proposed that lentinan enhances formation of lymphocyte-activating factor (interleukin-1 or IL-I), which results in the accelerated maturation of cells into effector cytotoxic T lymphocytes and natural killer cells. Lentinan augmentation of macrophage reactivity to macrophage-activating factor appears to result in enhanced formation of effector macrophages. Fruehauf and co-workers (1982) found that lentinan augmented IL-1 production by human monocytes and was able to stimulate IL-1 production by the leukemic cell line K-562, while Masuko and co-workers (1982) noted that it restored and potentiated the delayed hypersensitivity reaction in MM46 mammary carcinomabearing C3H/He mice. Sendo and co-workers (1981)observed that administration of lentinan to BALB/c mice enhanced natural killer cell activity in vitro against a radiation-induced lymphoma and a Molony murine leukemia virus-
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induced lymphoma, and natural cytotoxic activity against a methylcholanthrene-induced fibrosarcoma. Fachet and co-workers (1986) examined the influence of lentinan on the oxazolone-specific antibody response and delayed-type hypersensitivity reaction of BALB/c mice. Both the IgM and IgG humoral-immune responses were increased, but the delayed-type hypersensitivity reaction was only moderately enhanced. Lentinan treatment resulted in a considerable decrease in lethality by anaphylactic shock in both tumor-bearing and control animals. The effect of lentinan on granulopoiesis in BABL/c nude mice was investigated by Matsuo and co-workers (1987a,b).They concluded that mature T cells participate in regulation of granulopoiesis in vivo, and lentinan augments granulopoiesis, at least in part, via mature T cell populations. The in vitro studies of Abel and co-workers (1986, 1989) investigated the effect of lentinan on the pinocytotic and phagocytic activity of macrophages. Pinocytosis of HRP (horseradish peroxidase) and dextran by the murine macrophage cell line CaM4, which exhibits a lower basic pinocytic activity than peritoneal cells, was augmented up to 310 and l Z O % , respectively. Microbead phagocytosis by mouse peritoneal macrophages was amplified u p to goo%, suggesting a P-glucan receptormediated activation of pinocytosis and phagocytosis by lentinan. Cawley and co-workers (1987) studied the effect of lentinan on a number of early-phase host-defense mechanisms and on several clinically relevant sublethal infections in rats. It stimulated an increase in peripheral neutrophil numbers, accompanied by a decreased mobilization of these cells, and it demonstrated anti-inflammatory properties. Shiio and co-workers (1988d)found that lentinan administered subcutaneously (s.c.)to ICR/CRJ mice bearing sarcoma 180 solid tumors caused a regression of tumor growth and increased neutrophils, the footpad reaction to tumor antigen, and T cell and neutrophil chemotaxis. Thyroid-follicular epithelial cells (thyrocytes) have been shown to express a number of functions similar to monocytes. In addition to their functions as endocrine cells, they may also participate in the local immune responses under appropriate conditions. Although spontaneous production of thymocyte-stimulating activity (TSA) was not detected by Hirose and co-workers (1987) when grown in culture medium, TSA was demonstrated in culture supernatants after stimulation with the lentinan. Gergely and co-workers (1988) studied the in vitro effects of lentinan on cytotoxic functions of human lymphocytes in patients with solid tumors and chronic lymphocytic leukemia. Lentinan did not influence blastogenesis and lectin-dependent cell-mediated cytotoxicity, but did increase natural cell-mediated cytotoxicity of tumor-bearing subjects.
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4. Effect on Serum Proteins
A distinct correlation was seen by Chihara and Maeda (1982) between the anti-tumor effect of lentinan and the increase in serum protein components in mouse strains that are high responders to lentinan, but not in BALB/c mice which are low responders. The tumor-inhibition ratio for lentinan was 98.0% (Maeda et al., 1974).Maeda and co-workers (1986) separated and purified the inducing factors for acute-phase proteins and vascular dilatation and hemorrhage from lentinan. The former had a molecular weight of about 1.4 x lo5 and the latter consisted of two components with molecular weights of 3.4 x lo5 and 2.5 x lo5. Biological activities were markedly reduced by treatment with proteinase K or trypsin, indicating that they contain a peptide chain as an active part. One of the three kinds of mouse-serum proteins, ceruloplasmin, similar to human ceruloplasmin, was increased by the administration of lentinan (Itoh et al., 1980). A bioactive factor capable of stimulating the production of the acutephase transport proteins, haptoglobin, hemopexin, and ceruloplasmin was found by Suga and co-workers (1986) in mouse serum soon after lentinan treatment. The acute-phase transport protein-inducing factor (APPIF), which appears to be a peptide compound, was produced by macrophages and may regulate the productions of acute-phase transport proteins in hepatocytes. Appearance of APPIF is considered to be one of the earliest manifestations of the mode of action of lentinan, in addition to its augmented production of vascular dilatation and hemorrhage-inducing factor and IL-1. 5 . Effect on Enzyme Activity
Serum X-prolyl dipeptidyl-aminopeptidase activity, which is depressed in cancer patients, is clearly reduced in mice with Ehrlich carcinoma and sarcoma 180 and is slightly reduced in mice with methylcholanthrene-induced sarcomas. The reduced activity is completely reversed during tumor regression of sarcoma 180 by administration of lentinan (Kato et al., 1979). K. Sasaki and co-workers (1982, 1985, 1986) investigated the effect of lentinan on the hepatic drug-metabolizing enzymes in mice and found that in vivo it decreased cytochrome P-450 content and the activities of aminopyrine N-demethylase, aniline hydroxylase, and 7ethoxycoumarine 0-deethylase in the hepatic microsomes. In vitro it had no effect on aminopyrine N-demethylase or aniline hydroxylase activities. The depressing effect of lentinan on the increase in cytochrome P-448 content induced by 3-methylcholanthrene was much greater than its depression on the increase in cytochrome P-450 content induced by phenobarbital.
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The depression of hepatic microsomal enzyme systems varied with the strains of mice used. The potential use of lentinan as a hepatoprotectant was considered by Lu and Fang (1985). They found that serum glutamic-pyruvic transaminase (GPT) was inhibited in vivo after injection in lab animals and in vitro in the blood of humans and rabbits. Feher and co-workers (1989) studied the effects of lentinan on enzyme-induced lipid peroxidation, xanthine-xanthine oxidaseinduced cytochrome c reduction, and superoxide dismutase (SOD) enzyme activity. In low concentration it decreased SOD activity of lymphocytes and erythrocytes from healthy subjects. In higher concentrations, it increased the low superoxide dismutase activity of erythrocytes and lymphocytes of patients with cirrhosis of the liver. No antioxidant effect was observed in NADPH-induced and Fe3+-stimulatedlipid peroxidation and in a xanthine-xanthine oxidase system. Chen and Lu (1989) found that lentinan was a reversible inhibitor of ornithine decarboxylase. 6. Tumor-Host Systems
Using different murine hosts, Suga and co-workers (1984) confirmed the anti-tumor effect of lentinan in syngeneic and autochthonous tumor-host systems and its suppressive effect on 3-methylcholanthrene (MC)-induced carcinogenesis. They found DBA/2, SWM/Ms, and A/J mice suitable hosts for lentinan treatment. Possibly these strains of mice are most sensitive to delayed-type hypersensitivity and/or cytotoxic T cell response in which T cells and lentinan play an important role. Later investigations (Suga et al., 1989) showed the preventive effects of lentinan on metastasis or recurrence of DBAI2.MC.CS-1 and DBA/ 2.MC.CS-T fibrosarcoma, MH-134 hepatoma, and other murine tumors. Yoneda (1984) examined the effect of lentinan on pulmonary metastases in syngeneic mice bearing Lewis lung carcinoma (3LL). Shiio and co-workers (1987a) found that i.v. injection inhibited pulmonary metastases of 3LL, melanoma (BE),and fibrosarcoma (MC-CS-1)in mice transplanted with the tumor cell S.C.or i.v. as evaluated by tumor weight and number of metastasis in the lung. Rose and co-workers (1984) considered the effect against Lewis and Madison 109 (M109) lung carcinomas implanted in the footpads of syngeneic mice. They had greater success with lentinan alone, or lentinan combined with surgery, in treating the M109 lung carcinoma than in treating 3LL. Jeannin and co-workers (1988)tested the effect of lentinan in a model of colon cancer in rats. Lentinan inhibited the growth of carcinomatosis and increased life span. The effectiveness of lentinan was dependent on the number and frequency of the injections and the dose.
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Miyakoshi and co-workers (1984) indicate that lentinan can inhibit cancer in humans. The activation of killer T cells by a mixed lymphocyte culture was accelerated only when responder cells were mixed with both a suboptimum number of stimulator cells and lentinan. The interferon level in the peripheral blood circulation of cancer patients was elevated following lentinan administration, and natural killer activity of peripheral mononuclear cells was enhanced. Miyakoshi and Aoki (1984) found that augmentation of DNA synthesis of peripheral mononuclear cells (PMNC) occurred both in vitro and in vivo by adding or injecting lentinan. The coexistence of T cells, B cells, and adherent cells (mainly monocytes) was essential. 7. Toxicity and Age Dependence of the Host
Lentinan has only a slightly toxic side effect in in vivo application to animal models and human subjects. O’Hara (1980a,b) traced the fate of 3H-labeled lentinan after injection into mice, rats, and dogs. The radioactivity was predominantly incorporated in the liver, the spleen, and the mesenteric lymph nodes. Toxicity studies of lentinan have been performed by Moriyuki and Ichimura (1980),Ishii and co-workers (1980), and Shimazu and co-workers (1980) using rats and mice. The i.v. LD,, of lentinan in male and female rats was 250-500 mg/kg. Oral and S.C. LD,,s were > 2500 mglkg. The higher i.v. doses produced cyanosis, convulsions, and death. Other evidence of toxicity included enlargement of the spleen, nodules on kidneys, erythema of the ears, hemorrhages in the lungs and abdomen, enlargement of the mesenteric lymph nodes, and edema of the diaphragm and intestine. Kosaka and co-workers (1982) determined that lentinan administered to adrenalectomized and oophorectomized patients had no side effects and increased the patients’ survival; there were no toxic effects on adrenalectomized rats. The effect of lentinan on fertility and general reproductive performance of the rat and on pregnancy of the New Zealand white rabbit has been studied by Cozens and co-workers, (1981a,b,c,d). Reactions were generally dose related with no significant effects on the offspring. Toxiciticy studies of lentinan on the rhesus monkey (Sortwell et a]., 1981) and the beagle dog (Chesterman et a]., 1981) showed that a dose level of 0.5 mg/kg/day was without adverse effect. Shiio and co-workers (1987b) studied the effect of age on the antitumor activity of lentinan and found that it enhanced delayed cutaneous hypersensitivity similarly in aged as well as young mice, and is as effective as an anti-cancer immunopotentiator in aged as well as young animals.
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Tsukagoshi (1988) reported that in studies where lentinan was combined with tegafur for the treatment of inoperable and recurrent gastric cancer, the side effects included eruption and redness, mild pressure in the chest, nausea and vomiting, headache, sweating, flushing, dizziness, feeling of throat obstruction, and decrease of white and red blood cell counts and hemoglobin. All side effects were mild and transient. 8. Use of Lentinan in Combination Therapies According to Shiio and Yugari (1981) lentinan is a strong nonspecific immunopotentiator that will act effectively in living things with observed tumor immunity. However, the anti-tumor effects of lentinan vary with the experimental tumor, whether lentinan is used in combination with a therapeutic agent or another procedure, and the timing of administration in the course of treatment. Chang’s study (1981) of the protection against vesicular stomatitis virus (VSV), the Abelson virusinduced tumor, and the allogeneic trophoblastic tumor in mice underscored the importance of selecting the correct strain for study, and the necessity of determining the optimal conditions for enhancement, especially for boosting natural killer activity. a. Combination with Other Therapeutic Agents. Badger (1984) showed that lentinan in combination with chemotherapy increased survival 50% when compared to chemotherapy alone. Combination therapy (S. Abe et a]., 1982a,b, 1983, 1985) with bacterial lipopolysaccharide (LPS) was very effective against Ehrlich carcinoma in ddY mice and syngeneic mammary carcinoma MM46 in C3HIHe mice, but it was only slightly effective on solid-type MH134 hepatoma and colon 38 adenocarcinoma, and ineffective on ascitic L1210 in CDF, mice. On the other hand, combination with cyclophosphamide (CY) strongly inhibited the growth of solid-type MH134 and colon 38 adenocarcinoma even when administered after tumor inoculation. A combination of lentinan with LPS and streptococcus preparation OK-432 showed that all three components were needed for maximum anti-tumor activity in solid-type tumor MH134. Use of OK-432, CY, and/or lentinan plus LPS against Lewis lung carcinoma in C57BLi6 mice provided a model for combination therapy against weakly immunogenic tumors. Moriya and co-workers (1983, 1984) studied the anti-tumor effect of LPS alone and with lentinan using C3H/He mice bearing MI3134 tumor. In combination, the tumor growth was significantly inhibited as compared to that in the delayed-type hypersensitivity in tumor-bearing mice. Moriyama (1982) and Moriyama and co-workers (1981, 1982) found anti-tumor activity on C3H/He mice with MH134 related to dos-
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age and time of administration. Lentinan with mitomycin-C (MMC), 5fluorouracil (5-FU), cytosine arabinoside (ara-C), and particularly LPS inhibited the proliferation better than lentinan or chemotherapy alone. Ishimura and co-workers (1975) tested Corynebacterium parvum and lentinan, serotonin, or thyroid hormone for potentiation of host resistance. Shiio and co-workers (1983) reported on a synergistic effect with marinactan in inhibiting methylcholanthrene A-induced sarcoma (Meth A) growth in mice. Inagawa and co-workers (1986) showed that the combination of OK-432 and lentinan can be practically applied for endogenous tumor necrosis factor (TNF) induction in clinical trials of cancer therapy. Akimoto and co-workers (1984)found that administration of lentinan following a toxic dose of 5-FU induced protection from mortality in C3H/He mice. Interferon inducers apparently play an important role in the prevention of side effects of cell cycle-specific cytotoxic drugs like 5-FU without decreasing their anti-cancer activity. Matsuo and coworkers (1987a,b) suggested that lentinan could contribute to the recovery from some of the hematopoietic depression in clinical chemoimmunotherapy. Injection of lentinan 1 day after 5-FU resulted in prompt restoration of the leukopenia through the recovery of neutrophils, monocytes, and lymphocytes as well as prompt and marked rebound of granulocyte-macrophage progenitor cells. Jiang and co-workers (1985) observed a positive correlation in sarcoma 180 tumor-bearing mice of increases in CAMPlevel in the spleen, blood, and tumor tissue with tumor inhibition by lentinan (alone or in combination with sheep spleen RNA), Tricholoma matsutake polysaccharide, and sheep spleen RNA, in addition to their enhancement of the immunity function. S. Yamasaki and co-workers (1985) determined that nonspecific and specific immune effector induction was synergistically augmented by lentinan and purified recombinant IL-2. In vivo application of lentinan augmented the in vitro IL-2-triggered induction of lymphokineactivated killer cells (LAK) as well as the similarly induced natural killer cell (NK) activation against a wide range of murine solid tumors. Lentinan also increased the generation of cytotoxic T lymphocytes (CTL) from thymocytes against alloantigens in synergy with IL-2. K. Yamasaki and co-workers (1989) induced significant IL-2-activated killer activity in spleen cells of C57BL/6N mice bearing lung metastasis by injecting a combination of IL-2 and lentinan. Minaguchi (1986) found that lentinan decreased the incidence of cancer in rats treated with N-ethyl-N’-nitroso-N-nitroguanidine (ENNG). It also restored the chemotaxis by peritoneal macrophages,
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lymphocyte blastogenesis in the peripheral blood and mesenteric lymph nodes, and the percentage of T cells in the mesenteric lymph nodes. Shiio and Yugari (1987) studied the suppressive effects of combined use with a chemotherapeutic agent on pulmonary metastasis in mice (Lewis lung carcinoma) after surgical removal of the carcinoma. They found that combined therapy with tegafur or cyclophosphamide with lentinan was most effective when the former was given prior to surgery and the latter following surgery. In contrast, combined treatment with bleomycin and mitomycin C was most effective after surgery. Haranaka and co-workers (1987) found that the anti-tumor activity of recombinant human tumor necrosis factor (rhTNF) against Meth A sarcoma in mice and human tumors in vivo was enhanced when combined with lentinan. Takahashi and co-workers (1988) studied the local induction of a TNF-like cytotoxic factor (CF) in murine tissues (MH134 hepatoma) after administration of anti-tumor polysaccharides, including lentinan. Their findings suggest that CF induction is correlated with anti-tumor activity. Komatsumoto and co-workers (1988) found that a combination therapy of lentinan with UFT [2,4(1H,3H)-pyrimidinedione,5-flUOrO-l(tetrahydro-2-furany1)- with 2,4(1H,3H)-pyrimidinedione]was more effective than UFT alone in preventing the metastasis of primary mammary adenocarcinoma to the lungs of rats after surgical excision of the primary site. Shiio (1988a,b,c)studied the administration of a combination of lentinan and cyclophosphamide in the early growth stages of sarcoma 180 solid tumor in mice and found that the combination gave greater inhibition than either agent alone. An additive anti-tumor effect with cisplatin, adriamycin, carboquone, UFT, tegafur, and bleomycin was also observed. When lentinan was administered with cyclophosphamide, 5FU, or tegafur simultaneously, the suppressed growth of Lewis’s lung carcinoma was stronger than when treated with any of the agents alone. Similar results were observed in B,, melanoma and MM102 mammary carcinoma systems. Studies of lentinan and cyclophosphamide, or lentinan and 5-FU, in C3H/He mice bearing an autochthonous tumor showed neither singular nor simultaneous use was effective. However life was prolonged when administration of lentinan was started after cyclophosphamide or 5-FU treatment. Hasegawa and co-workers (1989) studied the effect of lentinan on the inhibition of mitomycin C-induced sister-chromatid exchanges in mouse bone marrow cells and determined that 23% could be inhibited. Lentinan is not only useful for cancer treatment as an immunopotentia-
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tor in combination with anti-cancer drugs, but may also prevent the increase of chromosomal damage induced by anti-cancer drugs. b. Combination with SurgicallEndocrine Therapy. Kosaka and coworkers (1984, 1985, 1986, 1987) studied the cumulative effects of lentinan and endocrine therapy on the growth of 7,lZ-dimethylbenzanthracene (DMBA)-induced mammary tumors of rats. Tumor growth was inhibited when lentinan injections were combined with surgical-endocrine therapy (adrenalectomy and ovariectomy) but not when combined with medicinal-endocrine therapy (tamoxifen treatment). Surgical-endocrine therapy was associated with an infiltration of macrophages and T lymphocytes into the mammary tumors, depletion of estrogen receptors and progesterone receptors in the tumors, and the lowering of blood prolactin levels. This combined therapy is being used clinically with women who have recurrent breast cancer. Patients treated with lentinan showed longer disease-free intervals and a much higher survival rate than controls. Shiio (1988d,e) found that the administration of lentinan after surgical tumor resection suppressed the natural metastasis of B,, melanoma, L1210, and LSTRAm tumors, and showed better results than those observed with the administration of lentinan before tumor resection. Postoperative administration of lentinan was also effective in prolonging the life of mice bearing a second tumor transplant in a nonnatural metastatic tumor system, such as MM102 and colon 26. Lentinan did not prolong life in mice with ascites sarcoma 180 tumor; when ascites tumor cells were transplanted i.p. into mice with solid tumor, it prolonged life and increased the immunity induced by the sarcoma 180 solid tumor, and the residual immunity after resection of the solid tumor. In this system, lentinan suppressed recurrence when administered either before or after surgery. c . Combination withX-Ray Therapy. Shiio and co-workers (1988a,b,c) found that X-ray irradiation and lentinan treatment of mice bearing solid-type sarcoma 180 had an additive effect with the administration of lentinan before or after X rays. C3H/He mice with syngeneic MM102 tumor transplanted S.C. in the footpad were used to study the timing of administration of lentinan. In combination with 2000-3000 rads of irradiation, tumor growth was decreased compared to groups that received radiotherapy or lentinan alone. Lentinan administration before or after irradiation had similar effects. A combination with X-ray therapy prolonged the life of BDF, mice bearing L1210 leukemia and sup-
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pressed the growth of KLN205 squamous cell carcinoma and the metastasis of Lewis lung carcinoma in mice. 9. Other Beneficial Effects a. Anti-parasite Activity. Byram and co-workers (1979) observed that administration of lentinan to thymus-intact mice by i.p. injection resulted in the formation of conspicuously enlarged lung granulomas in response to either Schistosoma mansoni or S. japonicum eggs or to antigen-coated beads. White and co-workers (1988) studied its effect on the resistance of CBA/H mice to Mesocestoides corti. Increasing prophylactic and therapeutic doses resulted in a marked reduction in the numbers of parasites in the peritoneal cavity, particularly in those mice that received lentinan therapeutically. Encapsulated parasites were observed to be dead or dying, and damage appeared to be mediated by increased numbers of macrophages and giant cells.
b. Antibacterial Activity. Sakamoto and co-workers (1983) investigated the effect of lentinan treatment on host resistance of malnourished rats against Listeria monocytogenes infection. Administration induced complement C3 elevation in vivo, and the C3 enhancement increased the resistance to infection. Iguchi and co-workers (1985) studied bacterial infections in mice with neutropenia caused by cyclophosphamide or fluorouracil. Administration strengthened resistance against infections with Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae by 10 to 100 times. Lentinan is effective in augmenting resistance against bacterial infections in a host immunocompromised by anti-cancer agents. Kawanobe and co-workers (1988) examined the ability of lentinan to increase host resistance to certain bacterial infections in malnourished rats through complement C3 activity. The administration of Znchlorophyllin and 2 mg/kg of lentinan enhanced C3 levels and C3b and C3bi formation. Apparently, phagocytic activity or the clearance capacity of macrophages is augmented through the interaction of macrophages and increased C3bi formation. Yano and co-workers (1989) found that lentinan could enhance the resistance of carp Cyprinus carpi0 to experimental bacterial infection of Edwardsiella tarda by activating the nonspecific immune system. c. Anti-fungal Activity.
H. Chen and co-workers (1987) investigated
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the therapeutic effects of lentinan against Candida infection. The peritoneal macrophages (PECs) from lentinan-treated mice showed a strong inhibitory activity against germ tube formation of C. albicans. Ohno and co-workers (1986) showed that it was effective in the augmentation of candidastatic activity of the murine macrophage cell line J177.1 in vitro. d. Antiviral Activity. Lentinan used in combination with 3'-azido3'-deoxythymidine (AZT) suppressed the surface expression of human immunodeficiency virus (HIV) antigen more strongly than AZT alone. Tochikura and co-workers (1987) showed that it can enhance the effect of AZT on replication of HIV in various human hematopoietic cell lines in vitro. Additional investigations (Tochikura et al., 1988, 1989) measured the effects of lentinan sulfate and E-P-LEM against human retroviruses. The substances almost completely blocked cell-free infection of HIV-1 and HIV-2 and inhibited cell-to-cell infection by HIV-1, HIV-2, and HTLV-I. Moreover, reverse transcriptase activity of avian myeloblastosis virus was inhibited. Yoshida and co-workers (1988) found that sulfated lentinan inhibited HIV-induced cytopathic effect and viral antigen synthesis in HIVinfected MT-4 cells. It showed >98% reduction of reverse transcriptase activity of avian myeloblastosis virus. Hatanaka and Uryu (1989) showed that sulfonated lentinan had anticoagulant and antiviral (HIV) activities, but complete sulfonation of the C-6 carbons and a high molecular weight were necessary for the biological activities. Lentinan sulfate with an S content of >13.9% effectively prevented HIV-induced cytopathic effects in an HTLV-Icarrying cell line (MT-4) in vitro at concentrations of >3.3 pgiml (Hatanaka et al., 1989). Tochikura and co-workers (1988) fractionated an extract of culture medium (LEM) and both the resulting product (E-P-LEM) and LEM were studied for their effect on the activity of HIV in vitro. The experiments were performed using either a cell-free infection system with MT-4 cells, or a cell-to-cell infection system with MOLT-4 cells, which induces multinucleated giant cells. E-P-LEM almost completely blocked both the cytopathic effect of giant cell formation and specific antigen expression due to HIV, whereas LEM before ethanol precipitation blocked the expression of HIV antigen in MT-4 cells only at a high concentration. Pretreatment of the virus with E-P-LEM before infection blocked HIV infection in the target cells. Thus, the inhibitory effect on HIV could be due to a blocking of the initial stages of HIV infection.
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H. Suzuki and co-workers (1989) showed that fractions from an extract of the mycelium culture medium (LEM) activated mouse macrophages, caused proliferation of bone marrow cells, and inhibited the replication of HIV virus in vitro. The EPS4 fraction was composed of water-soluble lignins containing minor amounts of protein (3.2%) and sugars (12.2%). The active principle in the fraction EPS3 is a highly condensed and polycarboxylated lignin which is denatured and solubilized by L. edodes from bagasse (H. Suzuki et ~ l . 1990). , e. Effect on Low Natural Killer Syndrome. Low natural killer syndrome (LNKS)is a newly proposed category of immune disorders being characteristically diagnosed by lowered NK cell activity against K562 target cells as a definite laboratory abnormality, with general clinical symptoms of remittent fever and uncomfortable fatigue, persisting without explanation for more than 6 months. It is independent of AIDS or the AIDS-related complex. LNKS patients responded well to the administration of lentinan, despite no responses to conventional fever treatments (Aoki et al., 1987)
f. Anti-diabetic Activity. Satoh et al. (1988) studied the effects of various biological response modifiers, including lentinan, on insulindependent (Type I) diabetes mellitus in nonobese diabetic (NOD) mice. Lentinan inhibited development. g. Radioprotection. Matsubara and co-workers (1988) studied the radioprotection action of metallothionein induction with lentinan in mice exposed to X rays. Increases in survival induced by pretreatment with heavy metals and immunostimulants were similar, but Zn used in combination with an immunostimulant appeared to produce optimal protection. Further studies (Matsubara et d., 1989) substantiated that lentinan enhanced survival of male ICR :Jcl mice subject to whole body X-irradiation. Chirigos and Patchen (1988) surveyed the ability of biological response modifiers to restore and/or counteract the suppressive effects of the radiomimetic drug cyclophosphamide and total-body irradiation. Patchen and co-workers (1988) studied the potential use of 1 7 immunoregulators for radioprotective efficacy in female C3H/HeN mice. Significant radioprotection, based on enhanced survival following wholebody irradiation, was observed for lentinan, which also stimulated he-
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TABLE I MEDICINAL BENEFITSOF THE SHIITAKE MUSHROOM Benefit
Compound
Antibiotic Anti-parasitic
Polysaccharide lentinan
Antibacterial
Polysaccharide lentinan
Anti-fungal
Polysaccharide lentinan
Anti-tumoI
Polysaccharide lentinan Nucleosides and/or Nucleic acid derivatives Polysaccharide lentinan
Antiviral
peptidomannan (KS-2) Double-stranded RNA Polysaccharide LAP1 Double-stranded RNA
Anti-diabetic Anti-thrombotic
Peptidomannan (KS-2) Polysaccharide AcZP Polysaccharide lentinan Lentinan sulfate LEM
Hy pocholesteremic (Hypolipidemic)
E-P-LEM EPS3 and EPS4 Eritadenine
Immunomodulatory
Polysaccharide lentinan
Radioprotection
Polysaccharide lentinan
Reference Byram et a]., 1979 White et al., 1988 M. Sakamoto et al., 1983 Iguchi et al., 1985 Kawanobe et al., 1988 Ohno et al., 1986 Chen et al., 1987 Satoh et al., 1988 Hokama and Hokama, 1981 Chihara et al., 1970 Dennert & Tucker, 1973 Hamuro et al., 1976 Fujii et al., 1978 Takehara et al., 1981 Sugano eta]., 1982 F. Suzuki et al., 1976 Takehara et al., 1979 F. Suzuki et al., 1979 Yamamura and Cochran, 1976b Tochikura et al., 1987 Tochikura et al., 1988, 1989 Tochikura et al., 1988 H.Suzuki et al., 1989 Tochikura et al., 1988, 1989 H. Suzuki et al., 1990 Kaneda and Tokuda, 1966 Tokita et al., 1972 Yamamura and Cochran, 1976a Suzuki and Ohshima, 1976 Tokuda and Kaneda, 1979 Kurasawa et al., 1982 Tsunoda and Ishida, 1969 Suzuki et al., 1976 Hamuro et a)., 1976 Chirigos and Patchen, 1988 Patchen et al., 1988
matopoiesis. Results indicated the potential use of immunomodulators for protection against acute radiation injury and hematopoietic enhancement alone may not be sufficient to enhance survival following irradiation.
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Ill. Patented Products and Processes
A. ANTI-HYPERTENSIVE AND ANTI-CHOLESTEREMIC COMPOSITIONS A germanium-rich beverage from L. edodes is both anti-hypertensive and anti-cholesteremic (Iizuka, 1982). A health food preparation containing shiitake has an anti-cholesteremic effect (Y. Abe and Kaneda, 1986). Manufacture of cholesterol-lowering and immunoactivating eritadenine was accomplished by fusing L. edodes and Collybia velutipes (Nippon-Food, 1987). B. ANTIBIOTICS
Various antibiotic substances have been obtained from Lentinula (Shiio et al., 1973). Lentinan can be used for Pseudomonas infection control in animals. Administration increased their survival 70% (Ajinomot0 Co., Inc., 1985). Lentiallexine (octa-7-en-3,5-diyn-l-o1) is obtained when L. edodes is cocultured with Trichoderma sp. (Mitsubishi Chem. Ind. Co., Ltd., 1988). C. VIRICIDESINCLUDING ANTI-AIDSAGENTS An anti-tumor viricide and fungicide for the treatment of verruca and collagenosis is produced from the mycelium and used culture medium of Basidiomycetes, preferably L. edodes. Xylose is the main component (Noda-Inst., 1983). Soluble protein extracted from L. edodes fruiting body (FBP)is useful as a viricide (Nikken Chem. Co., Ltd., 1986). A nontoxic extract from the nutrient medium and tissue medium of Basidiomycetes, such as L. edodes, is effective against viral hepatitis. The active components are a mixture of polysaccharides and cytokinin substances, mainly zeatin and zeatin riboside (Iizuka, 1986). Several compositions are used as anti-AIDS drugs. LEM-HT, extracted from cultured Lentinula mycelium, can control the reduction of T lymphocytes due to viral infection by stimulating macrophages and IL-1 activity (Iizuka and Maeda, 1988).Another is obtained from Basidiomycetes, especially L. edodes (Iizuka et al., 1990, 1992). A method for the inhibition of virus infection involves sterilization of devices with a dilute mycelial extract from L. edodes, which acts as a therapeutic agent against HIV virus and hepatitis B virus (Noda-Food, 1989). Lentinan sulfate was shown to be active against HIV when tested in MT-4 cells infected with HTLV-I11 (Yamamoto et al., 1989).A viricide composition to prevent the multiplication of HIV virus, herpes virus, and hepatitis
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B virus was obtained by fermentation of vegetable fiber by Basidiomycetes, especially L. edodes. The product of the extraction is in the form of the brown powder LEM (Noda-Food, 1990a). The polysaccharide fraction of an aqueous extract of a mycelial culture of L. edodes with a molecular weight of 1 x lo5-1 x lo6 is used in preparation of a drug for prevention or therapy of herpes simplex virus, cytomegalo virus, or Epstein-Barr virus infections (Nippon Chem.: Noda-Food, 1990; Koga et al., 1991). A similar compound suppresses replication of HIV virus, inhibits the adsorption of HIV virus onto host cells, and inhibits the activity of reverse-transcriptase. It is nontoxic with no side effects (Noda-Food, 1990b).
D. NEOPLASM INHIBITORS The first neoplasm inhibitor was identified as a glucan (Chihara et al., 1972). Emitanin (T. Suzuki and Ikegawa, 1977) and emitanin-1 (Yamamoto and Ikegawa, 1980) also have anti-cancer properties. An anti-tumor polysaccharide can be obtained cultivating L. edodes on bagasse (Japan Synthetic Rubber Co., Ltd., 1978). Anti-cancer polysaccharides are stabilized and anti-cancer activity is increased synergistically when the polysaccharides are dissolved in water in the presence of water-soluble high molecular weight compounds and monosaccharides. The addition of a water-soluble dextran increases the solubility of lentinan (M. Fujii et al., 1980). Liposaccharides isolated from bacteria (Proteus vulgaris) have been combined with lentinan for use as synergistic neoplasm inhibitors (Ajinomoto Co., Inc., 1981). E. IMMUNOREGULATORY SUBSTANCES
KS obtained from the cultured mycelium of L. edodes and refined KS-2-A enhance the host defense function (Ishida et al., 1979a; Kirin Brewery Go., Ltd., 1980). KS-2-B (Ishida et a]., 1979b, 1981) and KS-2D (Kirin Brewery Co., Ltd., 1981) are also effective interferon-inducing substances. A combination of human interleukin 2 (IL-2) purified from various cell lines and lentinan is useful as an anti-tumor agent (Yoshimoto et al., 1983,1988).Lentinan has been used as an immunotherapeutic agent for the control of neoplasm and infection (Ajinomoto Co., Inc., 1984). A tumor therapy has been described using murine monoclonal antibodies to carcinoma, melanoma, and pancreatic carcinoma as anti-tumor agents and lentinan as a macrophage activator to enhance the anti-tumor activity of the antibodies. Antibody-dependent macrophage-mediated
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cytotoxicity has been demonstrated using the antibodies and the lentinan-stimulated macrophages against various human tumors in vitro (Herlyn, 1986, 1992). An immunostimulant comprising an N-containing low molecular substance and sugar protein containing xylose is obtained from the mycelium culture medium of a Basidiomycete, such as L. edodes (NodaFood, 1984a; Sugano et al., 1984). This composition is useful for the treatment of cancer by intraperitoneal or oral administration. An extract composed of two peptoglycans from the mycelium and culture liquid enhances humoral and cell-mediated immunity to a wide range of diseases, e.g., hepatitis, influenza, and herpes virus infections, cancer, immunodeficiency disorders, and mycotic infections (Sugano et al., 1985). A preparation for treatment of kidney inflammation without reducing immunity, which consists of a saccharide and a protein, is obtained from the mycelium culture broth of Basidiomycetes, such as Lentinula, by fermentation in a culture medium rich in xylose (NodaFood, 1986). A high molecular weight immunostimulant containing mostly neutral sugar and protein extracted from the spawn of the fruit body of L. edodes grown in a solid medium containing cellulose is useful for the treatment of chronic-type hepatitis B and other immunodeficiencies (Noda-Food, 1987). F. ANTI-ULCER COMPOSITION
Ulcer-suppressing agents containing an extract of the mycelium of L. edodes may be administered orally, as suppositories, or by injection (Mitsubishi, 1983). G. ANTI-CLOTTING COMPOSITION
Pharmaceuticals contain lentinan for the prevention and therapy of disseminated intravascular clotting. The efficacy was demonstrated in rats with experimentally induced disseminated intravascular clotting (Res. Dev. Corp. of Japan, 1987).
H. ANTI-ASTHMA COMPOSITION A drug for asthma and treatment for cancer and skin disease contains the extract of a cultivated mycelium of a Basidiomycete, such as Lentinula. The mycelium cultured on a medium containing horse excrement contains saccharides, primarily xylose, and protein (Noda-Food, 1984b).
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I. BONE FORMATION ACCELERATOR An accelerator of bone formation contains lentinan. The pharmacological effects were shown in rats with bone damage (Res. Dev. Corp. of Japan, 1988).
J. DERMATOLOGICAL COMPOSITIONS A bathing composition employs the mycelium of a Basidiomycete, such as L. edodes. The method of preparation allows all of the pharmaceutically active ingredients contained in the mycelium, to be utilized (Nikkei Co., Ltd., 1986).A composition for external application to regenerate and revitalize damaged cells has been found useful in the cosmetic and pharmaceutical fields (Yamada and Yamada, 1992).
K. POSTOPERATIVE TREATMENT A prophylactic and therapeutic treatment of complications after lensectomy utilizes lentinan (Taiho Pharmaceutical Co. Ltd., 1990). L. ASSAYPROCESSES
P-1,3-Glucan has been used as part of the chain reaction-triggering substance in liposomes for a simple and highly sensitive lysis immunoassay. The change in viscosity was measured for detection of p-1,3glucan release from the liposomes to determine the antibody detection (Seiko Instruments & Electronics, Ltd., 1989). An anti-lentinan antiserum has also been employed in immunoassays (Ajinomoto Co., Inc., 1992). The concentration of endotoxin in an unknown sample can be determined by using the reaction of a horseshoe crab hemocyte lysate with endotoxin in a solution with a water-soluble polysaccharide containing p-1,3-glucosidic linkage, or a derivative containing the linkage, such as that found in lentinan (Matuura and Tsuchiya, 1993). IV. Discussion
In carefully controlled laboratory studies the shiitake mushroom has been shown to contain components effective in the treatment of cancer, heart disease, and diseases caused by viral infections. Animal models have demonstrated that these biologically active principles may exert totally different effects depending on the dose, route of administration, and the condition of the host. It is also apparent that similar types of
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S. C. JONG AND J. M. BIRMINGHAM TABLE I1 PATENTED PRODUCTS AND PROCESSES Product or process
Anti-asthma composition Antibiotics
Anti-clotting composition Anti-hypertensive/ anti-cholesteremic compositions Anti-tumor compositions (neoplasm inhibitors)
Anti-ulcer composition Antiviral compositions [including anti-AIDS agents)
Bone formation accelerator Dermatological compositions Immunoregulatory substances
Postoperative treatment Assay processes
Patent Noda-Food, 1984b Shiio et aI., 1973 Ajinomoto Co., Inc., 1985 Mitsubishi Chem. Ind. Co., Ltd., 1988 Res. Dev. Corp. of Japan, 1987 Iizuka, 1982 Abe and Kaneda, 1986 Nippon-Food, 1987 Chihara et al., 1972 T. Suzuki and Ikegawa, 1977 Japan Synthetic Rubber Co., Ltd., 1978 Yamamoto and Ikegawa, 1980 Ajinomoto Co., Inc., 1981 Fujii et ol., 1980 Mitsubishi, 1983 Noda-Inst., 1983 Nikken Chem., Co., Ltd., 1986 Iizuka, 1986 Iizuka and Maeda, 1988 Noda-Food, 1989, 1990a, 1990b Yamamoto et al., 1989 Nippon Chem.: Noda-Food, 1990 Iizuka et al., 1990, 1992 Koga et al., 1991 Res. Dev. Corp. of Japan, 1988 Nikkei Co., Ltd., 1986 Yarnada and Yamada, 1992 Ishida et al., 1979a, 1979b, 1981 Kirin Brewery Co., Ltd., 1980, 1981 Yoshimoto et ol., 1983, 1988 Ajinomoto Co., Inc., 1984 Noda-Food, 1984a, 1986, 1987 Sugano et al., 1984, 1985 Herlyn, 1986, 1992 Taiho Pharmaceutical Ltd., 1990 Seiko Instruments ?i Electronics, Ltd., 1989 Ajinomoto Co., Inc., 1992 Matuura and Tsuchiya, 1993
effects can be elicited by structurally diverse molecules. Two of the most promising and effective principles isolated are lentinan and LEM. Lentinan first extracted from the fruiting body of L. edodes is a pure P-1,3-glucan containing only glucose. It appears to act as a host-defense potentiator which can improve the physiological constitution of the
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host against cancer, as well as various kinds of infections, and restore or augment the ability of responsiveness of the host to bioactive substances, such as lymphokines or cytokines. Lentinan has been shown to exert prominent anti-tumor effects in murine allogeneic, syngeneic, and autochthonous hosts, to prevent chemical and viral carcinogenesis, to increase host-resistance to bacterial, viral, and parasitic infections, and is effective against HIV or AIDS infections. Remarkable life span prolongation has been achieved in patients with advanced and recurrent stomach, colorectal, and breast cancer. Lentinan is commercially available for clinical use. In 1987 it was the eighth top-selling anti-cancer drug in Japan with a 2.2% share of the market valued at $3 million (Fukushima, 1989). The whole extract of L. edodes mycelial culture (LEM)and its purified fractions have antiviral activities and immunomodulating functions. The active principle in the EP3 fraction and its lower molecular weight fraction (EPS4) has been identified as a highly condensed and carboxylated lignin. LEM inhibits the infectivity of HIV and cytopathic effects on virus-infected cells in vitro, enhances IL-1 production, activates murine macrophage functions, promotes proliferation of murine bone marrow cells, suppresses proliferation of rat ascite hepatoma AH414, and promotes seroconversion from HBe antigen to anti-HBe antibody in chronic hepatitis B patients. LEM has been shown to be effective in AIDS therapy and hepatitis B therapy by oral administration. The use of cultivated edible mushrooms, such as L. edodes, as a source of biologically active principles offers obvious advantages. Edibility increases the likelihood of a safe, tolerated principle, while cultivability assures an adequate supply. However, cultivation of the mushroom need not be a limitation when active principles can be derived from its mycelial cultures, as is the case with LEM. The biologically active components isolated and identified in edible fungi show great promise and should be further exploited for their therapeutic effects, either as dietary components or as purified drugs. The cure for the diseases that plague mankind, particularly cancer and AIDS, may lie within the biochemistry of edible mushrooms.
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Yeast Lipid Biotechnology Z. JACOB' Fermentation Technology and Bioengineering Discipline Central Food Technological Research Institute Mysore 570 013, India
I. Introduction 11. Yeasts as Potential Sources of Lipids 111. Importance of Yeast Lipids in Beverages and Foods A. Beer and Wine B. Dairy and Baked Products C. Oriental Foods and Pickles IV. Medical Importance of Yeast Lipids V. Modification of Lipids A. Fermentative Synthesis and Modification of Lipids B. Genetic Engineering Aspects of Yeast Lipid Modification C. Chemical and Biochemical Interesterifications VI. Commercial Significance of Yeast Lipid Biotechnology VII. Conclusion References
I. Introduction In vitro tissue culture (somatic, meristem, and shoot tip) techniques are now available for the clonal propagation of oil seed plants (Pandey, 1989a). In addition to the above, there are many examples in plant science of the genetic modification of oil seeds (rape, flax, sunflower, safflower, soybean) and transgenic seeds, which are now available to produce lipids of desired composition (Khatoon, 1991; Voelker et a ] . , 1992). Certain yeasts are also considered as potential lipid producers. Recent developments in yeast biotechnology are the results of the search for novel compounds, life-saving biopolymers, and modified food components and medicine (carbohydrates, protein, and lipids),which otherwise may not easily be synthesized by chemical pathways (Hodgson, 1991; Dixon, 1991).The integrated approaches of biotechnology, recombinant DNA technology, and fermentation technology have made the area of research on lipid biotechnology more challenging and attractive. During fermentation of various bioorganic substrates, the yeasts synthesize and store lipids intracellularly. Some species of yeasts such as Rhodotorula, Lipornyces, and Candida produce lipids closer to vegeta-
' Present address: Department of Medicine, Division of Oncology, V.C. 12-238,College of Physicians and Surgeons of Columbia University, New York, New York 10032. 185 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 39 Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ble oils and fats. Tapping of such alternate sources has assumed greater importance in the last few decades because of the widening gap between the total demand and production of vegetable edible oils and fats in many developing countries. Although many efforts have been made, limited success has been achieved in this regard mainly because of the lower productivity of the oleaginous biomass and because of many technological constraints in the downstream processing of the product. When comparing the time-consuming process of plant oil seed production to that of the yeast biomass, the latter has an edge over the former in the ease of cultivation in a shorter amount of time. However, yeasts and their biotechnological application in obtaining desired lipids have not received adequate attention as compared to the oil seeds. While granting the inherent loopholes of the use of yeasts for mass scale production of useful lipids, there has been a reorientation in thinking toward the production of value-added lipids or novel lipids, using the integrated approach of biotechnology. Recent attempts of insertion and expression of desired intraspecific traits have not fully fulfilled the goal of higher productivity of edible oils and modified lipids. This chapter considers the state of the art role of yeasts in lipid biotechnology and discusses their prospects in food and medicine. II. Yeasts as Potential Sources of Lipids
These eukaryotes produce beneficial products such as alcohol, beverages, single cell protein (SCP), and many biochemicals used in the food and pharmaceutical industries. As noted earlier, some yeasts also produce beneficial lipids and lipid-containing emulsifying compounds (Jacob 1989; 1992). The advantages of using yeasts as lipid producers are that (1) they produce lipids similar to vegetable oils and fats, (2) they can be grown reasonably well on cheap agroindustrial and food industrial wastes, (3) their lipids can be produced at a faster rate in bulk in large capacity reactors than the usual time-consuming agricultural practices, and (4) most of the potential lipid producers and their products seem to be relatively nontoxic to humans. Yeasts use carbohydrates as precursors to synthesize lipids by enzymatic pathways (Ratledge, 1982; Ratledge and Evans, 1988; Guerzoni et a]., 1985; Rattray, 1988; Holdworth et al., 1988). The lipid composition, quality, and amount vary from species to species according to the growth stage, optimal availability of essential nutrients, and the conditions of the reactor. However, normally, the ability to catabolize a specific substrate or precursor to a desired lipid component is limited. Increased productivity of a specific lipid component requires the control of specific enzymatic
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pathways. Specific lipid modifications may be possible by (1)rerouted metabolic pathways (stimulatory or inhibitory) under optimized conditions of growth in the presence of higher amounts of precursors or suitable substrates, (2) intraspecific cloning and expression of desired genes in the organism, (3) biochemical conversion of the postharvest lipids by enzymatic conversions or direct fermentation with a suitable microorganism, and (4) chemical conversion. Once the modifications are made by biochemical or chemical means the total lipid can be extracted by a suitable solvent system that is similar to vegetable oil extraction and from it desired components may be isolated by fractional extraction using supercritical CO, or by column separation. Ill. Importance of Yeast Lipids in Beverages and Foods
A. BEERAND WINE The primary contribution of brewer’s yeast to beverages is the production of ethanol and organoleptic and quality determining compounds such as aldehydes, ketones, lower fatty acids, and esters (Johnson et al., 1958; Wiseblatt, 1960). The quality of beer and wine depends on the type of fermentation, substrate supplied, and the yeast growth conditions (Kirsop, 1977, 1988; Berry and Watson, 1987). During vinification of grape juice, different species of Saccharomyces [chevaliere, carlsbergiensis, fructum) produce varying quantities of acetic, n-butyric, ncaprioc, n-caprylic, n-capric, 9-decenoic, succinic, formic, propionic, isobutyric, 2-methyl butyric, isovaleric, lactic, 2-hydroxycaproic, nperlargonic, and malic acids, higher alcohols (fuse1 oil), and esters (Margalithi and Schwartz, 1970; Berry and Watson, 1987). Many metabolic changes related to lipids also occur. During champagnization the wine yeasts proliferate and liberate diverse lipids, thus enriching the wine lipids (Kishkovskii et al., 1986). The improvement of sparkling and foaming properties of sparkling wine depends on the type of yeast and the chemical properties of the surfactants used for wine fortification (Razmadze, 1985). The performance of pitching yeasts during brewing also depends on the intracellular lipid content (Sayle, 1986). Optimum aeration results in the synthesis of an amount of lipid necessary for desirable fermentation (Ohno and Takahashi, 1986).Wort aeration, temperature, and oxygen supply strongly affect the lipid composition (Ohno and Takahashi, 1983). Oxygen is essential for unsaturation and cyclization of squalene to lanosterol. In the absence of oxygen, no phospholipids and triacylglycerols are synthesized. In oxygen-limited fermentations the toxic decanoic fatty acids are adsorbed by the yeasts (Munoz
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and Ingledew, 1989). Pitching yeasts are rich in unsaturated fatty acids like palmitoleic and oleic acids. The synthesis of oleic acid requires more oxygen and time (Ahvenainen, 1982). During beer fermentation, the intracellular unsaturated lipids help maintain the cell viability as well as the ethanol tolerance of the yeast (Rose, 1978). Taylor et al. (1979) reported that beer fermentation requires externally supplied lipids such as sitosterol, unsaturated fatty acids of spent grain lipids, and medium chain fatty acids as well as a higher oxygen supply. These factors cause favorable changes in the patterns of fatty acid composition, sterols, decreased content of esters, medium chain fatty acids, and increased fuse1 alcohol content. During wort production, the brewer’s yeast liberates more lipids with unsaturated fatty acids and sterols (Pfisterer et a]., 1977). The influence of yeast lipids on beer flavor is substantial and is growth related. Freshly synthesized fatty acids may be excreted and impart caprylic flavor to the beer and are primarily controlled by the oxygen supply. Aries et al. (1977) reported that in the presence of oxygen the fermented wort (produced by pitching yeast) contains a complex mixture of unsaturated fatty acids of varying chain lengths. During anaerobic fermentation, increased medium chain fatty acids are produced and a major fraction of them are excreted. Acetoin is also produced when ergosterol is fed in the presence of oxygen during brewing (Haukeli and Lie, 1976). In wine fermentation the lipid content of the yeast increases, regardless of the method of fermentation. However, maximum accumulation was noticed during the aerobic process and was the lowest in pressurized CO, conditions. Wine lipids contain C14 to C24 fatty acids. The major fatty acids are palmitic, stearic, palmitoleic, oleic, linoleic, and linolenic acids. The degree of anaerobiosis, temperature, CO, concentration, and ethyl alcohol formation affects the lipid content and its degree of unsaturation (Portnova, 1981). Abdurazakova et al. (1982) reported that the presence of unsaturated long chain fatty acids stimulates the production of lipase in Saccharomyces vini (wine yeast) and that maximum activity is observed at the exponential phase of growth. Chemiluminescence as well as the lipid content of the wine are also found to be affected by the oxygen supply during wine fermentation using S. vini (Magomedov and Portnova, 1977). Sparkling wine yeasts transfer lipids and proteins to the external medium and these depend on the nitrogen supply which affects the foaming characteristics (Razmadze et al., 1980). Commercially, the flavor of apple wine is enhanced by adding lipases obtained from Candida sp. or Rhizopus delamar (Tanabe Seiyaku Co., 1970). During wort fermentation, using S. cerevisiae, the extent of yeast growth is related to the cellular sterol levels, and the
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survival of yeasts is correlated with unsaturated fatty acid residues which in turn affect the cell physiology, particularly the membrane structure and function (Day et a]., 1976). B. DAIRYAND BAKEDPRODUCTS
As mentioned earlier, microorganisms are also responsible for desirable flavors in fermented and baked foods. Microorganisms utilize the supplied substrates and precursors and transform them to organic acids, aldehydes, ketones, alcohols, esters, and many other compounds, which, in totality, makes the dairy and baked products more appealing and acceptable. Bacteria and yeasts that possess lipolytic (fat splitting) activity are found in many proteinaceous and fatty foods (Davis, 1970; Scheibner, 1970). The action of bacteria is quite evident in imparting good sensory feeling to most of the fermented products, while the role of yeasts seems to be minimal. Little work has been reported so far on the contribution of different yeast species to the final flavor-determining components of dairy and baked products. Microbial lipases play an important role in the development of cheese flavors (Posorske, 1984). In situ synthesis of bacterial lipases has been reported in Dutch, Swiss, Gouda, and cheddar cheeses (Seitz, 1974). In Roquefort cheese, molds like P. roqueforti and Mucor mehii give a characteristic flavor and odor. The spores of P. roqueforti split the triacylglycerol to fatty acids which are further transformed to methyl ketones by an oxidase enzyme. It is the lipase activity of P. roqueforti that yields caprylic, capric, and caproic acids which may then be involved in the formation of methyl ketones (cheddar cheese flavor). However, in the ripening of Camembert cheese, various film yeasts (yeast-like fungus) belonging to the genera of Geotrichum apparently contribute a thin surface growth and reduce the acidity of the cheese before the main mold P. camemberti establishes its acivity. However, the involvement of yeasts and their production of flavor components (using the supplied lipids or precursors) in cheeses are relatively unknown. Except for a few instances, it is believed that yeasts have a smaller role than molds and bacteria. This observation does not seem to be correct. It was reported that lower fatty acids are Eormed as a result of carbohydrate metabolism. Higher fatty acids are produced by lipolysis by Micrococci, gram-negative bacteria (Reiter et a]., 1967), and probably to a certain extent by yeasts. Carini and Volonterio (1969) reported that all of the 20 strains belonging to 8 different species of Torulopsis isolated from Taleggio cheese possess intracellular milk fat hydrolyzing (lipolytic) activity. Peters and Nelson (1961) reported that
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supplementation with lipases produced by C. lipolytica could improve the quality of blue cheese. In limburger cheese production, the yeasts Debaromyces kloeckeri and C. mycoderma liberate organoleptic fatty acids (myristic, palmitic, palmitolein, stearic, and oleic acids). Baker’s yeast finds most of its utility in bread manufacture. This yeast is responsible for the fermentative synthesis of alcohol and carbon dioxide. Many bacteria are also involved in the process of dough raising. Both bacteria and yeast produce an array of chemical compounds that are responsible for the final flavor and taste. Distinguishing between the roles played by yeast and bacteria in the formation of such characteristics is difficult. However, the cumulative fermentative action of both causes chemical changes in a variety of fatty acids, organic acids, alcohols, ketones, aldehydes, and carbonyl compounds (Kohn et al., 1963).
c. ORIENTAL FOODSAND PICKLES Oriental foods like tempeh are among the richest sources of fatty acids and other organic compounds needed for human health. Herring et al. (1991) observed that when the mold Rhizopus sp. is forced to synthesize lipids de novo, an increased percentage of up to 21% of ylinolenic acid is present in fermented Indonesian tempeh. In addition to these mold species, many bacteria are also encountered during tempeh fermentation. However, in general there has been no special mention about the role of yeasts in the fermentation of oriental foods in reviews by Hesseltine (1965), Hesseltine and Wang (1967), and Saisithi et al. (1966).It is believed that yeasts have a smaller role in the development of flavor and odor than molds and bacteria. However, in miso fermentation the soybean oil (present in the lipid substrate) is digested by koji lipase to produce some fatty acids. Many osmophilic yeasts (Zygosaccharomyces major var miso, Z. sdoja var miso; spore-forming Saccharomyces zygopichia, Debaromyces, Zygosaccharomyces, Hansenula, Pseudohansenula, Pichia, and non spore-forming Torulopsis) are also involved in miso preparation (Hesseltine and Wang, 1967), but their role in flavor development is unknown. Alcohols contribute to the pleasant smell of miso. Some yeasts and bacteria also make films and affect the odor. The presence of osmotolerant yeasts in Marzipan, fermented wine, butter, and fat-based foods have been reported (Mohs, 1974),but their roles are not clear. Most of the yeasts found in margarine and butter are lipolytic in nature (Aeyraepaeae and Lindstrom, 1974). Certain yeasts (Candida citeromyces, Debaromyces, Endomycopsis, Hansenula, and Torulopsisf are also capable of oxidizing hydrocarbons (catalyzed by oxygen-dependent transformation by monoxygenase) to
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yield fatty acids or lipid-containing surfactants (Finnerty, 1984). The biotransformation of lipid-containing substrates to useful and stereospecific flavorants in the food industry has been reviewed by Welsh et al. (1989). Goldman and Perret (1965) produced 15- and 16-hydroxy palmitic acids by hydroxylating Torulopsis magnoliae and these were further cyclized to form lactones. Lactones contribute taste and flavor nuances to food. Labows et al. (1979) reported the production of lactones by the yeast Pityrosporum cultured on a lipid substrate (triolein, sebum, lecithin, oleic acid, and Tween 80) producing y-hexa-, y-hepta-, y-deca-, y-undeca-, and y-dodecalactones. Pityrosporum glaucum or P. platinis are capable of producing methyl ketones from coconut oil. So far, no ascertainable scientific description of the significance of the involvement of yeasts in pickle fermentation has been reported. It is believed that the general increase in fatty acids in pickles is attributable to the unsaponifiable fractions and the hydrolysis of lipids during fermentation. Keil and Weyrauch (1937) observed acetylcholine and lactylcholine accumulation in foods fermented by Bacterium acetylchoIini [syn. Lactobacillus plantarum, (Rowalt 1948)]. IV. Medical Importance of Yeast Lipids
Fat is a concentrated form of stored energy (9 caloriedg) supporting various metabolic activities of the body. Following the discovery of essentiality of fats in the diet by Burr and Burr (1929), it was shown that polyunsaturated fatty acids such as 0 - 3 and 0 - 6 fatty acids are essential for human health (Holman, 1968, 1982; Brown et al., 1938; Conner et al., 1992; Drevon 1992). Fatty acids are essential because the human body is incapable of synthesizing them. Long chain w-3 fatty acids (C18:4,C,o:4, C,,:,, C,,:,, and CZ2:Jare formed by the desaturation of a-linolenic acid (C18: 3 ) while linoleic acid (C18: acts as the precursor for 0 - 6 fatty acids (C,,:,, C,,:,, C,,:,, C,,:,, and CZzi5). Usually, excessive intake of diets rich in fat results in obesity and, in some caes, coronary atery diseases and certain types of cancer (Leibel, 1992). Deficiency of 0 - 3 and 0 - 6 fatty acids leads to many symptomatic features of diseases of brain, heart, retina, liver, skin, and failure of reproductive systems in man (Conner et al., 1992).This can be corrected by the administration of fat emulsions through food or intravenously in patients suffering from acute deficiency diseases (Kirby, 1992). Essential fatty acids are supplemented in our food through sources of vegetables, meat, and marine fish. Marine lipids (i.e, fish oil) are the major source of both 0-3 and 0 - 6 fatty acids. y-Linolenic acid (0-6 acid) is usually extracted from Primrose (Oenothera biennis), borage (Borago
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officinalis), and many species of Ribes. Some species of molds belonging to the genera Mortierella and Mucor are also good producers of w-3 and w-6 fatty acids. However, so far, no yeasts have been reported to produce such acids. The important w-3 fatty acids in the human plasma are alinolenic acid (C18:3) and its derivatives eicosanpentanoic and docosahexaenoic acids (Katan et al., 1991). It is already known that w-3 fatty acids serve as precursors for the synthesis of many essential compounds, thrombaxane B 3 , A3, prostaglandins E3,13, and leukotriene. Also, these substances have important physiological effects with regard to immune and platelet functions. Pharmacopeal requirements of different commercially available primary dried yeasts [S. cerevisiae and C. utilis (torula yeast)],for use in foods, mention only the proteins (- 40%) and vitamins 0.0012 mg%; riboflavin 0.0004 mg%, and (thiamine hydrochloride 0.0025 mg% nicotinic acid) and say nothing about lipid contents (Osol and Pratt, 1973). Such yeasts have not been considered a major source of essential fatty acids; however, other yeast species contain linoleic and linolenic acids in appreciable quantities (Table I). In general, linoleic acid occurs at higher levels than linolenic acid and the latter is absent in some yeasts. Leucosporidium species contain these acids as 80.23 to 87.35%of total unsaturated acids whereas some S. cerevisiae contain 35.96% (Table I ) . However, the actual utility as well as metabolism of yeast-derived linoleic and linolenic acids in the human body has not been demonstrated. It can be presumed that upon ingestion of such acids, they serve as precursors for the synthesis of elongated essential acids needed for the body metabolism.
-
V. Modification of Lipids
The use of modified lipids with desirable qualities in food and beverages has much commercial as well as nutritional importance. As mentioned earlier, derivitization of such products can be achieved by biotechnological means, fermentative synthesis using wild or genetically engineered organisms, and chemical or biochemical interesterification. Lipases, in general, are reasonably stable and stereoselectively catalyze substrate modification in four different ways: (1) ester hydrolysis, (2) ester synthesis, (3) transesterification, and (4) acyl transfer (Erdman et al., 1988). Some of the important general sources of lipases, other than yeasts, are listed in Table 11. A separate list of commercially important yeast lipases and their applications are given in Table 111.
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A. FERMENTATIVE SYNTHESIS AND MODIFICATION OF LIPIDS
Fermentation is the cardinal step in which the organism multiplies and accumulates lipids or excretes lipid-containing surfactants. The metabolic aspects of yeast lipids have been described by earlier workers (Ratledge, 1989). Yeasts can be grown in large quantities under submerged conditions using cheap and available carbon sources such as agroindustrial wastes, food industry wastes, and petrochemical industry wastes like n-alkanes. Lipid production patterns depend mainly on the type of oleaginous organism and its growth conditions (Ratledge, 1982, 1989; Yoon and Rhee, 1983a). In principle, once a good range of productivity of a particular product is achieved in batch culture, it is possible to increase productivity in continuous or fed-batch systems as the next step (Iwamoto, 1972). Standardization of fed-batch culturing seems to be a difficult task, especially with regard to the nitrogen and dissolved oxygen levels in the medium. However, Yamauchi et al. (1983)achieved a productivity of 21% while working with fed-batch culturing of Lipomyces starkeyi. It was also shown that a dissolved oxygen level of 40% is required at the lipid accumulation stage of the yeast Rhodotorula gracilis (Yoon and Rhee, 1983b; Choi e t al., 1982; Pan et al., 1986). Nevertheless, attempts in our laboratory using Rhodotorula gracilis CFR-1 (fed-batch culturing trials with an economically cheap cane molasses medium) were discouraging. Such trials yielded good biomass, but low oleaginicity. Changes in external environmental conditions such as temperature, pH, substrate, and oxygen can bring about changes in intracellular lipid composition. Oxygen is required for the conversion of stearic acid to oleic acid which, in turn, is converted to linoleic and linoleinic acids. Desaturation starts with a low oxygen supply and the production of major storage lipids occurs after the start of nitrogen, phosphorus, sulfur, and iron depletion from the medium (Pandey, 1989a). Even a small change in a single parameter brings about many sequential and interdependent biochemical changes in the pathway. The lipid metabolism of oleaginous yeasts as elucidated by Evans and Ratledge (1985) and Ratledge and Evans (1988) suggested that once the nitrogen is depleted more ATP is synthesized. The ATP:citrate lyase, one of the key enzymes involved in the pathway, cleaves citrate (found in the cytosol) to oxaloacetate and then to acetyl CoA, the basic building block in the synthesis of fatty acids. The synthesis of triacylglycerols takes place in the microsomes. Of the many controlling factors involved, higher C/N ratios affect the lipid productivity. Gill e t al. (1977) achieved more lipids [(- double
-
TABLE I POTENTIAL YEASTSAND THEIRLIPIDSTHATCONTAINLINOLEICAND LINOLENIC ACIDS r
04,
Fatty acids"
% Total ofb
(D
4
Organism
A
B
Debaromyces castelii D. hansenii Hansenula anomala H. anomala H. polymorpha Lipomyces kononenkoae Metschnikowia lunato Saccharornyces cerevisiae S. fibuligera Torulospora delbrueckii Yarrowa lipolytica Zygosaccharomyces rouxii
33.7 23.2 40.8 42.8 43.4 25.7 49.5 28.7
0.4 17.8 4.9 19.2 0.5 8.2
44.8 42.7 34.8 43.2
Traces
0.8
-
A&B~
C C
Reference
43.16 48.92 53.13 73.63 59.48 46.95 61.95 35.96
21 16.2 14 15.8 26.2 28.8 20.1 20.2
Moulin et al. (1975) Moul;n et al. (1975) Johnson and Brown (1972) Ng and Laneellee (1977) Dedyukhina et al. (1982) Hossack and Martins (1978) Malkhas'yan et al. (1983) Kovac et al. 11980)
59.14 47.49 59.79 51.30
33.9 20.1 47.8 15.8
Malkhas'yan et ol. (1983) Johnson and Brown (1972) Klug and Markovetz (1967) Watanabe and Takakuwa (1984)
Rhodotorula glutinis Yarrowa lipolytica Brettanomyces anomalus Candida albicans C. albicans C. guilliermondii C. humicola C. kefyr C. kefyr C. rugosa C. sake C. tropicalis C. utilis Cryptococcus ater Leucosporidiurn frigidurn L. frigidurn L. nivalis A. glutinis R. gracilis
49 51 33.7 26.8 33.5 39.1 61.3 36.1 31.9 48.5 33.8 32.8 54.1 48.7 40 25 42 53.1 34
3 1 0.4 19.8 24.7 3.6
Traces 14.9 16.7
nil 10.8 13.8 9.9 33 51 27 18
A, linoleic acid; B, linolenic acid. Total percentage of A and B, based on total unsaturated fatty acids Total percentage of saturated fatty acids.
65 60.46 43.16 60.9 77.8 51.13 71.52 62.42 57.17 57.8 51.98 50.53 79.69 74.17 84.61 87.35 80.23 62.69 65
20 14 31 23.6 25.2 16.5 14.3 19.3 15 16.1 14.2 35.1 14.8 21 9 13 14 15.3 20
Malkhas’yan et al. (1983) Malkhas’yan et al. (1983) Moulin et al. (1975) Nishi et al. (1973) Guarneri et 01. (1977) Jigami et al. (1979) Zotova et al. (1985) Moulin et al. (1975) Moulin et al. (1975) Iida et al. (1980) Kaneda and Smith (1980) Greshnykh et al. (1968) Johnson et al. (1972) Moulin et al. (1975) Watson et al. (1976) Watson et al. (1976) Watson et a]. (1976) Kaneko et al. (1976) Kessell (1968)
196
Z.JACOB TABLE I1 SOURCES OF LIPASES OTHERTHANYEASTS FOR THE PURPOSE OF FATSPLITTING Sources
Animal sources Porcine pancreas Gastric Plant sources Castor bean Wheat germ Microbial sources Molds Aspergillus niger Mucor jovanicus Rhizopus arrhizus R. delamer Bacterial Chromobocterium viscosum Pseudomonos sp. Unspecified organism
Specificitya
Remarks
A A
Commercially available Commercially available
A
Commercially available Commercially available
-
Mostly nonspecific B Commercially available B Commercially available B Commercially available B Commercially available B B B
Commercially available Commercially available Commercially available, splits triacylglycerols to diacylglycerols
A, positional and chain-length specificity: B, nonspecific
the amount), 50% (w/w)]in Candida lipolytica cells when the nitrogen supply was limited to 246 mg/liter than with normal conditions of fermentation. Similarly, other nutritional conditions, especially reactor conditions, also have a tremendous effect on the productivity and composition of the lipids as well. This point has been described in many of the earlier published reviews (Ratledge, 1989; Rattray, 1988). In Rhodotorula gracilis CBS 3043, the majority of triacylglycerols are produced during the stationary phase in a nitrogen-limited medium (Rolph et a]., 1989). The phospholipid concentration of the cells is adversely affected when the cells are grown in a carbon-limited medium, although there is little effect on triacylglycerol accumulation and the quality of lipids. However, in the earlier days of yeast lipid the expensive synthesis as well as a doubtful market introduction as an alternate source to vegetable oils and fats precluded large-scale production. This opinion has now changed because of the advent of biotechnological techniques and downstream processing which lead to the conclusion that such processes are economic if efforts have been concentrated on value-added products like y-linolenic acid or a cocoa butter substitute. When considering metabolic pathways, diverse modi-
TABLE I11 INDUSTRIALLY IMPORTANT YEAST ENZYMESAND THEIRAPPLICATIONS Organism
Nature of enzyme
No positional specificity and activity on long chain triacylglycerols. Hydrolyze almost all ester bonds and liberate all sorts of acyl chains on treatment. Olive oil and cocoa butter were used as substrates in this study (Benzonana and Eposito, 1971). Used in detergents. Commercially available. Meito Sangyo Co., Japan. Nonspecific (lipase) Commercially available from Sigma (United States) or Deisenhofen Specific (lipase) (Germany). ca.600,000 units/solid, enantioselective transesterification for producing L-methyl esters (Erdman et al., 1988). Economic fermentation, 1980 units/ml. Olive oil as substrate. Chain Specific (lipase) length specificity for C14 to C18 or C2O-C22 acyl groups (Lie and Lamberstein, 1986; Noguchi and Hibino, 1984). Specific (lipase) Chain length specificity to lower chain acyl groups and poor specificity for long chain acyl groups (Noguchi and Hibino, 1984). Nonspecific (Lipase-MY) Used in improving flavor of apple wine (Tanabe Seiyaku Co., 1970). Nonspecific (lipase) Thermostable, 55-60°C, used for fat spliting (Montet et al., 1985). Nonspecific (lipase) Thermostable, 55-60°C, used for fat spliting (Muderhwa et al., 1985). Used in detergents and in blue cheese manufacture (Peters and Nelson, Nonspecific (lipase) 1961). Lipase Enzymes from both yeasts are used in limburger cheese production, liberating myristic, palmitic, palmitoleic, stearic, and oleic acids from milk fat (Seitz, 1974). Specific (lipase) Specificity for sn-1 and sn-3 positions (Macrae and Hammond, 1985; Muderhwa et al., 1986). Specific (lipase) Specificity for polyunsaturated fatty acyl groups (Baldwin, 1986). Specific (lipase) Hydrolysis of long chain fatty acids at cis 9 or cis 9, 1 2 positions. Commercially available (Macrae, 1983; Jensen, 1974; Jensen and Pitas, 1976). Specific (pectic enzyme) Used in mechanical extraction of olive oil with higher FFA and lower peroxide values. Oil with acceptable qualities. (Servilli et ol., 1989).
Candida cylindracea (C. zeylanoides) Nonspecific (lipase)
C. cylindracea C. cylindracea
C. cylindracea
Candida sp. Candida sp. C. curvata C. deformans (Yarrowa lipolytica) C. lipolytica C. mycoderma Debaraomyces mycoderma Rhodotorula pilimanae
R. rubra Geotrichum candidum (yeast-like fungus) Cryptococcus albidus
Remarks, applications if any, and reference
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fications of intracellular fats may be possible (Noguchi et al., 1982). Ratledge (1970) reported the production of shorter chain length fatty acids (Cll to CIS)by Candida 107 and discussed the possibility of producing a cocoa butter substitute from yeast lipids. Normally, yeasts do not produce these special products, but could within limits synthesize compounds that are closely related. For example, let us analyze the case of a cocoa butter substitute. Natural cocoa butter contains 35% of stearic acid in the triacylglycerol. With this in mind many attempts have been made to produce a cocoa butter substitute by adopting different protocols. Noguchi and Hibino (1984) patented a process wherein they could produce a comparable content of stearic acid in species of Rhodotorula and Candida by supplementing stearic acid or its esters in the growth medium. In another attempt, Moreton (1985) rerouted the synthesis of stearic acid in yeasts (Candida 107, Trichosporon cutaneum, and Rhodosporidium toruloides) by supplementing sterculic acid in the medium. The sterculic acid inhibits the A-9 desaturase activity which results in more stearic acid synthesis. But the technique of inhibition of A-9 desaturase activity does not work with yeasts like Lipomyces and Saccharomyces cerevisiae. Certain groups of yeasts are also capable of producing industrially important lipid-containing biosurfactants of varying chemical nature. For general information interested readers may refer to Jacob (1992), ZajiC and Saffens (1984), and KosariC et al. (1987). Modification of oils and fats is also possible by fermentative growth and subsequent lipolysis with yeasts. Glatz et al. (1984) studied the modification of fats by fermentation using C. lipolytica. A yeast like C. curvata is capable of digesting and absorbing low-grade fats and oils. It produces lipids structurally related to the substrate supplied. During the growth of the yeast, it produces modest amounts of palmitic acid while the linolenic acid content is drastically reduced. The substrate is presumably hydrolyzed and resynthesised during deposition, resulting in an oil with an altered but nonrandom glyceride structure of triacylglycerol. According to Weete (1980), one should expect only reasonable alterations of fatty acids deposited in the cells. Mathew et al. (1990) studied the possibility of producing essential fatty acids by the hydrolytic action of microbial lipases on lipids. The mono- and diacylglycerols formed in the experiment were used as emulsifiers in ice cream, cakes, and puddings. Kajs and Vanderzant (1980) used S. lipolytica and C. utilis (food yeasts) for the emulsification of tallow. Their lipolytic enzymes degraded the tallow into fatty acids which served as a carbon source for their growth. It is amazing to see the enormous presence of lipolytic (lipase and
-
~
YEAST LIPID BIOTECHNOLOGY
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esterase) yeasts in our daily life. Alifax (1979) isolated 372 strains of yeasts from dairy, meat, bakery products, and cooked dishes. More than 70% of the strains possessed esterases, lipases, or both. The strong action of such enzymes produced by Candida curvata, C. deformans, C. humicola, C. parapsilosis, C. lipolytica, Crptococcus laurentii, Saccharomyces lipolytica, and T. cutaneum liberates fatty acids from butter oil, especially capric, caprylic, and lauric acids. Some yeasts possess isomers. Another interdifferential lipase activity against C, :, and C,: esting example is that of Endomycopsis biospora which can utilize fatty acids of rape seed and maize germ oil as the sole source of carbon. Candida lipolytica and T. cutaneum also have the capability to preferentially liberate oleic and myristic acids, respectively. As mentioned previously, many theoretical modifications are possible but only those that are physiologically acceptable will allow the organism to survive and grow. Inhibition of a particular step of metabolism may be achieved using substrate analogs and enzyme inhibitors. In this case, a number of problems need resolution. One of the constraints is the regulation of the mechanism that determines the level of intracellular accumulation of metabolites. Repressive or feedback adjustments have to be made to limit the amount of products like sterols in the cells. While doing so, however, it would be advantageous if other biosynthetic mechanisms were not disturbed.
B. GENETIC ENGINEERING ASPECTS OF YEAST LIPIDMODIFICATION Although lipids exist in various forms in nature, their commercial exploitation has been restricted because of constraints of availability and processing costs. There is then a good justification in exploring microbial sources employing gene manipulation techniques to produce many of the high value lipids. Gene manipulations are possible by mutant selection, hybridization, rare mating, spheroplast fusion, transformation, and gene cloning. Of these, mutant selection, spheroplast fusion, transformation, and gene cloning, through vectors or plasmids, are the generally employed techniques. Both plants and yeasts are mostly polyploid in nature. In contrast to the developments in plant genetic engineering, limited success has been possible with engineered industrial yeasts. In 1968 Larikova and Gal’tsova reported an almost twofold increase in total lipids and stearic acid content in yeasts treated with radiometric methyldichloroethylene and X rays. No further reports appear in the literature. Success in the development of engineered yeasts for lipid production seems to have failed because of the limiting stability of such recombinants, the lack of understanding of the metabo-
200
2. JACOB
lism of lipids under stressed conditions, and suitable techniques for the genetic characterization of various traits. One of the key enzymes in fat metabolism is fatty acid synthetase. It is a multienzyme with a molecular mass of 2.19 x lo6 Da and six protomers each with a and p subunits. The coded genes’ locations in the reported map are termed fasl and fas2 (Pandey, 1989b; Siebenlist et al., 1990). Pandey (1989b) reported a successful isolation of fas mutants from S. cerevisiae which received the fas complex from two oleaginous candidates, Rhodotorula gracilis and Candida sp. Further, a recombinant called 63a was obtained from a cross of both yeasts by protoplast fusion. The recombinant had the sugar tolerance (40%) and conversion efficiency (11-15%) of S. cerevisiae, and the lipid composition resembled that of palm oil. The production of such a microbial oil is not economical in terms of the capital cost of production or the value of palm oil in the world market. In another case, Hammond et al. (1981) produced lipids with palmitic acid similar to the cocoa butter substitute by growing mutants of C. curvata at 30°C. However, the same results were not obtained when the mutants were grown at higher and lower temperatures and at different pHs. Another serious disadvantage with such mutants is the instability of their desirable characteristics. Knowledge of stress conditions involved in turning on and off of the concerned genes to make appropriate new sets of enzymes and effector molecules is necessary in studies of oleaginous yeasts (Joseph, 1989). It seems that the stressed conditions also affect the rate of synthesis of other metabolites during lipid metabolism. According to Joseph (1989), acetyl-CoA is a key intermediate compound for the synthesis of carotenoids in Rhodotorula gracilis. This hypothesis was further strengthened when Joseph (1989, and unpublished data) observed that mutants of R. gracilis CFR-1 deficient in carotenoid pigments and ATP:citrate lyase produced only very insignificant quantities of lipids in addition to being devoid of carotenoid pigments. Sterols of yeasts are potential precursors for the chemical synthesis of hormones, growth mutators, drugs, and potent fungicides. Parks et al. (1984) described the tailoring of the yeast S. cerevisiae for sterol production by genetic manipulation. This yeast can be grown both aerobically and anaerobically and has a well-defined genetic mechanism that can be suitably manipulated to construct various combinations of markers, including many in the biosynthesis of lipids (Henry, 1982). Saccharomyces cerevisiae is not an oleaginous yeast, but recent developments may change this situation. Boulton and Ratledge (1981) observed a positive correlation between lipid accumulation in yeast and the presence of ATP:citrate lyase which, according to them, provides
YEAST LIPID BIOTECHNOLOGY
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a constant source of acetyl-CoA for fatty acid synthesis. Genetic determinants of ATP:citrate lyase could be introduced into S. cerevisiae by mutation and subsequent positive selection using resistance to polyene antifungal agents like nystatin (which make a complex with sterols in the cell membrane). However, the mutants thus obtained usually have a loss of single enzymic function in sterol biosynthesis. This indicates that many modifications may be possible, but only those that are physiologically acceptable will allow the organism to survive and grow. In addition to using sterol mutants to obtain desired end products, the inhibition of specific steps in enzymic pathways in wild types may be achieved using substrate analogs or inhibitors. In doing so, many practical problems may arise, especially while controlling repressive or feedback adjustments needed for a particular pathway.
c. CHEMICAL AND BIOCHEMICALINTERESTERIFICATIONS Modified fats have become essential food components of a myriad of sensory appealing, fast and convenient foods (mostly confectionary and baked foods). In the past, the hydrogenation of vegetable edible oils by inserting hydrogen into the double bonds of unsaturated fatty acids was found very useful in evolving plastic fats. Later it was found that the spatial rearrangement of fatty acids on the triacylglycerol by an acidolysis reaction or an ester-ester interchange yielded fats with unique properties. The products thus obtained are called speciality or modified fats. Cocoa butter fat, characterized by the high content of stearic acid (30-35%) and the predominance of l-palmitoyl-2-oleoyl-3-stearoyl glycerol, is a commercially important component of the food industry. Most soft soap and cosmetic industries rely on coconut oil for its medium chain length fatty acids (Clz and C1J. Other fats and derivatives of commercial importance are glycolipids as surfactants, carotenoids as food colorants, and poly-P-hydroxybutyrate with its unique plasticlike properties. High value fatty acids like y-linolenic acid (6,9,12octadecatrienoic acid), arachidonic acid (Cz0:J, eicosapentaenoic acid (Czo:J, and eicosahexaenoic (Czo acid are medically important. As mentioned earlier, seeds of Oenothera are the prime source of ylinolenic acid, and the total production is insufficient to meet the increasing demand. Now the question is whether these compounds can be synthesized through chemicallbiochemical means. Although the chemical interesterification reaction steps are easily performed, there is the serious disadvantage of nonspecificity yielding products with variable rheological properties. This unpredictable range of modifications can be
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Z. IACOB
achieved with the help of catalysts like sodium methoxide, metallic sodium, or sodium potassium alloys. These reactions do not involve costly equipment. Biochemical interesterifications are more useful in the sense that they give unique fats with rather specific and predictable rheological properties. Microorganisms use their extracellular lipases (glycerol ester hydrolase, EC 3.1.1.3) for digesting lipid substrates for metabolic activities. According to Macrae (1984), extracellular microbial lipases can be used for interesterification so as to obtain modified lipids which otherwise may not be easily obtainable by chemical interesterification. The reaction is reversible under certain conditions, and a particular organism’s own lipases may not have much significance in its own biosynthesis of modified oils and fats intracellularly (Tsujisaka et al., 1977). The natural substrates for lipases are triacylglycerols of long chain fatty acids. The enzyme acts at an interface between an insoluble substrate phase and an aqueous phase resulting in hydrolysis of a wide range of insoluble fatty acid esters. As mentioned previously, this is reversible. Consequently, hydrolysis and resynthesis of acyl glycerol groups occur when lipases are incubated with a mixture of triacylglycerols. This hydrolysis and resynthesis causes migration of fatty acyl groups between glycerol moieties and gives interesterified products. Extracellular lipases for interesterification can be grouped into two main categories according to their specificity of reaction (Brockerhoff and Jensen, 1974) (Table 11). The first group is nonspecific and the products formed have a random distribution of fatty acids as the acyl groups. This is similar to the chemical interesterification reaction. Enrichment of triacylglycerol is also possible by interesterifications. A mixture of triacylglycerol and free fatty acids as reactants are treated in the presence of lipase. The free fatty acids are exchanged with fatty acyl groups of triacylglycerol to give modified free fatty acid and triacylglycerol. Another possibility involving migration of fatty acyl glycerol and fatty acids also occurs during interesterifications. Hydrolysis and resynthesis of acyl glycerol and migration of fatty acyl groups between glycerol moieties occur when a mixture of triacylglycerols is treated with lipase. The second group is 1,3 specific. This is common with lipases of different molds such as Aspergillus niger, Mucor javanicus, and Rhizopus species (Okumura et al., 1976; Ishihara et al., 1975; Semeriva et al., 1967; Macrae, 1983). Interesterifications are performed in either stirred or packed bed reactors, depending on the type of operation (batch or continuous). For example, in a batch reaction, a mixture of palm oil mid-fraction and
YEAST LIPID BIOTECHNOLOGY
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stearic acid is dissolved in petroleum ether and stirred for 16 hr at 40°C with a catalyst. Enzymatic interesterification catalysts are prepared by coating macroporous inorganic particles with lipases. For example, the A. niger lipase is precipitated with acetone onto kieselguhr particles and is activated by hydration with a small quantity of water prior to the addition to the reactor. Kimura et al. (1983) reported the use of immobilized lipases of C. cylindracea on various organic as well as inorganic supports, and the hydrophobic matrices gave the highest activity in the hydrolysis of olive oil. In the chemical interesterification process, sodium metal or sodium alkoxide promotes the migration of fatty acyl groups between glycerol molecules. In enzymatic interesterifications, it is possible to minimize the rate of hydrolysis of fats by reducing the amount of water in the reaction system and then lipase-catalyzed interesterification becomes dominant (Coleman and Macrae, 1980; Matsuo et aI., 1980,1981;Tanaka et al., 1980).Enhanced interesterifications are also possible by providing a large area of interface and a minimum quantity of water in the supports. Supports include kieselguhr, hydroxyl apatite, and alumina. Catalysts are prepared by adding a solvent such as acetone, methanol, or ethanol to a slurry of the particles in a buffered lipase solution (Macrae, 1983). The solvent precipitated enzyme coats the particles and they are collected by filtration. They can be dried and stored. These dried particles contain low activity of lipases. Hydration with up to 10% of their weight with water activates the catalyst particles. Reactivation of supports is also done by coating it with diols or triols and free fatty acids or treatment with the reactants dissolved in petroleum ether or hexane. Erdman ef al. (1988) suggested the use of enzyme immobilization carriers such as VA-epoxy Biosynth and Duolite ion-exchange resins. Activation by diols or triols such as glycerol has also been recommended (Tanaka et al., 1980). It is interesting to note that the lipase of Geotrichurn candidum has a very marked specificity for hydrolysis of a particular type of long chain fatty acids containing a cis-double bond in the ninth position (Macrae, 1983; Jensen, 1974, 1983; Jensen and Pitas, 1976). Most of the site-specific lipolysis was found to be very slow. However, this disadvantage of slow reaction can be overcome using methyl esters of oleic, palmitoleic, linoleic, and linolenic acids (all of which has a cisdouble bond in the ninth position). During the interesterification, A 4 fatty acyl groups are selectively exchanged with other A-9 acyl groups in the mixtures of triacylglycerol or triacylglycerol and free fatty acids. For example, when olive oil + linoleic + stearic is treated with the
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lipase, the triacylglycerols are enriched in linoleate at the expense of oleate, while the saturated fatty acyl content of triacylglycerols remains substantially unchanged. In another study a slow process of interesterification at 40°C for 400 hr using stearate and Rhizopus lipase was reported. In this case, after an initial equilibration period of a few hours, the water was removed from the hydrated catalyst particles with the formation of a larger quantity of diacylglycerol and free fatty acids until steady-state conditions were attained. To date there are no authenticated reports of extracellular lipases which catalyze reactions only at the second position of acyl glycerols. Most of the extracellular lipases have little fatty acid specificity (Macrae, 1983). Lipolytic organisms with specific reactions at the desired sites of triacylglycerol need to be isolated. The report of the development of a rapid plate procedure by Collins and co-workers (1989) for the isolation and characterization of lipolytic microorganisms is noteworthy. With this technique microbial lipases can be conveniently detected by the digestion of a target lipid (emulsified in agar medium). The specific lipolytic activity is revealed by the cleared zones due to precipitation of liberated fatty acids by Ca2+in the agar medium. Lipolytic organisms of specific properties are also isolated from different natural sources like Elaeis quineensis fruits. Interestingly, higher levels of free fatty acids in palm oil are due to the presence of yeasts and molds which are lipolytic and exist in the mesocarp of fruits from the inception of the fruit development. Isolation of such types of microorganisms also could be used for insertion and expression of desired traits for novel lipases. VI. Commercial Significance of Yeast Lipid Biotechnology
The presently available biotechniques in the area of yeast biotechnology seem to be handicapped by uneconomic productivities. Nevertheless, it would be worthwhile to mention the significance of such products in food and medicine. Future commercial potentials for the production of tailor-made as well as modified, speciality fats would be expected to increase mainly because of the ever increasing nutritional and medical awareness, modern food habits, and prolonged life span of man. The importance of quality lipids with no rancidity and a higher degree of polyunsaturated fatty acids in foods has already been accepted as important nutritional criteria. Consumption of rancid lipids may lead to peroxidation and thus damage at the cellular level. With regard to essential fatty acids of importance in food and medicine (prostaglan-
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dins) and newer or novel derivatives of such products, lipids may be helpful in resolving many of the problems in nutrition science, and probably in the curing of many diseases. For such purposes, natural sources like yeasts have not been exploited yet. Genetically engineered yeasts can offer many positive promises for the future. Figure 1 represents prospects of lipid biotechnology in a nutshell wherein the role of yeasts seems to be quite significant. Higher productivity of different kinds of vegetable oil seeds is possible by adopting the latest agricultural practices in larger areas. As previously mentioned, cloning and expressing of desired intraspecific genes to the hosts by recombinant DNA techniques and tissue culture practices may help in raising more seedlings and thus larger-scale cultivation. Alternatively, lipids similar to vegetable oils can also be produced microbially by fermenting selected candidates under appropriate conditions using suitable and cheaply available substrates (agroindustrial wastes, whey, molasses, meat industry wastes, and petrochemical carbon sources like
FIG.1. Lipid biotechnology prospects.
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n-alkanes). Chemical or biochemical alterations of the postharvest microbial, plant, or animal lipids can be facilitated in reactors to yield modified lipids. Such a product can also be produced by genetically engineered yeasts or somatic cells possessing the qualities of high oleagicity and productivity and providing the natural conditions of biomass metabolism and lipid accumulation in reactors. Tailor-made microbial lipids can also be synthesized using yeasts in which the natural metabolic pathway is rerouted in a desired manner. This method cannot be considered as a fully natural process. In normal practice the results of such techniques are discouraging and unproductive. Although both plants and yeasts are polyploids, the former has an edge in the success of cloning and expression of desired genes. The question of whether lipids obtained by the natural metabolic pathway of a particular organism can be used as a carbon source for the synthesis of modified fats by genetically engineered organisms or by other types of wild organisms is not yet fully answered. Natural lipids produced by both wild as well as engineered candidates can be cycled for chemical as well as enzymatic conversions to modified products. It may also be possible to recycle these modified lipids as carbon sources for a particular candidate’s metabolism, thus obtaining novel products. It may be quite possible in the future to incorporate all of the desired traits in specific cells (somatic/mammalian) in producing tailor-made lipids in reactors. Additionally, a modified lipid can be further modified by any of the methods previously described to produce novel lipids which may find applications in food and medkine. -~ The logic of using yeasts as enriching agents to derive desired food products and/or to degrade biological wastes is understandable. The following examples reinforce this logic. Burkholder and Gervasini (1969) reported that the growth of C. lipolytica and G. candidum can be used for reducing fats in fish meat. After fermentation the final product has an increased protein content with appealing flavor characteristics. In another instance it was suggested that inedible tallow can be utilized as a carbon source for the production of SCP and metabolites (Tan and Gill, 1984). This was studied by Kajs and Vanderzant (1980) using S. lipolytica and C. utilis. Lipolytic activity of the enzyme produced by the yeasts helps in the breakdown of the tallow into simpler fatty acids which act as carbon sources for the production of SCP (Fiorentini et a]., 1976). Anelli et al. (1975) reported the production of SCP (containing all amino acids according to FA0 standards, except methionine) from the growths of C. lipolytica, Torulopsis holmi, C. mesentrica, and Cryptococcus albidus on fat wastes containing olein. When Friesian cows were fed torula yeasts the cows produced more
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milk with a higher level of lipids (0.08 to 0.29%) (Gheorghiu et al., 1976). However, when Toprina yeasts were fed to Friesian cows, although there was no significant increase in the total lipid content, the internal tissues were enriched significantly with C,,, C,,, and C,, : 2 fatty acids (Martillotti et a]., 1977). When the Ruse strain of S. cerevisiae was fed as fodder the lipids contained a large amount of a-tocopherols (6.32 pg/g) (Roshkova and Beshkov, 1974). Similarly, when Holstein cows were fed diets with yeast there was an increase of lipids and protein in the milk (Erdman and Sharma, 1989). In another report, yeast biomasses of Toprina and Liquipron were fed to cows and pigs and subsequently the milk and adipose tissues had odd-numbered fatty acids (Boniforti et al., 1979). Rys et al. (1975) reported the feeding effects of an n-paraffin- or molasses-grown yeast in pigs wherein an increased proportion of C,,, C,,, CI6: and C,,: and a decreased proportion of CI8:, and C,,:, fatty acids in the back fat were observed. It was also demonstrated that rats fed n-paraffin-grown Candida sp. did not produce any untoward effects (Yokoyama and Kaneda, 1972). Defatting of meats by microbial action is an interesting phenomenon. Glatz et al. (1984) suggested that lipolytic organisms could be used to degrade meat cholesterol. Another possible application of lipolytic yeasts (for example, Saccharornycopsis lipolytica which has the capability to degrade meat) would be in the degradation of meat-processing wastes. However, the feasibility or success of such applications remains to be seen.
,
VII. Conclusion
Some yeasts are potential producers of lipids similar to vegetable oils and fats. Research on their applications as a dietary supplement in food or essential pharmacological components in medicine has not progressed to desirable limits. When considering the uneconomic fermentative synthesis of yeast lipids in reactors, a reorientation in the approach to develop processes for value-added lipids for use in food and medicine seems to be productive. Modification of lipids using engineered or transgenic yeasts has not been successful so far. Since yeasts are polyploid in nature, similar to plants, they may be suitably engineered so as to synthesize novel lipids which may find utility in producing value-added oils and fats for use in the food and biomedicical industries. Rerouting of the yeast’s metabolic pathway for the synthesis of biomedically important polyunsaturated fatty acids has not received much attention. The cloning and expression of specific fatty acid synthetase genes to produce specific lipids still has a long way to go, and
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J p ect in, Pect in ase , and Proto pect inase : Product io n i Properties, and Applications TAKUO SAKAI, * TATSUJI SAKAMOTO," JOHAN HALLAERT,? AND ERICKJ. VANDAMME? *Department of Agricultural Chemistry College of Agriculture University of Osaka Prefecture Osaka 593, Japan +Department of Industrial Biochemistry and Microbiology Division of General and Industrial Microbiology University of Ghent B-9000 Ghent, Belgium 1. Introduction 11. Review of Pectin A. Nomenclature B. Chemical Constituents and Structure C. Occurrence and Function D. Properties E. Determination and Characterization F. Pectin Manufacturing: Chemical Extraction and Purification G. Applications of Pectin Ill. Classification of Pectic Enzymes A. Esterases B. Hydrolases C. Lyases IV. Role of Pectic Enzymes in Phytopathogenesis V. Applications of Pectinases A. Industrial Production of Pectinases B. Fruit Juice Industry C. Other Applications VI. Protopectin-Solubilizing Enzyme (Protopectinase) A. Assay of Protopectinase activity B. A-Type Protopectinase C. B-Type Protopectinase D. Applications of Protopectinase References
I. Introduction
Pectic substances are acid polysaccharides of high molecular weight that are widespread in the plant kingdom. The size, charge density, charge distribution, and degree of substitution of pectin molecules may be changed biologically or chemically. The chemical structure of pectin has been the subject of many scientific reports for more than 50 years 213 ADVANCES IN APPLIED MICROBIOLOGY,VOLUME 39 Copyright 5 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(De Vries eta]., 1981). Elucidation of this structure was and is important because of the function of pectin in the cell wall as a “lubricating” or “cementing” agent (Rees and Wight, 1969), its role during ripening of fruit (Knee, 1978), its role in food processing (Rombouts and Pilnik, 1978; Van Buren, 1979), and its role as nutritional fiber. Research in the field of “the pectic substances” has been carried out by scientists and technologists from a great variety of disciplines. In the very early days of pectin chemistry, when French botanists such as Fremy were commencing to investigate the role of pectic substances in plant physiology, other chemists of the same nationality were delving into the finer details concerned with flavor in wines and ciders (Charley, 1951).The pectins in these beverages were considered to be responsible for the “veloute” or “moeilleux” (softness or silkiness) which give the fruit product its particular character. At present, interest in the pectic substances is still continuing worldwide. The pectin production process consists mainly of an acid extraction, followed by a (partial) purification of the extract and, eventually, precipitation and drying. However, this process has several disadvantages: maceration of the pulp, difficult filtration of the residue, and corrosion of equipment. The main raw materials for pectin are the peel of citrus fruits and apple pomace (Towle and Christensen, 1973). Research was initiated with the aim of developing an improved alternative microbial pectin production process, by means of a specific enzyme. A major part of this chapter is concerned with this specific enzyme, called protopectinase. II. Review of Pectin
In his classical book “The Pectic Substances’’ Kertesz (1951) stated that fruit jelly making was practiced long before pectin was discovered. The first information on water-soluble substances with a strong jellying power, occurring in fruits, was presented by Vauquelin in 1790. In the next well-known scientific publication on these substances, Braconnot (1825) related the name of these substances to their jellying properties when he derived it from the Greek work “ T ~ X T O C T , ” meaning to congeal or solidify: “. . . en attendant, je propose le nom pectique, de T ~ X T O C T , coagulum, pour distinguer ce nouvel acide de ses congeneres.” A. NOMENCLATURE
In the past, a number of confusing terms have been proposed for various pectic substances. In 1944, the Committee for the Revision of the Nomenclature of Pectic Substances, a former subdivision of the
PECTIN, PECTINASE, AND PROTOPECTINASE
215
American Chemical Society, finally accepted the following definitions (Table I): “Pectic substances” is a group designation for those complex colloidal carbohydrate derivatives which occur in, or are prepared from, plants and contain a large proportion of anhydrogalacturonic acid units which are thought to exist in a chain-like combination. The carboxyl groups may be partially esterified by methyl groups and partially or completely neutralized by one or more bases. “Pectinic acids” are the colloidal polygalacturonic acids containing more than a negligible proportion of methyl ester groups. “Pectinates” are either normal or acid salts of pectinic acids. “Pectic acids” is the group designation applied to pectic substances mostly composed of colloidal polygalacturonic acids and essentially free from methyl ester groups. “Pectates” are either normal or acid salts of pectic acids. “Protopectin” is applied to the water-insoluble parent pectic substance which occurs in plants and which upon restricted hydrolysis yields pectin or pectinic acids. Still, these definitions are rather vague. For example, there is no indication about the minimal polygalacturonic acid content required, nor about the minimal polymerization degree. Apart from the definitions based on composition and structure, there is also a definition based on composition and application of some pectic substances as gelifying agents. The general term “pectin” (or pectins) designates those water-soluble pectinic acids of varying methyl ester content and degree of neutralization which are capable of forming gels with sugar and acid under suitable conditions. Pectins with a rather high methoxyl contents show their jellying power only in the presence of a relatively high sugar and acid content, whereas gel formation by pectins with a lower methoxyl content is also possible without sugar in the presence of certain metallic ions. For this reason, two groups of jellying pectic substances are distinguished: the high-methoxyl pectins (>50%) and the low-methoxyl pectins. The term “high-methoxyl pectins’’ is often abbreviated to “pectins.” A schematic survey of the interrelationship of pectic substances is presented in Fig. 1. TABLE I NOMENCLATURE FOR PECTIC SUBSTANCES Pectic acid Polygalacturonic acid (pectate) Pectin Pectic acid partially esterified (pectinate] with methanol and containing some neutral sugars Pectic substance fixed in the plant Protopectin tissue
TAKUO SAKAI ET AL.
216
PROTOPECTIN I
I
1
+
Acid or enzymes
Warm alkali
Cold alkali
PECTATES
PECTATES
1
Nonfibrous, nonviscous Low molecular weight Insoluble due to alkaline earths from peel
PECTIN
I
Alkali, enzymes, or strong acid
Fibrous, viscous High molecular weight Insolubledue to alkaline earths from peel
I
I
Acid
PECTINATES
c
Strong acid
m enzymes
and I or pectinic acid
Mild acid
P
Nonfibrous Low molecular weight
Alkali
PECTIC ACID Fibrous High molecular weight
Cold alkali
i
Enzymes PECTATES Nonfibrous NH3 and alkali metal salts soluble
PECTATES Viscous, fibrous NH3 and alkali metal salts soluble
a-GALACTURONIC ACID
FIG.1. Interrelationship of the pectic substances (from Campbell and Palmer, 1978).
B. CHEMICAL CONSTITUENTS AND STRUCTURE 1. General Properties
Pectin is a heteropolysaccharide with galacturonic acid and methanol as the main components, with some neutral sugars attached. Pectin molecules are formed by a-l,4-glycosidic linkages between the pyranose rings of D-galacturonic acid units. The pyranose rings of D-galacturonic acid probably occur mainly in the chair L form, corresponding to the most stable conformation of D-galaCtOSe. As both hydroxyl groups of Dgalacturonic acid at carbon atoms 1 and 4 are on the axial position, the Some polymer resulting from such linkage is trans-1,4-po~ysaccharide. pectins, such as those from flax, tobacco, and sugar beet, also contain
217
PECTIN, PECTINASE, AND PROTOPECTINASE
some acetic acid. The carboxyl groups of pectin are partially esterified with methanol, and the hydroxyl groups are sometimes partially acetylated. Many pectin preparations contain other sugar units along with galacturonic acid, such as galactose, arabinose, or rhamnose, probably in accompanying polysaccharide linked as a side group to the polygalacturonic chain. Although the solubilization of pectic polysaccharides by water and their selective precipitation with alcohol was described by Vauquelin as early as 1790, the elucidation of their structure has proved particularly difficult and is still by no means completed (Cook and Stoddart, 1973). From a chemical point of view, pectin usually, and perhaps invariably, exists as a branched heteropolysaccharide in which the backbone is based on linear sequences of a-1,+linked D-galacturonate residues (Morris et a]., 1980). The carboxylic acid groups can be neutralized by mono- or divalent ions such as K + , Na+, and Ca2+ or may be partially esterified with methanol. Some of the hydroxyl groups on C, or C, may be acetylated (Fig. 2). The measure of esterification of pure galacturonic acids may be indicated by the methoxyl-(CH,O-)content or by the degree of esterification, i.e., the number of esterified carboxyl groups calculated as the percentage of the total number galacturonic acid units. When the carboxyl groups in pure polygalacturonic acids are all esterified, the methoxyl content is 16.32% and the degree of esterification is 100%. Esterification of pectinic acids, extracted from natural sources, is seldom higher than 70% (Table 11). Similarly, the acetylation can be expressed by the acetyl content or by the degree of acetylation (Table 11). The backbone, consisting of polygalacturonic acid, is also periodically interrupted by the insertion of a-L-rhamnopyranose residues. These residues are present in segments of the structure a-Dgalactopyranosyluronic acid-(l-+2)-a-~-rhamnopyranosyl-(l+4)-a-~galactopyranosyluronic acid (Lau et al., 1985; Aspinall and Cottrell, 1970; Aspinall et al., 1968a,b; Aspinall, 1981).The effect of the presence
H
OH
C4H
f-W 0
FIG.2. Hypothetical part of a pectin molecule (from Pippen et a ] . , 1950).
218
TAKUO SAKAI ET AL. TABLE I1
DEGREEOF ESTERIFICATION AND DEGREEOF ACETYLATION OF SOMEPECXICSUBSTANCES’ Source of pectin
Esterification (YO)
Acetylation (Yo)
Apple Potato Sugar beet Pear Mango Citrus fruits Sunflower
71 31
4 14 20 14 4
55 13 68 64 17
3 3
Data from Voragen et al. (1986)
of rhamnose is to cause a T-shaped “kink” in the chain (Fig. 3) (Rees and Wight, 1971; Rees, 1972; Albersheim, 1976). Neutral sugars other than L-rhamnose occur exclusively in side chains of pectins. D-Galactopyranose and L-arabinofuranose occur most frequently; D-xylopyranose, o-glucopyranose, and L-fucopyranose are less common units, while rarely found sugars like D-apiose, 2-O-methyl-Dxylose, and 2-O-methyl-~-fucoseare usually very minor, but widespread, constituents of pectins (Darvill et a]., 1978; Aspinall, 1981; Barrett and Northcote, 1965). The branching occurs through the C-2 (Ovodov et al., 1971) or C-3 atom (De Vries et a]., 1983; Aspinall et a]., 1968a) of galacturonic acid or through the C-4 (Aspinall and Fanous, 1984; Stevens and Selvendran, 1984) or C-3 atom (Darvill et al., 1978) of rhamnose. It was stated that
OH
FIG.3. The presence of rhamnose and the resulting T-shaped kinking of the pectin molecule (from Barford et al., 1986).
PECTIN, PECTINASE, AND PROTOPECTINASE
219
the rather rare sugars occur in short (one to three units) side chains, substituting the galacturonic acid skeleton, while arabinose and galactose form oligo- and polysaccharides substituting the rhamnosyl units. Also, the pectic polymers of primary cell walls have a relatively higher proportion of oligosaccharide chains on their backbone, and these side chains are much longer than those of the pectins of the middle lamellae (Selvendran, 1985). Enzymatic breakdown of pectic substances followed by analysis of the fractions revealed that these side chains were not distributed regularly along the galacturonan chain but concentrated in so-called “hairy regions,” leaving important parts of the backbone unsubstituted. Especially, the galacturonate residues in the hairy regions are esterified with methanol (Fig. 4) (De Vries et al., 1982; Rouau and Thibault, 1984; Axelos et al., 1989; Konno et a]., 1986; Thibault, 1983). Pectinic acids that lack neutral blocks will generally be referred to as “Type 11.” Detailed structure of a pectin is described in Fig. 5 (Cook and Stoddart, 1973). In the plant cell wall, the side chains link the pectin molecules to proteins, hemicelluloses, and cellulose to form the insoluble protopectin (Figs. 6 and 7).
Relatively short arabinan, galactan, or arabirogalactansidechains
-
Relatively Long arabinan, galactan or arabinogalactansidechains
Highly branched rharnnogalacturonan (primary cell wall)
FIG.4. Schematic representation of some structural aspects of pectins from the middle lamella (A) and primary cell walls (B) (from Selvendran, 1985).
220
TAKUO SAKAI ET AL
FIG.5. Detailed structure of a pectin [from Cook and Stoddart, 1973).
It is also suggested that acidic and neutral pectins carry ferulic acid on the nonreducing ends of the neutral arabinose and/or galactosecontaining domains. The pectins carry approximately one feruloyl residue per 60 sugar residues. Possible roles of feruloyl pectin are in the regulation of cell expansion, in disease resistance, and in the initiation of lignification (Fry, 1983).
Rharnnogalacturonan
FIG.6. Molecular structure of the primary plant cell wall (from Adler-Nissen, 1987).
PECTIN, PECTINASE, AND PROTOPECTINASE
Extension helix
221
Cellulose microfibril
Extension nonhelical region
Xyloglucan latches
Intermolecular isodityrosine cross link
Pectin
FIG.7. Three-dimensional view of polymer arrangement in the plant cell wall (from Wilson and Fry, 1986).
The molecular mass of pectic substances from various sources has been the subject of many investigations. The reported values vary from about 10,000to 400,000 (Table 111).Apart from existing significant differences, the results are also influenced by the method of extraction and technique of measuring molecular mass.
TABLE 111 MOLECULAR MASSOF SOMEPECTICSUBSTANCES" Source
Molecular mass
Apple and lemon Pear and prune Orange Sugar beet pulp
200,000-360,000 25,000-35,000 40,000-50,000 40,000-50,000
a
Data from Fogarty and Kelly (1983).
222
TAKUO SAKAI ET AL.
Finally, it must be clearly stated that there is no such thing as a uniform pectic substance. This is illustrated by the many variations in molecular mass; in degree of esterification and acetylation; and in quantity, type, and distribution of the non-uronide components. The pectic substances seem to be more heterogeneous than first assumed (Neukom et al., 1980). 2. Protopectin Protopectin was already defined as the “original” water-insoluble parent pectic substance which occurs in plants and which yields pectin or pectinic acids upon restricted hydrolysis. In most plant tissues, only this insoluble form of pectin occurs. The main exceptions to this rule are the ripe(ning) fruits. The many hypotheses about how insoluble protopectin is composed out of soluble pectic substances are summarized by Joslyn (1962).They include the reasons for the insolubility of protopectin: 1. The very large molecular weight of protopectin in comparison to that of pectin. 2. Mechanical enmeshing of the filamentous pectin macromolecules by one another and with other high polymers (cellulose, hemicellulose, lignin) of the cell wall. 3. Ester bond formation between the carboxylic acid groups of pectin and the (alcoholic) hydroxyl groups of the other cell wall constituents. 4. Lactone bond formations within the entangled pectin molecule. 5. Salt bonding between the carboxyls of pectic substances and basic groups of proteins. 6. Polyvalent ion bonding (Caz+,Mg2+,Fez+)between the carboxyls of the different cell wall constituents. 7. Secondary valence binding (i.e., H-bonds, hydration bonding, and molecular cohesion) between pectic substances.
c. OCCURRENCE AND FUNCTION Pectic substances are prominent structural constituents of (primary) cell walls in non-woody tissues, next to cellulose, several hemicelluloses, and protein (Fig. 8) (Brillouet, 1987). In addition, they are the sole polysaccharides in the middle lamella responsible for cell cohesion (Pilnik, 1981). Pectic polysaccharides occur mainly as water-insoluble protopectin. Their synthesis, beginning from UDP-D-galacturonic acid and taking place in the Golgi system (Karr, 1976), is performed mainly during the early stages of growth, in
PECTIN, PECTINASE, AND PROTOPECTINASE
223
: : Miwofibrils (organizedphase) : : ;Continuous matrix (amorphous phase)! I
,
I
,
2 a,
I
Primary wall
Secondary wall
Lumen
-5 BI U
FIG.a. Distribution of materials in the "mature" cell wall. The arrows indicate direction of increasing relative concentration (from Northcote, 1958).
young enlarging cell walls. Compared with young, actively growing tissues, lignified tissues are low in content of pectic substances. Furthermore, primary cell walls of graminaceous monocotyles have a low content of pectin compared to those of dicotyles (Jarvis et al., 1988). The average pectin content of several plant tissues is shown in Table IV. Texture of vegetables and fruits is strongly influenced by the type of pectin present. One of the most characteristic changes during the ripenTABLE IV PECTINCONTENT OF SEVERAL TISSUESO Tissue
Pectic substances (%)
Apple Bananas Peaches Strawberries Cherries Peas
(fresh) (fresh) [fresh) (fresh) (fresh) (fresh) (dry matter) Carrots Orange pulp (dry matter) (dry matter) Potatos Tomatos (dry matter) Sugar beet pulp (dry matter] Data from Chenoweth and Leveille (1975).
0.5-1.6 0.7-1.2 0.1-0.9 0.6-0.7 0.2-0.5 0.9-1.4 6.9-18.6 12.4-28.0 1.8-3.3 2.4-4.6 10.0-30.0
224
TAKUO SAKAI ET AL.
ing of fleshy fruit is softening. This change is attributed to enzymatic degradation and solubilization of the (proto) pectic substances (Labavitch, 1981; Soda et al., 1986; Pressey, 1988; Barbier and Thibault, 1982; Dick and Labavitch, 1989). However, during ripening the neutral sugar composition of the extractable pectin does not change (De Vries et al., 1981). In processing certain vegetables, for example, cauliflower, excessive softening is prevented by adding Ca salts. As a consequence, insoluble pectates and pectinates are formed, giving a firm texture to the vegetables. Similarly, the mechanical properties of cossettes cut from sugar beet can be improved by adding lime to the diffusion water. The addition of lime to sugar beet tissue at lower temperatures causes demethylation of the pectin in the cell walls of the beet tissue, allowing Ca2+to cross-link the pectin as a stable insoluble matrix. This permits alkaline diffusion with less disintegration of the pulp (Camirand et a]., 1981).
D. PROPERTIES Pectic substances are insoluble in most organic solvents. They do dissolve in water, dimethyl sulfoxide, formamide, and (warm) glycerol. The solubility in water decreases with increasing polymerization degree. Solubility is increased by all factors diminishing possibilities of intermolecular association. These factors can be of a sterical ( e g , the presence of substituents) or a chemical (e.g., charges) nature. Mostly, solubilization is proceeded by a slow swelling. Aqueous solutions of 1 to 2% (w/v) already have a relative high viscosity. This viscosity is proportional with molecular mass and is also influenced by degree of esterification (Pippen et a]., 19531, ionic strength, pH, and temperature. Depending on their degree of esterification, pectic substances are precipitated from aqueous solutions with water-miscible organic solvents or with cations. In acid solutions, the degree of esterification and/or the polymerization degree decreases. Deesterification is dominant at low temperature, whereas a high temperature enhances depolymerization. Also, the neutral sugar content decreases as these side chains are more sensitive to acid. On the average, the neutral sugar content decreases compared to the rhamnose content. The increase in the relative amount of rhamnose compared with other sugars in the heated tissue indicates possible degradation in the “hairy region.” In alkaline solutions, at low temperature, saponification of the methyl ester groups occurs readily. However, depolymerization is strongly enhanced by a rise in temperature. Such high alkali sensitivity is unique as polysaccharides are usually alkali resistant. Even more
PECTIN, PECTINASE, AND PROTOPECTINASE
225
significant is that degradation is not the result of hydrolysis of the glycosidic bonds in the classical manner but rather the result of a pelimination cleavage of glycosidic linkages (Neukom and Deuel, 1958). This reaction only occurs at glycosidic bonds adjacent to an esterified carboxyl group (Fig. 9). Pectates are indeed very much more stable at high temperature toward alkaline or neutral degradation than pectinates (Albersheim et al., 1960). Cross-linked pectin chains form insoluble polymers having ion-exchange properties. They are very selective for calcium and heavy metal ions, e.g., Zn2+,Cu2+,and Fe3+.The most unique and outstanding physical property of pectins is their ability to form gels with sugar and acid. Gels can be divided into two groups: Those containing a rather high sugar content (60-70%) and those with a lower sugar content. In the case of the former, high-methoxyl pectins (>50%) are used, while low methoxyl pectins are used in the case of the latter. Highly esterified pectin gels are obtained when, besides a sufficient high concentration, two other conditions are met: (1)Electrostatic repulsion between pectin molecules has to be decreased by repressing dissociation of carboxyl groups. Consequently, pH will play a determining role, and (2) Sugar, e.g., sucrose, or a similar carbohydrate, e.g., polyalcohols, is present in sufficient amounts. High polyol concentration decreases water activity leading to interchain interactions (Michel et a]., 1984). The hypothetical structure of a pectin-sugar gel is shown in Fig. 10. For preparations of a low ester content, gels are usually formed by the controlled introduction of calcium ions. According to Harvey (1960) the presence of calcium promotes the formation of gels by forming strong ion associations with carboxyl groups of neighboring pectin chains (“salt bridges”). However, Morris et al. (1980, 1982) have shown that the primary mechanism of this gelation involves extended chain sequences which adopt a regular twofold conformation and dimerize with specific interchain chelation of Caz+ (“egg-box” binding). Each Ca2+ ion takes part in nine coordinative links with an oxygen atom (Fig. 11).
H FIG.9. P-Elimination in a uronic acid unit in pectin [from Albersheim et al., 1960).
TAKUO SAKAI ET AL.
226
H I
H molecule
/O.
'H,
/H 0'
'H-0-
I
H H FIG.10. Hypothetical structure of a pectin-sugar gel [from Doesburg, 1965).
Apart from the degree of esterification, pH, and concentration of sugar or acid, the presence of side chains and/or groups, the degree of polymerization, the temperature, and the presence of ions also play an important role in gel formation. However, gelation of sugar beet pectins is greatly inhibited by the presence of acetyl groups. These substituents change the surface structure of the polymer (Solms and Deuel, 1951), cause sterical hindrance, and prevent the free carboxylic acid groups to form H bonds (Pippen et al., 1950). However, the presence of feruloyl
t
FIG.11. Pectins of low methoxyl content showing egg-box binding (0, non Caz+-bound oxygen atom; 0 , Caz+-bound oxygen atom) (from Thibault, 1980).
227
PECTIN, PECTINASE, AND PROTOPECTINASE
groups at the end of the neutral sugar side chains offers a third way for a gelling process, in addition to classical calcium gels of “low methoxyl” pectins and sugar acid gels of “high-methoxyl” pectins (Thibault, 1986). Indeed, sugar beet pectins can be cross-linked through their feruloyl groups and produce gels if the pectin concentration is greater than about 1%. Ammonium persulfate (Thibault et al., 1987; Thibault, 1986) and peroxide/peroxidase (Rombouts and Thibault, 1986a,b) are effective agents for this cross-linking reaction (Fig. 12). In conclusion, it can be stated that many of the unique physical properties of pectic substances are chiefly associated with the carboxyl
vH3 rvH3
a
OH
+
OH
w2- p H 3 F ___)
*
CH CH
HO-CH
HO
\
I
CH’
COOP
COOP
I
cCH
‘7’4 n
COOP
HO
OH
OH
r v H OCH, 3 @
CH \ CH
xc: -I
I
COOP
CH
AOOP
(2) FIG.12. Cross-linking of sugar beet pectins. (a) by ammonium persulfate (from Thibault et al., 1987). F, ferulate or feruloyl; P, H or pectic chain. (b) by hydrogen peroxidel peroxidase (from Markwalder and Neukom, 1976).
228
TAKUO SAKAI ET AL.
group of the galacturonic acid residues. Complete esterification of commercial pectin by chemical means totally modifies its acidic, viscosity, and gel-forming properties. For example, changes in pH have no significant effect on viscosity of solutions of totally esterified pectins. E. DETERMINATION AND CHARACTERIZATION
It was already stated earlier that pectin is a complex heteropolysaccharide with properties depending on a (varying) composition. As indeed the properties of pectin are strongly influenced by its composition, numerous techniques for analysis of pectin were developed. Pectins are usually characterized by (1)their content or uronide material, (2) their degree of esterification (DE), and (3) their degree of polymerization (DP) or some quality connected with it (viscosity, gel strength). The measurements usually made to express these characteristics give average values only (Van Deventer-Schriemer and Pilnik, 1976). On some occasions, the following analyses are also performed: (1)degree of acetylation, (2) neutral sugars, and (3) jellying power. 1. Anhydrogalacturonic Acid (AGA] Content Determinations of pectic substances in situ are qualitative, mostly by use of staining agents. For example, ruthenium red is used to obtain a molecular visualization of pectin (Hanke and Northcote, 1975). In dealing with preparations of pectic substances, the determination of polygalacturonide content is most important. In determining this AGA content, four methods have been widely used. The first method used was the decarboxylation method according to Lefevre and Tollens (1907). When uronides are boiled with hydrochloric acid (12%) they react as C,H,O$j + C,H,Oz
+ COZ + 2HzO
Then, furfural or CO, is determined gravimetrically. This method was modified by Whistler et al. (1940) and McCready et al. (1946), while Vollmert’s (1949) method, using hydroiodic acid for the decarboxylation, permits simultaneous determinations of uronide and methoxyl contents. Among colorimetric methods, the carbazole-sulfuric acid method of Dische (1950) and modified by McComb and McCready (1952), McCready and McComb (1952), and Furutani and Osajima (1965) is used most often. For estimating uronic acids in chromatographic fractions, this reaction is the most satisfactory method, but 2 hr are required for the full
PECTIN, PECTINASE, AND PROTOPECTINASE
229
development of color and, with certain compounds, the color is partially suppressed by salts (Bitter and Muir, 1962). At present, the titrimetrical method of Deuel (1943) is not frequently used any more because of interference by minerals or free acids. However, the necessary reagents and equipment are inexpensive. The most recent method [Blumenkrantz and Asboe-Hansen, 1973) is based on the appearance of a chromogen when uronic acids, heated to 100°C in concentrated sulfuric acidltetraborate, are treated with metahydroxydiphenyl. This method has been automated by Thibault (1979) and modified further by List et al. (1985). Ahmed and Labavitch (1977) used this procedure to determine uronide content of plant cell walls. 2. Degree of Esterification (Me0 content)
Here, titration (with alkali) is less problematic because the determination is carried out in two steps: titration before and after saponification. Also, methanol released on alkaline deesterification can be determined, either colorimetrically [Wood and Siddiqui, 1971; Klavons and Bennett, 1986) or gaschromatographically (McFeeters and Armstrong, 1984). The latter also used their method to measure the methoxylation of pectin in situ. The oxidation-reduction method of Laver and Wolfrom (1962) relies on the conversion of Me0 to methyl iodide. The neutralization and gas chromatographic methods have major advantages of simplicity and shortness of time of completion over the hydroiodic acid method (Walter et a]., 1983). Recent developments include 'T-NMR [ Fishman et al., 1984;Grasdalen et al., 1988) and analytical pyrolysis techniques (Barford et al., 1986). 3. Degree of Acetylation To determine the degree of acetylation by titration, the pectin sample (dissolved) has to be saponified and either steam distilled (Pippen et al., 1950) or extracted with an immiscible solvent (e.g., butanol:chloroform, 4 : 1) (Kertesz and Lavin, 1954). The reaction between esters and hydroxylamine to produce hydroxamine acids has also been applied successfully for the analysis of acetyl content in pectin (McComb and McCready, 1957) [Fig. 13). Pectin hydroxamine acid forms an insoluble complex with ferric ions and acetohydroxamic acid forms a soluble red complex. After filtration, the intensity of the red color is determined colorimetrically (at 520 nm). The Hestrin method [Downs and Pigman, 1976) is based on the same reaction. A procedure for the simultaneous quantitative analysis of methoxyl and acetate groups in pectin has been developed, using HPLC on a
1
-0
1
0 It
C-O-CH,
r
0 II
C-N
,H
+z?
+ 2n CH3CONHOH + n CH3W
0-
H
0 I O=C-CH3 _In
Acetylated pectin
n
L
Pectin hydroxamic acid
FIG.13. The hydroxamine acid reaction (from McComb and McCready, 1957).
Acetohydroxamic acid
PECTIN, PECTINASE, AND PROTOPECTINASE
231
cation-exchange resin in the protonated form and refraction index detection (Voragen et a]., 1986). 4. Neutral Sugars
Mostly, pectic substances are hydrolyzed to component sugars and, after conversion to alditol acetates of silylates, these are determined by gas chromatography (Blakeney et al., 1983; Voragen et al., 1983). 5. Molecular Mass
Generally,the estimation of the degree of polymerization or the molecular mass is the most difficult problem in the analysis of pectic substances. The results of estimations of the number of reducing end groups have been shown to be unreliable, since these results are affected by minute amounts of ballast materials. Viscosimetry has been used most frequently to determine molecular mass. However, it must be taken into account that the viscosity of a solution of pectinic acids depends on such things as molecular mass, concentration, degree of esterification, pH, and presence of electrolytes. Christensen (1954) calculated M, from viscosity measurements on commercial high-methoxyl pectins: based on the intrinsic viscosity [q],found by extrapolating qsp/c (qsp = specific viscosity, c = concentration) to c = 0.This method has been modified by Smit and Bryant (1967) to determine the M, from one measurement (of viscosity). To eliminate the complications caused by the complex colloidal properties of pectin, Schneider and Fritschi (1936) converted the pectinic acids into water-insoluble nitropectins and used acetone as the solvent for the viscosity measurements. Other techniques, although less applicable by routine, include ultracentrifugation, electron microscopy, osmometry (Jordan and Brant, 1978), light scattering (Chapman et al., 1987; Jordan and Brant, 1978; Sorochan et al., 1971), gel filtration (Anger et a]., 1977), HPSEC (Sjoberg, 1987; Fishman et al., 1989), and HPLC (Strubert and Hoverman, 1978). 6. Jellying Power
The jellying power is the most important property of pectins and, consequently, is used to grade commercial pectins. There are numerous devices for testing the consistency of jellies. Results obtained with such instruments are, however, usually expressed in arbitrary units as it is not possible to calculate the rigidities directly, i.e., in absolute units (Campbell, 1938). The jellying power of pectins is usually measured by estimating the strength of gels which have been prepared under accurately described conditions. The methods of deter-
232
TAKUO SAKAI ET AL.
minating gel strength can be divided into two large groups (IFT Committee, 1959). This was done to devise standard methods for the determination of the grade strength of pectins as commercially supplied for jam manufacture (Report of the Pectin Subcommittee, 1951). Methods belonging to a first group quantify the jellying power by measuring a controlled deformation of the jellies within their limit of elasticity. A simple apparatus, the rigidometer, has been developed for determining jelly grades of commercial pectins. It measures the modules of rigidity of gels (Owens et a]., 1947). Cox and Higby (1944) determined the percentage sag, or slump, occurring when a test jelly is removed from its supporting container and inverted upon a glass plate. The “BLOOM gelometer” measures the force required to push down the gel surface for 4 cm. In second group method of measurement, the elastic limits of the jellies (the “breaking strength”) are exceeded and the jellies ruptured. For example, the “Delaware jelly tester,” developed by Tarr (1926) and Baker (1926), measures the force required to push down a gel surface until the gel breaks. The jellying power of high-methoxyl pectins is described in relation to their sugar carrying power. The jelly grade is the number of parts (by weight) of sugar that one part of pectin will convert to a jelly under standard conditions. F. PECTIN MANUFACTURING: CHEMICAL EXTRACTION AND PURIFICATION
Data concerning pectin consumption are given in Table V. Today, the chief raw materials for the production of pectin are by-products from the manufacture of fruit juices: apple pomace (dried) and citrus residues (peel). The raw materials for pectin production are wastes from other operations, and the quality of the pectin produced is often determined by physical and chemical operations in the primary industry (Charley, 1951). TABLE V CONSUMPTION, PRICES, AND MARKETS OF PECTIN‘ Consumptionb (tondyear)
Prices
Product Low methoxyl pectin High methoxyl pectin
6000 8000
7.15-11.00 7.92-8.80
a
Data from Yalpani and Sandford (1987). For the United States, during 1983-1985
($W
Markets
(lo6 $/year) 22
PECTIN, PECTINASE, AND PROTOPECTINASE
233
Until recently, chemical extraction has been the only way to produce pectin. This extraction is performed by acid hydrolysis. Conditions vary but generally a pH in the range of 2.0-3.0 is used for 0.5-5 hr within a temperature range of 70-100°C. The solid to liquid ratio is normally about 1: 18. In some countries the use of mineral acids is prohibited and these are replaced by citric, lactic, or tartaric acids. The pectin extract is separated from the pomace using hydraulic presses and/or centrifugation. Sometimes, gelatinization of starch takes place and this necessitates an enzymatic treatment with amylases. Subsequently the extract is filtered again and then finally concentrated to a standard setting strength. In preparation of powdered pectins the concentrated liquor is treated with organic solvents or certain metallic salts to precipitate the polymers. The pectin precipitate is collected, dried, and ground. Commercial pectins are standardized products. This is done to ensure that the users always get the same gel strength. Standardization can affect the chemical structure of pectin as esters can be partly saponified or acid groups can be amidated. High-methoxyl pectin only forms gel above a soluble solids (sugar) content of about 55%. Low ester pectin forms gels in the presence of (calcium) ions, irrespective of soluble solids. A scheme of the commercial production of pectin is represented in Fig. 14.
G. APPLICATIONS OF PECTIN 1. In the Food Sector
Pectin is first and foremost a gelling agent (E440) and is used to give a gelled texture to foods, mainly fruit-based foods. About 80% of the world production of high-methoxyl pectin is used in the manufacture of jams and jellies, to make up for their “deficiency” of natural pectins. Indeed, under these conditions, i.e., a high sugar concentration and a low pH, it is the best gelifying agent available (Nelson et al., 1977). Pectin establishes a texture that retains a uniform distribution of fruit particles during transportation, gives a good flavor release, and minimizes syneresis. The pectin concentrations used vary from 0.1 to 0.4% in jams and jellies (Pectin, 1987). Low ester pectins are often used in fruit preparations for yogurt in order to create a soft, partially thixotropic gel texture, sufficiently firm to ensure uniform fruit distribution, but still allowing the fruit preparation to be easily stirred into the yogurt. The pectin may further reduce-especially when combined with other plant gums-color migration into the yogurt phase of the final product.
TAKUO SAKAI ET AL.
234
I
1
I
1 Treat with acidified isopropanol I
&
I
Rinse with is0 ro anol
in isopropanol
1-
+
(Grind to pass 60 mesh screen
1
Standardize, package, and sell as slow-setting pectin (HM)
FIG.14. Production and standardization of pectin (from Nelson et al., 1977).
Gelation (by pectin) also provides stabilization of emulsions, suspensions, and foams. This is demonstrated in the fruit drink concentrates. In recombined or instant juice products, pectin gelation restores the sensorial properties to those of the fresh juice. In dairy products, the pectin reacts with the casein, preventing the coagulation of the casein at a pH below the isoelectric pH (4.6) and allowing pasteurization of the sour milk products to extend their shelf life. Another application of pectin is in confectionary fillings. Also, the possibility of using pectin for the production of single cell protein in a modified “Symba process” was reported (Fellows and Worgan, 1986, 1987a,b).
PECTIN, PECTINASE, AND PROTOPECTINASE
235
2. In the Pharmaceutical Sector
In a number of (liquid) pharmaceutical preparations the ability of pectin to increase viscosity and stabilize emulsions and suspensions is utilized. Pectin, belonging to the chemically heterogeneous group of substances referred to as “dietary fiber” (Aspinall and Carpenter, 1984), is further reported to possess a number of valuable biological effects. It acts as a general “intestinal regulator” and a detoxifying agent, but the most well known effect is its anti-diarrhea effect (Chenoweth and Leveille, 1975). This probably explains the ancient use of the diet of scraped apples as a home remedy against diarrhea (Birnberg, 1933). Indeed, “an apple a day keeps the doctor away.” There are several hypotheses to explain these effects. It is suggested that pectin, or its degradation products, moves rapidly to the large intestine and exerts a bacteriostatic effect against several pathogens (Werch and Ivy, 1941; Campbell and Palmer, 1978). Pectin, being a colloidal carbohydrate, acts as a lubricant in the intestines, coating the mucosa with uncharged polysaccharide and promotes normal peristalsis without causing irritation. This makes it a standard additive to baby foods. The detoxifying action is probably a consequence of the binding of metal ions to pectin (fragments of) (Kohn, 1987). Pectin could also be used as a carrier for pharmaceuticals (Heinzler et al., 1987). It also decreases the toxicity of pharmaceuticals (some) and prolongs their activity without lessening their therapeutic effect (Pilnik and Voragen, 1970). More recently, pectin-gelation microglobules were developed for potential use in regional cancer chemotherapy as an intravascular biodegradable drug delivery system (Bechard and McMullen, 1986). Pectic substances also seem to show hemostatic and antifibrinolytic effects (Barth and Rumpelt, 1947; Fogarty and Kelly, 1983). Work has also been done to show that pectin administered orally is somewhat effective in reducing cholesterol levels in the blood. Probably, the absorption of bile acids is decreased and, as a consequence, more cholesterol has to be converted to bile acids (Baig et al., 1980; Kay and Truswell, 1977; Keys et al., 1960; Lin et al., 1957; Pfeffer et al., 1981). It is hoped that the use of pectin-enriched foods will aid in the prevention and treatment of arteriosclerosis (Panchev et al., 1989). 3. The Cosmetical Sector The applications in the cosmetical sector only utilize the “ordinary” properties of pectin. Examples are the numerous gels (hair) and pastes.
236
TAKUO SAKAI ET AL.
Ill. Classification of Pectic Enzymes
Basically three types of pectic enzymes exist: pectinesterase, which only removes methoxyl residues from pectin, a range of depolymerizing enzymes (pectinase), and protopectinase, which solubilizes protopectin to form pectin (Table VI). Pectinases are distinguished under three headings (Enzymic Commission, 1944) according to the following criteria: (1)Whether pectin, pectic acid, or oligo-D-galacturonate is the preferred substrate, (2) Whether they act by transelimination or hydrolysis, and (3) Whether the cleavage is random (endo-, liquefying, or depoly-
TABLE VI CLASSIFICATION OF PECTICENZYMES
Pectinesterase (PE) (Pectin methylhydrolase, EC 3.1.1.11) Catalyzes deesterification of the methoxyl group of pectin forming pectic acid. Depolymerizing enzymes Enzymes hydrolyzing glycosidic linkages: Polymethylgalacturonase (PMG) Endo-PMG, causes random cleavage of a-l,4-glycosidic linkages of pectin, preferentially highly esterified pectin. Exo-PMG causes sequential cleavage of a-1,4-glycosidic linkages of pectin from the nonreducing end of the pectin chain. Polygalacturonase (PG) glycanohydrolase], catalyzes Endo-PG [EC 3.2.1.15, poly(l,4-a-D-galacturonide) random hydrolysis of a-l,4-glycosidic linkages in pectic acid (polygalacturonic acid). Exo-PG [EC 3.2.1.67, poly(l,4-a-~-galacturonide) galacturonohydrolase], catalyzes hydrolysis in a sequential fashion of a-1,4-glycosidic linkages in pectic acid. Enzymes cleaving a-1,4-glycosidic linkages by transelimination which results in galacturonide with an unsaturated bond between C, and C, at the nonreducing end of the galacturonic acid formed. Polymethylgalacturonate lyase (PMGL) Endo-PMGL [EC 4.2.2.10, poly(methoxygalacturonide)lyase], catalyzes random cleavage of a-l,4-glycosidic linkages i n pectin. Exo-PMGL, catalyzes stepwise breakdown of pectin by transeliminative cleavage. Polygalacturonate lyase (PGL) Endo-PGL [EC 4.2.2.2, poly(l,4-a-D-galacturonide)~yase], catalyzes random cleavage of a-1,4-glycosidic linkages in pectic acid by transelimination. catalyzes sequential Exo-PGL [EC 4.2.2.9, poly(l,4-a-o-ga~acturonide)exo~yase], cleavage of a-1,4-glycosidic linkages in pectic acid by transelimination. Protopectinase The enzyme solubilizes protopectin forming highly polymerized soluble pectin.
237
PECTIN, PECTINASE, AND PROTOPECTINASE
merizing enzymes) or end-wise (exo- or saccharifying enzymes). The modes of action of the different types of pectic enzymes are illustrated in Fig. 15. However, no distinction is made between endo- and exoenzymes. The cup-plate method of Dingle et al. (1953) is a frequently used qualitative method for detecting microbial pectinase activity. A supernatant of a microbial culture is inserted into wells cut in an agar-pectin gel. If zones of activity appear around the cups, one may expect pectinase activity. A bioassay specific for polygalacturonase (PG) activity is described by Mussel1 and Moore (1969).This assay is based on fresh weight loss of cucumber pericarp tissue. Hildebrand (1971)tested many Pseudomonas sp., and also other plant pathogens, for pit formation on polypectate gels and used the results in the differentiation of the species. Another method for detecting polygalacturonase activity uses ruthenium red staining of colonies on polygalacturonate-agarose plates. Ruthenium red was shown to penetrate beneath the surface layers of the gel only in the regions surrounding a colony where degradation of polygalacturonate had occurred (McKay, 1988). A similar procedure is suitable in locating pectic enzymes in polygalacturonate-agarose overlays into which pectic enzymes diffuse from electrophoresis gels (Collmer et a]., 1988).
Protopectin
1
Protopectinase
-oo eozofi -06 Polymethylgalacturonate(Pectin)
O H H
H
I
OH
OH
OH
OHOH
O H
H O H
CoocHi
Polymethylgalacturonate lyase
H
c-3
I
-
+@
cooa4
w n
o n
cooa4
M
Polyrnethylgalacturonase
Pectinesterase
1
Polygabcturonate (Pectic acid) H
CM
-
0
H
+ m
0
OH
ic
Q
n
aa
M
o
o
-
-
-oQ
O
rm
H
u r n
O
OH
o
0
O H
Cm
H
l i b 4
Polygalacturonate lY-
=
o
O H
im
Q
+ H
OH
M
H
FIG.15. Mode of action of pectic enzymes.
o H
M
! i Q
Polygalacturonase
n
too
238
TAKUO SAKAl ET AL.
However, these methods can only be used for the quantitative determination of one specific enzyme. A. ESTERASES Pectinesterases (PE) are formed by fungi, bacteria (Table VII), yeasts, and higher plants. Pectinesterases, especially those from higher plants, are highly specific enzymes. In many cases they saponify almost exclusively the methyl ester groups of pectic substances. McDonnell et al. (1950) examined several fungal enzymes and found that rates of hydrolysis of the ethyl ester of pectic acid range between 6 and 16% of the rates obtained with the natural methyl ester. Some PEs attack pectin only at the reducing chain end while others attack the nonreducing end (Miller and Macmillan, 1971). Also, PEs seem to hydrolyze only methyl ester groups adjacent to free carboxyl groups. The enzymes then proceed in a linear fashion along the substrate. The result is a blockwise distribution of free carboxyl groups and esterified carboxyl groups (Fig. 16) (Speiser and Eddy, 1946). This also accounts for the high calcium sensitivity of enzymatically deesterified pectinic acids as compared with pectinic acids saponified in acid or alkaline milieu. In some cases, Caz+ (and also Na+) stimulate PE activity. In binding the pectic acid ( = reaction product), Ca2 prevents the PE from binding with it which would result in inhibition of the enzyme. It has also been reported that incomplete hydrolysis of methyl esters by PE can be caused by the inhibition of enzyme activity by the side chains of neutral sugars in the pectin molecules (Matsuura, 1987). The synthesis of certain pectinesterases (e.g., some Aspergillus niger strains) is repressed by glucose, even in the presence of the inducer (Maldonado et al., 1989). pH values at which PEs are active range from 4 to 8. The pH optimum for PE activity of fungal origin is generally lower than that of bacteria. Care must be taken not to confuse nonenzymatic deesterification with enzymatic activity at pH values above 7.0. The activity of these enzymes can be followed by determinating the increase of free carboxylic acid groups or by measuring the liberation of methanol into the solution. Free carboxyl groups can be measured by titration (Kertesz and Lavin, 1954). This has been simplified by the introduction of continuous, automatic titrations (Hagerman and Austin, 1986). Forster (1988) defines one unit of enzyme activity as the amount of enzyme which causes a decrease in pH of the reaction mixture of 0.1 in 30 min. The methanol may be distilled off and estimated by oxidation to formaldehyde as described by Holden (1945). Methanol can also be +
239
PECTIN, PECTINASE, AND PROTOPECTINASE TABLE VII OCCURRENCE OF PECTIC ENZYMESIN SOMEMICROORGANISMS~ ~
Source
PE
PG
Bacteria
Bacillus sp. Bacillus sp. No. RK9 Bacillus subtilis Bacillus polymyxa Bacillus purnilus Bacillus sphaericus Bacillus stearothermophifus Erwinia aroideae Erwinia carotovora Pseudomonas sp. Pseudomonos ff uorescens Pseudornonas marginalis Xanthomonas sp. Xanthomonas campestris Xanthornonas cyanopsidis Clostridiurn multiferrnentans C1ostri di urn aumntibutyricurn Clostridium felsineurn Cytophaga johnsonii Cytophaga deprimata Cytophaga albogilva Streptornyces nitrosporeus FUlgi Trichoderma koningii Trichoderrna pseudokoningii Cercospora orachidicoia Cephalosporiurn sp. Aspergillus niger Aspergillus sojae Aspergillus saita Fusarium culrnorum Fusariurn oxysporurn Fusariurn solani Penicillium expansurn Penicilliurn italicum Penicilliurn digitaturn Penicillium chrysogenum Rhizoctonia fragariae Ahizoctonia solani Ahizopus arrhizus
+ + +
+ + + +
+
+
+
+ + + + + +
+ +
+
+ + +
PGL
PMG
+ + + + + + + + + + + + + + + + + + + + + +
+
PMGL
OG
OGL
i
+
+
+ + +
+ + +
+ + +
+
+ +
+
PE, pectinesterase; PG, polygalacturonase;PGL, polygalacturonatelyase; PMG,polymethylgalacturonase; PMGL. polymethylgalacturonate lyase; OG, oligogalacturonase;OGL, oligogalacturonide lyase. (Data from Fogarty and Kelly, 1983.)
240
TAKUO SAKAI ET AL.
COOCH, COOCH, COOCH, COOCH, COOH I
1
I
I
I
COOH
COOH
COOH
I
I
I
FIG.16. Mode of saponification by pectinesterase (from Doesburg, 1965).
measured chromatographically (McFeeters and Armstrong, 1984) or colorimetrically (Wood and Siddiqui, 1971). B. HYDROLASES
1. Endopolygalacturonases (Endo-PG) These enzymes are produced by numerous fungi and bacteria (Table VII), by a few yeasts (Luh and Phaff, 1951; Ravelomanana et al., 1986), and also by higher plants and some plant-parasitic nematodes (Riedel and Mai, 1971). In general, by the action of PG, pectic acid is broken down into mono-, di-, and trigalacturonic acid. These end products may be produced by a “single chain multiple attack” mechanism, in which case they can be detected rapidly, or by a “multi-chain attack” mechanism, where the mono-, di-, and trimers accumulate only after further hydrolysis of the initial depolymerization products (higher oligogalacturonates) (Fogarty and Kelly, 1983). The former reaction is performed by the PG from Colletotrichum lindemuthianum (English et al., 1972; Albersheim, 1976) while the latter is in agreement with the action pattern of Kluyveromyces fragilis (Phaff, 1966). Endopolygalacturonases are specific for pectic acid. If the degree of methoxylation increases, the rate and extent of hydrolysis decreases. Free carboxyl groups seem to be necessary for catalytic activity (Jansen and McDonnell, 1945; Koller and Neukom, 1969). The rate of splitting of the glycosidic bonds also decreases with the shortening of the substrate chain. Still, many PGs are able to degrade the trimer, at a much lower rate (Rexova-Benkova and MarkoviC, 1976). Three different patterns of action toward low molecular substrates are known. The character of the active center constitutes the determining factor. Generally, endo-PGs are optimally active at a rather low pH (4.0 to 6.0) and at a temperature of 30-40°C. Kaji and Okada (1969) even described an endo-PG with an optimum for catalytic activity at pH 2.5. Other specific properties of the enzyme, as for example the need for coenzymes, have not been reported. Calcium ions influence the activity of polygalacturonase. However,
PECTIN, PECTINASE, AND PROTOPECTINASE
241
in some cases the activity was inhibited, while in other cases it was stimulated (Perley and Page, 1971). PGs can be produced constitutively (Wimborne and Rickard, 1978) or inducibly (Bhaskaran and Prasad, 1971; De Lorenzo et al., 1987). In other cases, the activity is only slightly enhanced (Bashan et a]., 1985). Induction or stimulation is mostly caused by low concentrations of pectins or oligo- and monomeric fragments thereof (Cooper and Wood, 1975). Brookhouser et al. (1980) reported that some microorganisms (e.g., Rhizoctonia solani) produce different froms of endo-PG and that the predominant form produced during pathogenesis differs from the single-peak form produced in culture. Some PGs are sensitive to catabolic repression (Horton and Keen, 1966; Keen and Horton, 1966; Hsu and Vaughn, 1969; Maldonado et al., 1989). Other are inhibited in vivo, mostly by a protein present in the host (Barmore and Nguyen, 1985; Collmer and Keen, 1986; Mahadevan et a]., 1965), sometimes by tannins or phenolic compounds present in the host tissues (Prasad and Gupta, 1967). The formerly mentioned inhibition by a protein is competitive. The activity of PG is also influenced by substitutions on the pectic acid molecule (Jansen and McDonnell, 1945). Over the years, mainly two methods for the determination of PG activity have been developed. Methods of a first group are based on the determination of reducing groups, released as a consequence of substrate hydrolysis by PG. The colorimetric method by Nelson (1944) or the iodometric method by Jansen and McDonnell (1945) can be used. However, viscosity measurements have also found widespread use for determinating pectinase activity (Cappellini, 1966; Bateman, 1972)
% reduction in viscosity = To - TJT,
-
T,,
where To,T,, and T, represent the flow time (in a capillary viscosimeter) in seconds for the reaction mixture without enzyme, the test mixture, and water, respectively. However, viscometric methods have met with limited success. This is due to the requirements for strictly standardized conditions, since the viscosity of solutions of pectic substances depends on pH, temperature, buffer, and ionic strength. The unit of enzyme activity is mostly selected as that amount of enzyme required for attaining a certain decrease of viscosity per unit time. 2. Exopolygalacturonases (Exo-PG)
Exopolygalacturonases occur less frequently. They are produced by fungi and some bacteria. Two types of exo-PG can be distinguished. Fungal exo-PGs (e.g., from Coniothyrium diplodiella) produce monogalacturonic acid as the main end product and have pH optima from 4.0
242
TAKUO SAKAI ET AL.
to 6.0. This enzyme is called galacturan 1,4-a-galacturonidase or exoPG 1. Bacterial enzymes, however [e.g., from Erwinia aroideae or Selenomonas ruminantium (Heinrichova and Wojciechowicz, 1989)], produce digalacturonic acid as the main end product. They are mostly designated as exo-poly-a-galacturonidase or exo-PG 2. Both enzymes, however, degrade pectic acid from the nonreducing end (Fig. 17). With respect to other, general characteristics (e.g., constitutivity, inhibition, and repression), exo-PG resembles endo-PG. Here too, the increase of the number of reducing groups can be followed to determine enzyme activity. Exopolygalacturonase activity can be detected more specifically on the basis of the release of digalacturonic acids. This can be done by spotting samples on TLC plates and developing them with a benzidine solution (Gothoskar et al., 1955). Exo- and endo-PG activity can be differentiated by measuring both increase of reducing groups and reduction of viscosity. An endo-enzyme is characterized by a strong reduction in viscosity (e.g., 50%) without a significant release of reducing groups ( 2 4 % ) .To obtain a 50% viscosity reduction, an exo-enzyme has to hydrolyze 20% of the glycosidic linkages (Nasuno and Starr, 19681. 3. Oligogalacturonases (OG) An oligogalacturonate hydrolase, free of transeliminase and unsaturated oligogalacturonate hydrolase activities, has been isolated from the cell extract of a Bacillus species by Hasegawa and Nagel (1968). The pH optimum is 6.0-6.5. The hydrolase is highly specific for saturated oligogalacturonides, attacking it from the nonreducing end of the molecule. A similar activity has been isolated from the mycelium of Aspergil-
1
Exq-PG 2 ( E )
Exo-PG 1 (C)
0-0+o-o+e-
No reaction
4 O
O
-
4
Exo:PG 1 (C)
O
+R
~
O
-
R R
ExoiPG 2 ( E )
O
f R ~
FIG.17. Action of exo-PG of Coniothyrium diplodieiia (C) and Erwinia oroideae (E) on saturated pectic acid (from Fogarty and Ward, 1974). 0,unsaturated galacturonic acid unit.
PECTIN, PECTINASE, AND PROTOPECTINASE
243
lus niger (Hatanaka and Ozawa, 1969). In addition, Nagel and Hasegawa (1968) also described an unsaturated oligogalacturonate hydrolase. 4. Polymethylgalacturonases (PMG)
Although several articles on PMGs have appeared in the literature (Perley and Page, 1971; Finkelman and ZajiC, 1978), the existence of these enzymes is still in question. Polygalacturonase preparations, contaminated with PE, can be mistaken for PMG-containing preparations. Also, if the substrate is not completely esterified, PG or PGL could hydrolyze the glycosidic bonds in these areas. Assay methods similar to those used for determining PG activity can also be applied here, with exception of course of the substrate to be used. C. LYASES
Lyases (or trans-eliminases) perform a nonhydrolytic breakdown of pectates and pectinates, characterized by a trans-eliminative split of the pectic polymer. The lyases break the glycosidic linkage at C-4 and simultaneously eliminate the H from C-5 (Ayers et al., 1966). In some aspects, e.g., induction and catabolic repression (Hubbard et al., 1978; Kurowski and Dunleavy, 1976), lyases resemble hydrolases. This can also be concluded from data in the extensive review article by Linhardt et al. (1986). The methods, used for determination of PG activity, are also suitable here. To detect lyase activity specifically, one assay has been widely and frequently used; measuring the increase in light absorption by reaction mixtures at 230 or 235 nm. At this wavelength, the double bond produced on trans-eliminative cleavage of pectin substrates absorbs maximally. The unsaturated di- and oligouronides also react with thiobarbituric acid to form red chromogens with a maximum absorption at 545-550 nm (Hasegawa and Nagel, 1962; Albersheim et al., 1960). 1. Endopolygalacturonate Lyase (Endo-PGL)
Endopolygalacturonate lyases are produced by several bacteria and fungi (Table VII). On some aspects, PGL can be distinguished clearly from PG. Their pH optima are significantly higher, ranging from 8.0 to 10.0. In addition, all endo-PGLs are activated by Ca2+(e.g.,Lyon et al., 1986) and, in some cases, also to some extent by other divalent cations like Mg2+, Co2+,and Sr2+.It is suggested that a pair of galacturonic acid chains, linked together with a salt bridge, may be the true substrate.
244
TAKUO SAKAI ET AL.
Generally, pectates are good substrates for endo-PGL, but some enzymes exert optimal activity on pectins with a specific degree of polymerization (Pilnik et al., 1973). The main end product from polygalacturonic acid is unsaturated diagalacturonic acid. Lesser amounts of unsaturated trigalacturonic acid and saturated mono- and digalacturonic acids are formed (Moran et al., 1968a). Also, PGL activity decreases as the polymerization degree decreases. 2. Exopolygalacturonate Lyases (Exo-PGL)
Exopolygalacturonate lyases release unsaturated oligogalacturonates from the reducing end of the polymer. The smallest substrate they can hydrolyze is the trimer. Just like endo-PGL, they have a high pH optimum and are activated by the addition of divalent cations (Macmillan and Phaff, 1966). 3. Oligogalacturonide Lyase (OGL)
Some phytopatogenic bacteria, e.g., Erwinia carotovora (Moran et al., 1968b) produce an OGL. Oligogalacturonide lyases are cell-bound enzymes degrading oligogalacturonates or unsaturated oligogalacturonates by removing unsaturated monomers from the reducing end of their substrates by the trans-elimination process. 4. Polymethylgalacturonate Lyase (PMGL)
The PMGLs are the only pectinases proven to be able to hydrolyze pectin. These enzymes are found in some fungi, but rarely in bacteria (Sone et al., 1988). Riedel and Mai (1971) also detected PMGL activity in aqueous extracts of a population of Ditylenchus dipsaci, a plant parasitic nematode. All PMGLs are endo-acting enzymes that cause a rapid drop in viscosity. pH optima range form 5 to 9, and Ca 2 + does not stimulate enzyme activity. The preferred substrates are highly esterified pectins; polygalacturonates are not attacked. Here too, activity decreases with decreasing chain length. IV. Role of Pectic Enzymes in Phytopathogenesis
Evidence to prove the role of pectic enzymes as a cause of (fungal) plant diseases has been accumulating (Ikotun and Balogun, 1987; Morris et al., 1980). These enzymes cause tissue maceration by degrading the pectic substances of the middle lamellae. Endopolygalacturonase and endo-PGL are considered to be the primary enzymes for maceration (Barmore and Brown, 1979). In some cases, the pectic enzymes also convert the pectic polymers of the host plant to a utilizable substrate
PECTIN, PECTINASE, AND PROTOPECTINASE
245
for pathogen growth during pathogenesis (Bateman, 1972). However, the importance of microbial PG in pathogenesis has been established not only in plant diseases characterized by rapid and extensive degradation of host cell walls, but also in some diseases where only a minimal breakdown of cell wall polysaccharides occurs during penetration and colonization of host tissue (Cervone et al., 1987). Collmer and Keen (1986) distinguished several steps during the interaction of a pectinolytic pathogen and a potential host: (1)The entering pathogen possesses structural genes encoding pectic enzymes with particular physical and catalytic properties. (2) These genes are expressed in a characteristic manner in the infected tissue. (3) The enzymes are exported from the pathogen cytoplasm to the host tissue environment. (4) In some tissues the enzymes encounter inhibitors or protected substrates. In other tissues the enzymes are active and cleave structural polymers in the primary cell wall and middle lamella, facilitating pathogen penetration and colonization. Because of the accessibility of pectic polymers in the primary cell wall to enzymatic attack and the consequent rapid release of pectic inducers, pectic enzymes are the first polysaccharidases to be induced when fungi are cultured on isolated cell walls, and the first to be produced in infected tissues. V. Applications of Pectinases
A. INDUSTRIAL PRODUCTION OF PECTINASES It is very difficult to find reliable and detailed information about the commercial production of pectinases. Probably all producer strains are Aspergillus species. Research for additional pectinase producers is hampered by the fact that only a limited number of microorganisms are approved for application in the food industry. The preparations available mostly contain mixtures of PE, PG, and PGL activity (Zetelaki, 1976). The relative amounts of the respective enzymes produced vary considerably with the particular strain used, with nutrient composition, and various environmental factors. In fact, there are three different industrial methods used to produce microbial enzymes; the surface-bran culture (Koji) method, the deep-tank (submerged) process, and the two-stage submerged process. According to Rombouts and Pilnik (1980), most pectinases are still produced by the surface method, carried out in rotating drums, although in general the submerged process is more widely used because of its easier control. A crucial factor in pectinase production is the composition of the medium. Details about such media are considered to be strictly confidential and
246
TAKUO SAKAI ET AL.
are not released by the manufacturers. In general, the medium will be a mixture of carbohydrates (glucose, molasses, CSL, and starch hydrolysates), N sources (NH,+ salts, CSL, DDS, and yeast extract), and minerals. If the enzyme is not produced constitutively, an inducer also has to be added. For reasons of economy, pectin is not used much in production media, but it is substituted by dried sugar cossettes (Zetelaki, 1976), citrus peel, or apple pomace. Control of pH is also very important. The highest enzyme production is achieved when the pH value drops from an initial value of about 4.5 to a more or less constant value of 3.5 during the course of the fermentation, which usually takes 3 to 6 days. At extreme pHs (7) a marked inactivation occurs (Schroder and Muller-Stoll, 1962). At the end of the fermentation, the enzymes are extracted from the semisolid medium and mycelium, the dilute enzyme solution is concentrated, and the enzymes are then precipitated with organic solvents or inorganic salts. Following precipitation, the enzyme cake is centrifuged or filtered and then dried at low temperatures or spray-dried. Subsequently, it is ground to a particular particle size and used to prepare commercial enzyme formulations. Some preparations are sold as liquid concentrates. Pectinases are produced by a number of companies in Europe (NovoNORDISK, Miles Kali-Chemie, Swiss Ferment Co.), the United States (Miles laboratories, Rohm and Haas Co.), and Japan (Kikkoman Shoyu Co.). Rombouts and Pilnik (1980) estimated that the worldwide food enzyme production represents a value of about U.S. $45 million, of which perhaps one-quarter relates to pectinases. The development of this enzyme industry has been related with the fruit juice industry. Indeed, because of their low pH optima, pectinases are particularly suitable to be used in this sector. Preferably, it should also be possible to use them at elevated temperatures.
B. FRUITJUICE INDUSTRY
The majority of the pectinase preparations are used in the fruit processing industry. In the beginning of the 1930s publications in Germany and the United States reported on the application of pectinase preparations in apple juice processing in order to facilitate filtration, remove turbidity, and prevent cloud-forming. Also, when apples are of poor pressing quality because of variety or storage, the amount of juice released is low but by treating the pulp with pectinases the juice yield is increased (De Vos and Pilnik, 1973). Almost as old as the application in clarification is the application in extraction. At first, pectinases were
PECTIN, PECTINASE, AND PROTOPECTINASE
247
added to black currants to facilitate extraction by pressing and were later added to other soft fruits like raspberries and black cherries. After crushing, these crude fruit juices are often very viscous and sometimes slightly gelified. It is also very difficult to separate remaining solids from the juice. By adding pectinases, however, viscosity drops and it becomes possible to extract juice by pressing. Some preparations are used to produce juices by liquefaction. In this process, cell walls are dissolved by a combination of pectinases and cellulases and a yield (juice) of up to 100% can be obtained (Dorreich, 1983; Janda, 1983). Also, mechanical desintegration is sometimes replaced by enzymatic maceration (Rohm, 1969). Maceration results in the release of single cells, leaving intact cell walls, and thereby also releasing flavor compounds, pigments, and active ingredients in an intact state (Silley, 1986). Problems in clarification of fruit juices are caused mainly by the presence of pectic substances which suspend toward insoluble (pulp) particles. After treatment with pectinases, these particles can be separated by sedimentation or filtration. This technique is also used in wine production. An additional advantage is the improved liberation of anthocyanins as a consequence of tissue degradation and breakdown of anthocyanin-pectin complexes. As a result the “color yield” increases and the production time shortens (Pilnik, 1981). Pectinases induce clarifying, filtration improving, colour liberating, and yieldenhancing effects (Grampp, 1982). In contrast, for some fruit juices turbidity is wanted. In these cases, the native enzymes must be inactivated, mostly by heat. This prevents well-known quality defects such as cloud loss in citrus juices and gelation of concentrates, caused by citrus pectinesterases through deesterification of juice pectin which subsequently precipitates or gels as calcium pectinate or pectate.
C . OTHERAPPLICATIONS
Pectinases are also involved in the retting process (Ali, 1958).Retting is a fermentation process in which certain bacteria (e.g., Clostridium, Bacillus) and fungi (e.g., Aspergillus, Penicillium, Cladosporium) decompose pectins of the bark and release the fiber (Chaudhury, 1953). This process plays an important role in the production of many important textile fibers such as flax,hemp, and jute. Novo-NORDISK developed an enzyme preparation, “flaxzyme,” which accelerates and improves this process, Fogarty and Ward (1972) were the first to report
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TAKUO SAKAI ET AL.
the potential application of pectinase producing organisms or their enzymes to treat commercial softwoods in order to render them more amenable to treatment with preservatives. VI. Protopectin-Solubilizing Enzyme (Protopectinase)
The enzyme that catalyzes the solubilization of protopectin was originally named protopectinase by Brinton et al. 1927. They proposed that this term should be applied to an enzyme that hydrolyzes or dissolves protopectin, causing plant cells to separate from each other, a process which is usually called maceration. The term of “protopectinase” superseded the older term “pectosinase,” with which it was synonymous. However, further research on pectic enzymes showed that the decomposition of protopectin was due to the action of a system of enzymes, including pectinesterase, endo-polygalacturonase, endo-pectate lyase, and pectin lyase. Kaji (1956, 1959) found a microbial enzyme in the culture filtrate of Clostridiurn felsineurn that catalyzes the breakdown of middle lamella pectic substance from the bark of gampi (Wikstremia sikokiana Fr. et. Sav.) to a soluble pectic substance. The enzyme activity that liberates pectin from plant tissues was also found in the culture filtrate of Aspergillus japonicus by Ishii (1976). He found that the enzyme reaction was catalyzed by the combination of endo-polygalacturonase and endo-pectin lyase. Karr and Albersheim (1970), studying Pectinol R-10, a mixture of enzymes produced by Aspergillus niger, isolated an enzyme that liberated a pectic substance from protopectin but degraded pectic acid only to a limited extent. Details about the enzyme were not studied. In these reports, the protopectin-solubilizing enzyme has been regarded as an enzyme that macerates plant tissues, and little was known about enzymes that liberate highly polymerized pectin (pectinliberating enzymes). In 1978, the first study on pectin-liberating enzymes was reported by Sakai and Okushima. A microorganism was detected that produced a protopectin-solubilizing enzyme, which liberated water-soluble and highly polymerized pectin from protopectin. They also reported that protopectin is solubilized by restricted hydrolysis and called such enzymes “protopectinase” (PPase). Since then, several PPases, which are classified into two types depending on their reaction mechanism, have been isolated (Sakai and Okushima, 1982; Sakai and Yoshitake, 1984; Sakai et a]., 1984; Sakai and Sakamoto, 1990). One type PPase reacts with the polygalacturonic acid region of protopectin (inner site) and the other on the polysaccharide chains that may connect the polygalacturonic acid chain and cell wall constitutents
PECTIN, PECTINASE, AND PROTOPECTINASE
249
(outer site), as shown in Fig. 18.Sakai and co-workers called the former A-type PPase and the latter B type PPase. These enzymes are described here in more detail. ACTIVITY A. ASSAYOF PROTOPECTINASE PPase activity is assayed by measuring the amount of pectic substance liberated from protopectin by the carbazole-sulfuric acid method (Furutani and Osajima, 1965). The reaction mixture contains 10 mg of protopectin, 100 pmol of acetate buffer [AcB) containing 50 pgiml bovine serum albumin, pH 5.0, and 50 p1 of enzyme solution, in a total volume of 1.0 ml. The reaction mixture is incubated beforehand for 1 hr at 37°C. The reaction is started by the addition of the enzyme solution, and the mixture is kept for 1 hr at 37°C. The reaction is stopped by cooling the
Neutral sugar sidechain
I reaction with A-type protopectinase
I I I
reaction with B-type protopectinase
I
M
4 % A /-
High molecular weight pectin
Low molecular weight pectin
FIG 18. Schematic illustration of structure of protopectin and reaction mode with Atype and B-type protopectinases.
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TAKUO SAKAI ET AL.
reaction mixture in an ice bath. The control blank is run with the use of a heat-denatured enzyme solution. After the reaction, the mixture is filtered on filter paper. To a test tube containing 250 p1 of filtrate of the reaction mixture, 3 ml of chilled 32 N H2S0, solution is introduced, followed by 250 p l of 0.2% carbazole in ethanol. This step is done in an ice bath. The assay mixture is heated at 75°C for 20 min and cooled to room temperature, after which the optical density at 525 nm is measured. The pectin concentration is measured as D-galacturonic acid from a standard assay curve with D-galacturonic acid. One unit of PPase activity is defined as the activity that liberates pectic substance corresponding to 1 pmol of D-galacturonic acid per milliliter of reaction mixture at 37OC in 1 hr, and the specific activity is expressed as units per milligram of protein. Protopectin used in routine experiments can be prepared from lemon (Citrus limon Burm) peel by the following procedure. The albedo layer of the peel is scooped out, pooled, washed with distilled water until the water-soluble substances that react with carbazole-sulfuric acid are washed off, and then lyophilized. The dried protopectin preparation is stored in the refrigerator. In the experiments with B-type PPase, protopectin treated with ethylenediaminetetraacetic acid (EDTA)is used. The EDTA-treated protopectin is prepared from protopecitn as obtained earlier. The protopectin is washed with 50 mM EDTA until EDTA-soluble pectic substances are washed off completely, washed with distilled water, and lyophilized. B. A-TYPEPROTOPECTINASE
Two types of A-type PPases are known: one has polygalacturonic acid hydrolyzing activity (A, type), and the other has polygalacturonic acid transeliminase activity (A2type). 1. A,-Type PPases
a. Occurrence. Some A,-type PPases are found in the culture filtrate of yeasts and yeast-like fungus. They have been isolated as crystals form culture filtrates of Kluyveromyces fragilis IF0 0288(Sakai et al, 1984), Galactomyces reessii L. (Sakai and Yoshitake, 1984), and Trichosporon penicillatum SNO 3 (Sakai and Okushima, 1982); they are called PPase-F, -L, and -S, respectively.
b. Purification of PPases. PPases are extracellular proteins and they are purified from culture filtrates in basically the same way (Sakai,
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251
1988). The purification procedure for PPase-F is the most complicated, and will be described here. Step 1: Production of PPase-F. Kluyveromyces fragilis IF0 0288 is used for the production of PPase-F. The yeast is maintained on agar slants of a medium containing 2% glucose, 0.6% peptone, and 0.5% yeast extract, pH 5.0. For enzyme production, the yeast is aerobically cultured in a medium (40 liters) containing 3% glucose, 0.6% peptone, 0.2% yeast extract, and 0.08% Silicone KM-70 (an antifoaming agent), pH 5.0, at 30°C. Production of the enzyme begins after about 5 hr of cultivation, and reaches a maximum at 15 hr of cultivation. The culture filtrate (37 liters) is concentrated by evaporation at reduced pressure at 30°C (to 1.5 liters) and is used for enzyme purification. Step 2: CM-Sephadex C-50 Column Chromatography. The concentrated culture filtrate is dialyzed thoroughly against 20 mM AcB, pH 5.0, and then put on a CM-Sephadex C-50 column (3 x 50 cm) equilibrated with 20 mM AcB, pH 5.0. The column is washed thoroughly with 20 mM AcB, pH 5.0, and the enzyme is then eluted with 350 ml of a linear gradient of NaCl at from 0 to 400 mM in the same buffer, at the flow rate of 20 ml/hr. The fractions containing enzyme activity are pooled and concentrated to about 5 ml by evaporation at reduced pressure at 30°C. Step 3: Sephadex G-75 Column Chromatography. The concentrated enzyme solution is chromatographed on a Sephadex G-75 column (2.2 x 80 cm) equilibrated with 20 mM AcB, pH 5.0, containing 200 mM NaC1, and elution is done at the flow rate of 6.7 ml/hr. PPase activities are recovered in two peaks (Fig. 19a). Most of the activity is recovered in fraction I1 (F-11),which is concentrated to 2 ml by evaporation at reduced pressure at 30°C. The chromatography is repeated once more and the enzyme solution obtained is concentrated to 10 ml. Step 4: Crystallization. Solid ammonium sulfate is added to the enzyme solution until faint turbidity is observed. After being left for 1 week in a refrigerator, the enzyme forms needle-like crystals (Fig. 20). The crystallization is repeated two more times. From 37 liters of culture filtrate, about 50 mg of crystalline enzyme is obtained, with a recovery of about 40%. The enzyme preparation is homogeneous on the criteria of electrophoresis and sedimentation analysis. Fraction I (F-I), which is eluted at around the void volume of the Sephadex G-75 column, is chromatographed again on a Sepahdex G-100 column (2.5 x 60 cm) with 20 mM AcB pH 5.0, containing 200 mM NaCl as the solvent and the flow rate of 2.7 ml/hr. The activities are recovered in two peaks (Fig. 19b). The molecular weight of F-1-1 is high, so Sephadex G-200
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TAKUO SAKAI ET AL. FI-lb
FI-la
Fraction number
’ 0
30
50
K r - y
70
Fraction number (5rnVfraction) FIG.19. Chromatograms of protopectinases from K. fragilis IF0 0288 on Sephadex columns. (a] G-75; (b) G-100;(c ) G-200. -0-, protopectinase activity; ---0---, protein (Sakai, 1988).
is used instead of Sephadex G-100; the column (1.6 x 80 cm) is equilibrated with 20 mM AcB, pH 5.0, containing 200 mM NaCI. By eluting with the same buffer at a flow rate of 0.7 ml/hr, activity is recovered in two peaks (Fig. 19c). Thus, this strain seems to produce at least four PPases of different molecular weights.
c. Properties of the PPases. Some physical and biological properties of the three PPases (PPase-F, -L, and -S) are shown in Table VIII. These three PPases are similar in biological properties as well as in molecular weight (about 30,000), but not in specific activities. Table IX shows the amino acid composition and carbohydrate content of PPases. Amino acid compositions of these enzymes are different. The antiserum to PPase-S gives precipitation lines with PPase-L and -S, but it does not react with PPase-F (Sakai, 1988). Amino acid sequences at the N-terminal and the 27 residues long fragments in PPase-
PECTIN, PECTINASE, AND PROTOPECTINASE
Protopectinase-F
253
Protopectinase-L
Protopectinase-S FIG.20. Photomicrographs of crystals of protopectinases.
L and -S are identical (Fig. 21) (Sakai, 1988). PPase-F is not homologous to these two enzymes.
d. Catalytic Properties. The enzymes have pectin-releasing effects on protopectins from various origins. This is called PPase activity. The enzymes catalyze the hydrolysis of polygalacturonic acid; they decrease viscosity while slightly increasing the reducing value of reaction medium containing polygalacturonic acid (Sakai, 1988). Because of these findings, the enzymes are classified endo-polygalacturonases [EC 3.2.1.15; poly(l,4-a-~-galacturonide)glycanohydrolase].
254
TAKUO SAKAI ET AL. TABLE VIII PHYSICOCHEMICAL AND BIOLOGICAL PROPERTIES OF PROTOPECTINASES Properties
Molecular weight By electrophoresis By gel filtration By sedimentation S2O.W
E;& nm Isoelectric point N-terminal amino acid Optimum pH Optimum temperature ("C) Inhibitor pH stability Activity (U/mg) Protopectinase Polygalacturonase K , value (mgfml) For protopectin For polygalacturonic acida
PPase-F
PPase-L
PPase-S
40,000 33,000 32,800 2.99s 10.0 5.0
40,000 30,000 29,300 3.77s 11.9 8.4-8.5
40,000 30,000 29,300 3.66s 9.20 7.6-7.8
Aspartic acid 5.0 60
Glycine 5.0 55
Hgz+,Hg+, CaZ', Hgz+,Hg', Ag', Ba2+,Ca2+,PbZ+ Ba", Coz+
Glycine 5.0 50
Hg2+,Hg+,Ca2+, Ba2+,Coz+
2-8
3-7
3-7
556 2,053
3,945 16,219
5,770 21,107
90 6.6
50 7.7
30
9.0
a Polygalacturonic acid, having a mean polymerization degree of 130, was used for the determination of the K, value.
The hydrolysis of galacturonic acid oligomers is different for different enzymes. Figure 22 shows the mode of action in the hydrolysis of galacturonic acid oligomers and gives the K, and V,, values for the reaction. Three patterns of action toward galacturonic acid oligomers are known for endo-polygalacturonases (Demain and Phaff, 1954; Pate1 and Phaff, 1959, 1960; Mill and Tuttobello, 1961; Nasuno and Starr, 1966; Koller and Neukom, 1969; Rexova-Benkova, 1973; Kimura et al., 1973). PPase-S is novel in its action pattern toward oligogalacturonic acids. The K, and V, values change with the substrate chain length; the K, values tend to decrease and the V, values tend to increase with increasing chain length. V,,, is very different with trigalacturonic acid and tetragalacturonic acid. In contrast, the number of methoxyl groups in the substrate affects the molecular weight of the reaction products; the molecular weight of the reaction products increases as the number of methoxyl groups in the substrate galacturonic acid increases (Fig. 23).
PECTIN, PECTINASE, AND PROTOPECTINASE
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TABLE IX AMINOACIDAND SUGAR COMPOSITIONS OF PROTOPECTINASES Amino acid residuesa per molecule of protopectinase ~~~~~~
~
Amino acid
PPase-F
PPase-L
PPase-S
Lysine Histidine Argin in e Tryptophan Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half -cystine Valine Methionine Isoleucine Leucine Tyrosine Pheny lalanine Suaar
15 4 4 5 40 31 32 10 5 30 10 4 13.
16 6 4 9 35 19 29
13
1 13 11 4
7 1Ob
17
6 42 17 1 18 0 20 11 5 8 9C
5 6 5 37 30 39 20 6 34 16 6 16
1 21 11 4 10 3b
Calculations based on a molecular weight of 30,000 Determined as mannose. Determined as rhamnose.
e. Postulated Mechanism of Protopectinase Activity. On the basis of the kinetic properties of the enzyme action as a polygalacturonase, the mechanism of PPase activity seems to be as follows: the enzyme reacts with the pectin molecule in protopectin at sites with three nonmethoxylated galacturonic acid chains or more (actually, four or more
PPase-F PPase-L PPase-S PPase-SEl P Pase- SE2 PPase-SE3
D-S-G-T-L-S-G-K-T-AGG-G-L-S-N-?-A-T-V-T-V-N-N-V-?-V-P-AG-
G-G-A-?-V-F-K-D-AQ-S-A-I-AG-K-A-S-?-?-S-I-T-LB-N-F-A-V-PG-G-A-?-V-F-K-D-AQ-S-A-I-AG-K-A-S-S-S-S-I-?-LQ-N-FG-G-A-C-V-F4C-D-A-Q-S-A-IA-G-K-AG-G-A-?-V-F-R-D-A*-S -A-I- A-G-K-K-S G-?-A-?-V-Fg-D-A-K-S-A-I-A-G-K-K FIG.21. N-terminal amino acid sequences of A,-type PPases.
256
TAKUO SAKAI ET AL. Enzyme
Substrates
Reaction products
ooo -0-00 oooo/oooo
KmhM) >I02
'00 00
Protopectinase-F
Vmar(AM/unit/min)