Environmental Biodegradation Research Focus

Environmental Biodegradation Research Focus

Wang

ENVIRONMENTAL BIODEGRADATION RESEARCH FOCUS

ENVIRONMENTAL BIODEGRADATION RESEARCH FOCUS

B.Y. WANG Editor

Nova Science Publishers, Inc. New York

Copyright © 2007 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Environmental biodegradation research focus / B.Y. Wang (editor). p. cm. Includes index. ISBN-13: 978-60692-562-1 1. Biodegradation. I. Wang, B. Y. QH530.5.E58 2008 628.5--dc22 2007031240

Published by Nova Science Publishers, Inc.

New York

CONTENTS Preface Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

vii Biodegradation of Polysaccharide Sourced from Virulence Factor or Plant and Pathogenic Cell Wall Constituent and its Application in Management of Phytopathogenic Disease Xianzhen Li and Xiaoyi Chen Biodegradation or Metabolism of Bisphenol A in the Environment Jeong-Hun Kang and Yoshiki Katayama From Planting to Harvest: Environmental Dissipation of the Herbicide Molinate and Proposal of a Clean-Up Methodology Célia M. Manaia and Olga C. Nunes

1

49

77

Natural Attenuation of High Concentrations of Organic Pollutants by Biodegradation in Soils L. Reijnders

101

Microbial Polyaromatic Hydrocarbon (PAH) Biodegradation in Submerged Sediment Environments Yinjie J. Tang and James Carothers

127

Microbial Degradation of 2-Benzothialzole Derivatives: A Review A. Bunescu, P. Besse-Hoggan, M. Sancelme, A. Cincilei, G. Mailhot and A.-M. Delort

159

Biodegradable Aliphatic Polyesters Derived from 1,3-Propanediol: Current Status and Promises George Z. Papageorgiou and Dimitrios N. Bikiaris

189

Aerobic Biodegradation of Fish-Meal Wastewater from Lab Scale to Large Scale Joong Kyun Kim and Geon Lee

217

vi Chapter 9

Chapter 10

Chapter 11

Chapter 12

Index

Contents Methods in Study of Biodegradation of Water Insoluble Polymer Materials Marek Koutny and Anne-Marie Delort Biodegradable Synthetic Octacalcium Phosphate Bone Substitute Takahisa Anada, Hideki Imaizumi, Shinji Kamakura and Osamu Suzuki

239

259

Biodegradation of Phenol and Resorcinol by a Halotolerant Penicillium Ana Lúcia Leitão

273

Kinetics and Metabolic Pathway of Melatonin Biodegradation by a Bacterium Isolated from the Mangrove Sediment Xiang-Rong Xu, Hua-Bin Li, Ji-Dong Gu and Xiao-Yan Li

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303

PREFACE This new book is devoted to leading-edge research on environmental biodegradation which is the destruction of organic compounds by microorganisms. Microorganisms, particularly bacteria, are responsible for the decomposition of both natural and synthetic organic compounds in nature. Mineralization results in complete conversion of a compound to its inorganic mineral constituents (for example, carbon dioxide from carbon, sulfate or sulfide from organic sulfur, nitrate or ammonium from organic nitrogen, phosphate from organophosphates, or chloride from organochlorine). Since carbon comprises the greatest mass of organic compounds, mineralization can be considered in terms of CO2 evolution. Radioactive carbon-14 (14C) isotopes enable scientists to distinguish between mineralization arising from contaminants and soil organic matter. However, mineralization of any compound is never 100% because some of it (10–40% of the total amount degraded) is incorporated into the cell mass or products that become part of the amorphous soil organic matter, commonly referred to as humus. Thus, biodegradation comprises mineralization and conversion to innocuous products, namely biomass and humus. Primary biodegradation is more limited in scope and refers to the disappearance of the compound as a result of its biotransformation to another product.Compounds that are readily biodegradable are generally utilized as growth substrates by single microorganisms. Many of the components of petroleum products (and frequent ground-water contaminants), such as benzene, toluene, ethylbenzene, and xylene, are utilized by many genera of bacteria as sole carbon sources for growth and energy. Chapter 1 - Plant cells can initiate own defense reactions to resist plant diseases on attacked by phytopathogen, in which the infection will not proceed further if such responds occur in a timely manner. Therefore the thoughtful application of the plant defense mechanisms will help plant more effectively protect against pathogen infection. Some oligosaccharides have been demonstrated to be elicitor- or antimicrobe-active. Most of these active oligosaccharides are degraded enzymatically from polysaccharide sourced from the structural constituents of plant or fungal cell walls, as well as exopolysaccharide of virulence factor of pathogens. The elicitor and antimicrobial activity greatly depends on molecular weight or degree of polymer, charge distribution, branch form, terminal groups, etc. of oligosaccharide molecules. The well-known oligosaccharides include β-glucan oligosaccharides, chitooligosaccharides, oligogalacturonides, xyloglucan-derived oligosaccharides, oligoguluronates, xanthooligosaccharide, and alginooligosaccharide. The production of oligosaccharides can be performed by both chemical and enzymatic methods, whereas the enzymatic degradation of polysaccharides is beneficial to the preparation of the

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active oligosaccharides. The enzymes involved in the degradation of polysaccharides are dependent on the carbohydrates being depolymerised and requirements to the structural feature of end products. The enzymatic degradation of polysaccharides can be managed artificially to form specific products, but the chemical degradation cannot be controlled and the hydrolysates of polysaccharides usually are their constituent units. In this chapter the biodegradation of polysaccharides including glucan, chitin/chitosan, pectin, carrageenan, xylan, xyloglucan, xanthan and alginate has been discussed. The process for polysaccharide degradation was evaluated, such as enzymes and its sources, specificity of the enzymes, enzymatic route for degradation, depolymerization and its effect on the oligosaccharide nature, preparation of bioactive oligosaccharide. The roles of oligosaccharides in plant disease resistance were also discussed generally in this chapter. Chapter 2 - Recently, there has been increasing interest in the effects of endocrine disruptors on organisms. Bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane; CAS Registry No. 80-05-7) is an endocrine disruptor with estrogenic activity and acute toxicity to aquatic organisms. BPA is made by combining acetone and phenol and is used mainly as a material for the production of epoxy resins and polycarbonate plastics. Due to intensified usage of these products, exposure of organisms to BPA via several routes, such as the environment and the food chain, has increased. BPA contamination in the environment occurs through several routes, such as migration from human wastes and effluent from wastewater treatment plants. BPA exposed to the environment can be biodegraded or metabolized by microorganisms (bacteria, fungi and plankton), plants, invertebrates and vertebrates (fish, amphibians and mammals). Biodegradation or metabolism is a very important step for removing or detoxifying BPA in the environment or organisms. Although some metabolites of BPA may exhibit enhanced estrogenicity or toxicity, in general, BPA biodegradation or metabolism by organisms leads to detoxication of BPA. However, excessive BPA doses cause bioaccumulation if detoxification pathways are saturated. In this chapter the authors describe 1) contamination routes of BPA, 2) biodegradation or metabolism of BPA by organisms, and 3) bioaccumulation of BPA in organisms, with the main subject of this chapter being the biodegradation or metabolism of BPA by organisms. Chapter 3 - The deliberated application and intensive use of pesticides in the environment has been leading to the contamination of air, soils, surface and ground water, and living organisms. The environmental contamination of the trophic chain with pesticides has serious negative impacts on the biological diversity and possible implications on the public health. The monitoring of environmental contamination with pesticides and the implementation of decontamination processes may contribute to minimize the impact of intensive agricultural practices. This study was conducted in a rice field situated in central Portugal, where molinate is supposed to contaminate surface and underground waters. Molinate content was monitored in water samples collected before, during and after molinate application and the results showed that molinate was dissipated in the environment, reaching concentrations of 3.9 µg l-1 in underground water and 15.8 µg l-1 in the river receiving tail waters. The feasibility of clean-up methodologies based on adsorption and/or biodegradation processes to remove molinate from these waters was assessed. At a laboratory scale, these clean-up processes led to reductions of the molinate concentration to values close to the legally recommended limits (< 2 µg l-1). Given the inability of the autochthonous microbiota to degrade molinate contaminating the agriculture effluents, the implementation of a biodegradation process requires the use of

Preface

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an exogenous molinate degrading culture. The aerobic biodegradation of molinate in the rice field waters was assayed using a defined mixed bacterial culture, previously isolated from an industrial effluent. This mixed culture is able to mineralize molinate under a wide range of operating conditions removing between 55 % (1 mM molinate at 15 °C) and 80 % (1 mM molinate and complex nutrients at 30 °C) of the initial total dissolved organic carbon. Given the low concentrations, and hence the low bioavailability, of molinate in agriculture effluents, the use of an adsorption step was considered a valuable auxiliary tool to improve the clean-up of contaminated waters. Resin Amberlite XAD-4 and activated carbon showed efficient molinate removal. The bio-regeneration of these materials, using the above mentioned mixed culture, permits the decontamination of the adsorbents, both for future reuse or for final disposal. Chapter 4 - In view of its relatively low cost, monitored natural attenuation by biodegradation is increasingly relied upon to clean up pollution of soils caused by landfills, industrial activities and major transport and storage related spills. Current policy tends to aim at reducing soil pollution to levels reflecting tolerable risk for specific recipients within a reasonable time frame. Natural attenuation by biodegradation of high concentrations of major pollutants that preferentially partition to the particle fraction of soils tends to be poor. These pollutants include hazardous hydrocarbons, highly halogenated hydrocarbons and nitroorganics. Relying on natural attenuation of these compounds, when feasible at all, leads to exceeding tolerable risk levels for a long time. Better perspectives exist for natural attenuation by biodegradation of pollutants that partition to a significant extent to the aqueous phase, especially for low molecular weight organic aromatics and chlorinated solvents. Dependent on conditions in the aquifer and the presence of suitable micro-organisms, there can be substantial biodegradation of a variety of hydrocarbons, organochlorines and oxygenates. In some cases natural attenuation has been found to bring down high levels of pollution to levels meeting current standards of tolerable risk. Predictions whether in the future levels reflecting tolerable risk can be attained, are uncertain. Uncertainty is especially large in case of expanding plumes. There are uncertainties that beset current modelling to predict future concentrations and there is uncertainty about what in the future will be considered tolerable risk. Even when it is supposed that current standards will be applied indefinitely, uncertainties related to present modelling and sampling often prevent certainty that these standards will be met in the future. In practice, natural attenuation is often falling short of attaining promised outcomes. This means that often interventions aimed at enhanced remediation will be necessary to achieve tolerable risk within a reasonable time frame. These may include enhanced biodegradation. Chapter 5 - Polycyclic aromatic hydrocarbons (PAHs) in submerged sediments can have potentially carcinogenic effects on human health through the food chain. PAH compounds persist in submerged sediment because of their very low aqueous solubilities, tendencies to adhere to sediment particles and recalcitrance to biodegradation. This chapter covers research topics important for understanding PAH bioremediation in submerged sediments: 1. microbial processes under anaerobic or aerobic sediment conditions; 2. the effects of adding inexpensive environmentally benign substances to stimulate biodegradation or improve PAH bioavailability; 3. methods to characterize microbial, chemical and physical properties in sediment sites; 4. A mathematical model linking the understanding of chemical, physical, and biological activities occurring in the sediment field. Physical capping is frequently used to treat PAH contaminated submerged sediment sites when the site is large volume and has a

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low contaminant concentration. Recent research suggests that including a physical cap with in situ bioremediation can reduce the volume of cap required to secure a site and provides a potentially economical way to rapidly remediate contaminated sediment sites. Chapter 6 - This review is focused on one particular family of pollutants, 2-benzothiazole derivatives. This group of xenobiotics containing a benzene ring fused with a thiazole ring is manufactured worldwide. After a short presentation of benzothiazole structures and their industrial applications, the fate of benzothiazoles in the environment is described both in natural waters and in wastewater treatment plants. Then data available on the toxicity of benzothiazoles are reported. The main part of this review is devoted to the microbial degradation of these compounds: i) using activated sludge and mixed cultures, ii) in soils, iii) using pure cultures. In that later case, detailed pathways of biodegradation are described for benzothiazole, 2-hydroxybenzothiazole, 2-mercaptobenzothiazole, 2-aminobenzothiazole and methabenzthiazuron. Special attention is made on methodology used to establish these pathways, namely Nuclear Magnetic Resonance (NMR). Finally photodegradation processes are described because molecular mechanisms are often closely related to those of biodegradation processes and lead to common products in the environment. To conclude, the possibility of combining these two approaches is discussed. Chapter 7 - Among biodegradable polymers, polyesters derived from aliphatic dicarboxylic acids and diols are of special importance. Polyesters of 1,3-propanediol were overlooked till recently, since the specific monomer was not available in quantities and price that might enable production of polymers. However, in recent years more attractive processes have been developed for the production of 1,3-propanediol from renewable resources. Nowadays, research on biodegradable poly(1,3-propylene alkanedioate)s, such as poly(propylene succinate) (PPSu), poly(propylene adipate) (PPAd) and poly(propylene sebacate) (PPSe), has gained an increasing interest, due to their fast biodegradation rates and their potential uses in biomedical or pharmaceutical applications, such as drug delivery systems. The odd number of methylene units in the diol segment is responsible for the lower melting points, lower degree of crystallinity and higher biodegradation rates of the specific polymers compared with their homologues based on ethylene-glycol or 1,4-butanediol. In this chapter synthesis and properties of the 1,3-propanediol based aliphatic polyesters and especially their biodegradation characteristics are reviewed. Specific attention has been paid to preparation of related copolymers and blends with other important polymers, since these techniques may offer routes for optimizing properties and produce tailor-made materials. Copolymerization of 1,3-propanediol with mixtures of aliphatic or even aromatic acids, leads to linear polyesters with improved or balanced biodegradation and mechanical properties. Blends with other biodegradable polymers have been studied recently. Finally, potential pharmaceutical applications of poly(1,3-propylene alkanedioate)s as solubilizing and stabilizing carriers for drugs are exemplified. Chapter 9 - Increasing waste disposal problems from polymer wrapping materials have resulted in constant endeavors to replace recalcitrant materials with biodegradable alternatives. The biodegradability of these materials can often simply be limited, or the processes involved are relatively slow, causing complications when applying standard methodologies and thus promoting the development of customized testing protocols. Moreover, some properties of these materials, especially their water insolubility, require further adaptations to conventional methods. This chapter brings together data found in literature, along with the personal findings and experiences of the authors. A broad variety of

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experimental methods has been used and described in literature, which are dependent on authors’ expertise or the availability of particular techniques in their laboratory. Here, a comprehensive overview of current and the most prominent techniques is provided. These include spectroscopic techniques to examine changes in the material; NMR, MS and separation methods for investigating compounds released from the material; electron microscopy methods; optical fluorescence microscopy that enables surfaces of the material and eventual microbial colonization to be visualized; different methods for biomass quantification and indicators of metabolic activity; and various ways of monitoring carbon dioxide production. The relevance of the methods for studying biodegradable synthetic polymer materials was analyzed, compared and then critically evaluated. Original microphotography and original data have been introduced to illustrate the text. Chapter 10 - Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. In fact, several studies using physical techniques demonstrated that OCP is involved as a transitory intermediate phase to biological apatite crystals in enamel, dentine and bone. The authors previous studies demonstrated that synthetic OCP facilitates bone regeneration, compared to synthetic hydroxyapatite (HA), including non-sintered stoichiometric or non-stoichiometric HA, and sintered HA ceramic, when implanted in murine and rabbit bone defects. Synthetic OCP can be replaced with newly formed bone in conjunction with its simultaneous biodegradation. Crystallographic OCP-apatite conversion of the implanted OCP advances gradually during the bone regeneration. OCP is known to be a thermodynamically less stable salt under physiological condition than HA and β-tricalcium phosphate (β-TCP); the latter is a well known biodegradable bone substitute ceramic. The biodegradability predicted from the solubility isotherm is actually reproduced in the implantation of these synthetic calcium phosphate compounds into bone defects. OCP-apatite conversion induces various physicochemical reactions on the crystal surfaces, including bio-molecule adsorption, and may be involved in the stimulatory effect to enhance osteoblastic cell differentiation in vitro and bone formation in vivo. Chapter 11 - Many industries are known to generate wastewater enriched in phenolic compounds. These include petrochemicals, basic organic chemical manufacture, coal refining, pharmaceutical and tanning. Consequently, these compounds are commonly encountered in industrial effluents and surface water. Due to its high toxicity as shown by ecotoxicological studies, several methods have been reported for the removal of these pollutants from wastewater. Additionally to this toxicity problem some of these industrial effluents are likely to generate highly saline wastewaters. The discharge of such wastewaters containing at the same time phenol and phenolic compounds and high salinity without prior treatment is known to negatively affect the aquatic life, agriculture and potable water. Biological treatment with halotolerant/halophilic microorganisms is considered advantageous over the other physical and chemical methods as it leads to complete mineralization of phenolic compounds, is one of the safest, least costly and most socially acceptable. Halotolerant microorganisms are well known for their great versatility to remove pollutants, under saline and non saline conditions. Penicillium chrysogenum is an economically important ascomycete used as producer of penicillin. However, little attention has been paid to the ability of this microorganism to transform or metabolize compounds that are pollutants. This article presents a different approach to a classic problem. It employs an individual test on marine organism of trophic level 2 to validate bioremediation process by halotolerant fungus.

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A biodegradation process is an effective method of remediation if the toxicity of the system decreases. The purpose of this study was to compare cell growth and biodegradation of single phenol and resorcinol at high initial substrate levels by halotolerant strain, P. chrysogenum CLONA2, under saline and non saline conditions, adapted with either phenol or resorcinol, in a batch system, and to study the inhibitory and enhanced effect during the biodegradation of phenol and resorcinol. Single and binary substrate experiments were performed. HPLC analysis shows that halotolerant strain, Penicillium chrysogenum CLONA2, degraded up to 300 mg/l of both xenobiotics compounds in mineral salts medium with 58.5 g/l of sodium chloride. When phenol and resorcinol were together in low concentrations (< 15 mg/l and < 30 mg/l, respectively), phenol enhanced resorcinol degradation. P. chrysogenum CLONA2 metabolized phenol faster than resorcinol when present as the sole carbon source. The acute toxicity of phenol and resorcinol, individually and in combination, to larvae of the Artemia franciscana has been verified after and before bioremediation process with P. chrysogenum CLONA2. Resorcinol was more toxic than phenol. The authors findings indicate that mixtures of resorcinol and phenol had an effect more toxic in A. franciscana than individual phenolic compounds. Chapter 12 - Melatonin (MLT) is a hormone produced primarily by the pineal gland. It can be found in animals and humans as well as a number of bacteria, fungi and plants. Since it has been widely used as the healthcare product in the world market, MLT entered the various environmental compartments by different ways. Recent research found that MLT may suppress the production of testosterone, decrease semen quality, and affect sexual activity and reproduction of animal and human. The fate of melatonin in environment has attracted increasing public and scientific concerns in recent years. In this chapter, biodegradability of MLT was studied for the first time. A strain that can efficiently degraded melatonin was isolated from the mangrove sediment. This strain, a gram-negative bacterium identified as Shewanella putrefaciens, can grow on melatonin as sole sources of carbon and energy under aerobic conditions. The growth was greatly enhanced by the addition of a small amount of yeast extract. Effects of melatonin concentration, pH, temperature and salinity on MLT biodegradation were studied, respectively. The experimental results showed that 50 mg l-1 melatonin could be degraded within 2 d under the optimal condition (pH 7.0, salinity 15‰ and temperature at 37 °C). The process of MLT biodegradation was monitored by highperformance liquid chromatography with ultra-violet detection. The biodegradation of melatonin could be fitted to a first-order kinetic model. The major metabolites of melatonin biodegradation were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry, and a preliminary metabolic pathway of melatonin was proposed. The results obtained are helpful to understand environmental behavior of MLT, and also could be used for the bioremediation of MLT-contaminated site, such as the wetland of the Mai Po Natural Reserve in Hong Kong.

In: Environmental Biodegradation Research Focus Editor: B. Y. Wang, pp. 1-47

ISBN: 978-1-60021-904-7 © 2007 Nova Science Publishers, Inc.

Chapter 1

BIODEGRADATION OF POLYSACCHARIDE SOURCED FROM VIRULENCE FACTOR OR PLANT AND PATHOGENIC CELL WALL CONSTITUENT AND ITS APPLICATION IN MANAGEMENT OF PHYTOPATHOGENIC DISEASE Xianzhen Li∗ and Xiaoyi Chen Department of Bio & Food Engineering, Dalian College of Light Industry

1. ABSTRACT Plant cells can initiate own defense reactions to resist plant diseases on attacked by phytopathogen, in which the infection will not proceed further if such responds occur in a timely manner. Therefore the thoughtful application of the plant defense mechanisms will help plant more effectively protect against pathogen infection. Some oligosaccharides have been demonstrated to be elicitor- or antimicrobe-active. Most of these active oligosaccharides are degraded enzymatically from polysaccharide sourced from the structural constituents of plant or fungal cell walls, as well as exopolysaccharide of virulence factor of pathogens. The elicitor and antimicrobial activity greatly depends on molecular weight or degree of polymer, charge distribution, branch form, terminal groups, etc. of oligosaccharide molecules. The well-known oligosaccharides include βglucan oligosaccharides, chitooligosaccharides, oligogalacturonides, xyloglucan-derived oligosaccharides, oligoguluronates, xanthooligosaccharide, and alginooligosaccharide. The production of oligosaccharides can be performed by both chemical and enzymatic methods, whereas the enzymatic degradation of polysaccharides is beneficial to the preparation of the active oligosaccharides. The enzymes involved in the degradation of polysaccharides are dependent on the carbohydrates being depolymerised and

2

Xianzhen Li and Xiaoyi Chen requirements to the structural feature of end products. The enzymatic degradation of polysaccharides can be managed artificially to form specific products, but the chemical degradation cannot be controlled and the hydrolysates of polysaccharides usually are their constituent units. In this chapter the biodegradation of polysaccharides including glucan, chitin/chitosan, pectin, carrageenan, xylan, xyloglucan, xanthan and alginate has been discussed. The process for polysaccharide degradation was evaluated, such as enzymes and its sources, specificity of the enzymes, enzymatic route for degradation, depolymerization and its effect on the oligosaccharide nature, preparation of bioactive oligosaccharide. The roles of oligosaccharides in plant disease resistance were also discussed generally in this chapter.

2. INTRODUCTION The chemical pesticides have successfully been used to control plant diseases due to their quick and effective management. However their incessant and indiscriminate use will have harmful effects on human health and environment. In recent years a large number of synthetic pesticides have been banned because of their undesirable attributes such as high and acute toxicity, long degradation periods, accumulation in food chain and an extension of their power to destroy both useful and harmful pests [Annon, 1996; Barnard et al., 1997]. Besides, many pathogenic microorganisms and insect pests have developed resistance against chemical pesticides, which seriously hinders the management of diseases of plant [May, 1985; Williams and Heymann, 1998]. Therefore, an urgent requirement for searching biological control agents to manage phytopathogen has been intensified in recent years. Plants are capable of initiate various defense reactions, such as the production of phytoalexins, the expression of antimicrobial proteins, the generation of the reactive oxygen species, and the reinforcement of cell walls, when they are attacked by pathogens. Therefore it is believed that the thoughtful application of the plant own defense mechanisms can lead to more effective protection against phytopathogens [Somssich and Hahlbrock, 1998]. Carbohydrates are the most abundant materials among the polymers in nature, which have been thought only to play roles as energy storage molecules or structural elements in cell walls for a long time. However, they have recently been actively studied as important biological macromolecules due to its great structural diversity for encoding biological information. Oligosaccharide signals have been shown to regulate the defensive or symbiotic processes in plants [Coté and Hahn, 1994; Darvill et al., 1992], and possess versatile functional properties such as antitumor activity, immune-enhancing effects, antimicrobial activity, and growth stimulation on probiotics [Hirano and Nagao, 1989; Lee et al., 2002; Suzuki, 1996; Suzuki et al., 1986]. An oligosaccharide is any short chain of sugar residues interconnected by glycosidic linkages. Oligosaccharides can be produced artificially by the enzymatic fragmentation of polysaccharides. Some oligosaccharides have been demonstrated to be elicitor- or antimicrobe-active, which can stimulate plant defense responses and may help plants resist infective disease [Shibuya and Minami, 2001]. ∗

Correspondence should be sent to Dr. Xianzhen Li, Department of Bio & Food Engineering, Dalian College of Light Industry, Ganjing Qu, Dalian 116034, PR CHINA; Tel: (86) 411 86314195; Fax: (86) 411-86323646; Email: [email protected]

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3

The first bioactive oligosaccharide was degraded from the fungal cell walls of phytopathogen Phytophthora sojae, which was able to induce the synthesis of phytoalexins in plant cells to prevent fungal infection [Sharp et al., 1984a]. Other active oligosaccharides derived from the cell walls of plant and pathogen were subsequently produced and isolated, which have shown important roles in plant protection. Most of the known oligosaccharides identified as having elicitor or antimicrobial activity are from the structural components of fungal or plant cell walls, as well as the virulence factor of exopolysaccharides by partial degradation. The well-known active oligosaccharides include β-glucan oligosaccharides, chitin/chitosan oligosaccharides, pectic oligosaccharides, xyloglucan-derived oligosaccharides, oligoguluronates, xanthooligosaccharide, alginooligosaccharide, etc. [Fry et al., 1993b; Liu et al., 2005]. Their bioactivities are greatly dependent on the degradation degree, the charge number and distribution, and the branch form and end groups, in which only a few oligosaccharides exhibit elicitor or antimicrobial activity. In this chapter we will review the biodegradation of polysaccharides for preparation of bioactive oligosaccharides by different microorganisms and their enzymes, including enzymes and their sources for biodegradation, process for enzymatic degradation, production of bioactive oligosaccharides, oligosaccharide nature and its effect on bioactivity, function involved in plant defensive responses, roles in plant disease resistance.

3. POLYSACCHARIDE FRAGMENTS FROM FUNGAL AND PLANT CELL WALLS OR VIRULENCE FACTORS ACTED AS ELICITORS OR ANTIMICROBES Oligosaccharides released from fungal and plant cell walls or some virulence factors of phytopathogen are powerful signaling elicitors, capable of acting at very low concentrations to convey information to the plant under attack. In response to this information, plant defense response was activated, leading to the induction of genes that encode enzymes responsible for the synthesis of phytoalexins and other defensive compound. Some of the oligosaccharides with bioactivity were illustrated in Figure 1. The first oligosaccharide identified as having elicitor activity was isolated from a fungal pathogen of soybean, Phytophthora sojae [Sharp et al., 1984a]. This branched glucan heptasaccharide, composed of β-1,3-and β-1,6-linked glucose residues, is the smallest possible unit from the fungal cell wall capable of inducing the synthesis of phytoalexins by plant cells, an antimicrobial molecules for resistance on microbial infection. Subsequently, the other structural components of the cell walls of pathogenic fungi have also been shown to elicit defense responses in plants such as chitin/chitosan oligosaccharides [Coté and Hahn, 1994]. In addition, polysaccharide fragments released from plant cell walls play an important role in signaling a plant defensive response. Oligogalacturonides were released from the pectic component of plant cell walls via the enzymatic degradation with polygalacturonase and pectate lyase, which can elicit a broad spectrum of plant defense responding reactions, including the increased synthesis of phenylalanine ammonia-lyase (PAL), chalcone synthase, chitinases, β-glucanase, and protease inhibitors [Bishop et al., 1984; Coté and Hahn, 1994;

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Glc

Glc

α -1,3

β -1,6

Glc

GlcN

β -1,6 β -1,6 β -1,6 Glc Glc Glc Glc β -1,3 β -1,3 Glc Glc Glucan heptasaccharide

α -1,3

β -1,4

α -1,3 α -1,3 α -1,3 Glc Glc Glc Glc α -1,3 Glc Hexosaccharide

GlcN

β -1,4

GlcN

β -1,4

GlcN

Chitosan oligosaccharides GlcNAc

β -1,4

GlcNAc

β -1,4

GlcNAc

β -1,4

GlcNAc

Chitin oligosaccharides GlcA

α -1,4

GlcA

α -1,4

GlcA

α -1,4

GlcA

α -1,4

GlcA

α -1,4

GlcA

Pectin oligosaccharides (oligogalacturonides) β -1,4 β -1,4 β -1,4 GlcN GlcN GlcN GlcN α -1,6 α -1,6 α -1,6 Xyl Xyl Xyl α -1,2 Gal α -1,2 Fuc Xyloglucan nonasaccharide Figure 1. Structure of some bioactive oligosaccharides derived from fungal and plant cell walls. Abbreviations: Glc, glucose; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; GalA, galacturonic acid; Xyl, Xylose; Gal, galactose; Fuc, fucose.

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5

Ryan and Farmer 1991]. Another oligosaccharide signal derived from plant cell walls is a xyloglucan oligosaccharide. This bioactive oligosaccharide was released from plant cell walls by cleavage of xyloglucan catalyzed by endo-β-1,4-glucanase [Fry et al., 1993a]. Xyloglucan-derived oligosaccharides were also reported to influence on defense reactions and elicit the release of the related rotting enzymes that can hydrolyze the structural constituents of the host plant further [Slováková et al., 1994]. Xanthooligosaccharide, produced by the degradation of xanthan via a series of xanthan-degrading enzymes, is another interesting oligosaccharide, which has been demonstrated to have elicitor and antibacterial activity [Liu et al., 2005; Qian et al., 2006]. Xanthan is an extracellular heteropolysaccharide produced by a phytopathogenic bacterium Xanthomonas campestris pv. campestris [Rogovin et al., 1961], which was involved in the pathogenicity caused black rot lesions to form on cruciferous plants [Daniels et al. 1987]. Similar oligosaccharide also includes alginooligosaccharide derived from alginate, a virulence factor of phytopathogen Pseudomonas syringae [PeñalozaVázquez et al., 2004].

1) Mechanism of plant defense response When plants are attacked by pathogenic microorganisms, they have the ability to initiate various defense reactions themselves against pathogenic organisms infection, such as the induction of secondary metabolic enzymes like PAL for phytoalexin formation, the reinforcement of cell walls by deposition of callose, the synthesis of hydrolytic enzymes like chitinase and β-1,3-glucanase for broken cell walls, a rapid formation of the plant hormone ethylene, and the release of reactive oxygen species [Alvarez et al., 1998; Bell, 1981;Boller, 1995; Hahlbrock and Scheel, 1989;Mehdy, 1994] (Figure 2). If these reactions occur in a timely manner, the infection will not proceed further. However, if these defense reactions occur too late or are suppressed, the infection process will proceed successfully, leading to various plant diseases happen [Somssich and Hahlbrock, 1998]. The recognition of phytopathogens by plant cells depends on the perception of elicitors generated by the pathogen. Fungal or plant cell wall fragments and molecules secreted by the pathogen can induce signaling cascades that activate a cellular response to minimize injury to host plant [Blumwald et al., 1998; Dixon et al., 1994]. It is well known that the presence of oligosaccharides on the cell membrane of pathogen can act as elicitors. And the receptors for oligosaccharide elicitors were also demonstrated to occur on plant cell membranes [Cheong and Hahn 1991; Yoshikawa et al. 1983]. Upon infection by a phytopathogen, plants can percept its invasion and commonly activate a variety of defense mechanisms. For example, the challenged plants will synthesize β-glucanase and chitinases instantly, catalyzing the partial degradation of the cell wall of plant or pathogen to form β-glucan, chitosan/chitin oligosaccharides [Flach et al., 1992]. Such oligomers released by degradation of fungal and plant cell walls acted as elicitors in turn can facilitate additional plant defense mechanisms [Frindlender et al., 1993], such as cell wall fortification [Bradley et al., 1992], defense-related gene expression [Jabs et al., 1997; Levine et al., 1994], and phytoalexins synthesis [Bradley et al., 1992; Jabs et al., 1997]. Maurhofer et al. [1994] reported that the induction of systemic resistance by Pseudomonas fluorescens was correlated

6

Xianzhen Li and Xiaoyi Chen

pathogen

pathogen

pathogen

nuclear

pathogen

pathogen

pathogen

pathogen

Plant nuclear

mRNA

1

2

pathogen

4

3

Plant mRNA

5

More defense response Pathogen Oligo-glucan Cell death

Pa tho

ge n

Chitooligosaccharide

Plant

Oligogalacturonide 4 Chitinase

1 Phytoalexin

2 Active oxygen species

3

Glucanase

5

Pecticn lyase

Figure 2. Plant defense response on infected by phytopathogen or elicitor treatment.

with the accumulation of β -1,3-glucanase and chitinase at the site of penetration of fungal hyphae. These enzymes acted on the fungal cell walls resulting in the degradation and the loss of inner contents of cells, and leading to the destabilization of the organism [Benhamou et al., 1996; Ji and Kuc, 1996]. The fungal cell wall elicitors have been reported to elicit various defense reactions in greengram [Ramanathan et al., 2000]. So the pathogen invasion can be minimized by produced elicitors in the infection process. It has been proved that there are two types of defense mechanism occurred in plant cells. One is endogenous defense mechanism as described above, which can be induced in response to the attack of pathogens. Another is exogenous defense response, which is initiated by the foreign elicitor treatment. So expression of β-glucanases and chitinases can be activated not only upon the challenge of plant tissues by microbes, but also upon exposure to certain elicitors [Kirsch et al., 1993; Kombrink and Hahlbrock, 1986; Mauch et al., 1984]. Structural polysaccharide fragments from plant or pathogen cell walls can serve as elicitors in many plant species [Lee et al., 1999]. Induction of plant defense genes by prior application of biological inducers is called induced resistance, which can prevent plants from the infective disease of pathogen or virus. It is well known that the defense genes are inducible genes and appropriate stimuli or signals are needed to activate them. In many plant species, resistance may be induced against pathogens by means of pretreatment with some of oligosaccharides. Once resistance was induced, the plant expressed a number of inducible defense responses or mechanisms that usually coincides with the accumulation of pathogenesis-related proteins [Gatz, 1997; Greenberg, 1997; Hunt and Ryals, 1996; Keen, 2000; Lawton and Lamb, 1987]. PAL is a key enzyme in the production of phenolics and phytoalexins [Daayf et al., 1997]. PAL activity could be induced in plant-pathogen interactions and exogenous elicitor treatment [Ramanathan et al., 2000]. Other defense enzymes include pathogenesis-related

Biodegradation of Polysaccharide Sourced from Virulence Factor…

7

proteins such as peroxidase, which is a key enzyme in the biosynthesis of lignin [Bruce and West, 1989]. Increased activity of cell wall bound peroxidases has been elicited in different plants such as cucumber, rice, and tomato due to pathogen infection [Chen et al., 2000; Mohan et al., 1993; Reimers et al., 1992]. Thaumatin-like proteins belong to PR-5 family, showing antifungal activity and enhancing resistance to pathogen infection [Datta et al., 1999]. Induction of defense proteins makes plant resistant to pathogen invasion [Van Loon, 1997]. Therefore the induction of plant resistance mechanisms by application of elicitors has been suggested as an alternative approach for crop disease control [Benhamou, 1996; Cartwright et al., 1977; Gatz, 1997; Hunt and Ryals, 1996].

2) Mechanism of antimicrobial activity Some oligosaccharides can bind to the microbe’s carbohydrate-binding proteins to prevent bacterial attachment on host cells or clear bacteria already attached [Zopf and Roth, 1996]. The mostly accepted mechanism to explain the possible antibacterial actions of chitooligosaccharides has been proposed by EI-Ghaouth et al. [1992]. It was asserted that chitooligosaccharide reacted with the pathogen cell surface to alter the permeability characteristics of microbial cell membrane, and further prevented the entry of materials or caused the leakage of materials, and finally led to the death of bacteria. More adsorbed chitooligosaccharide will result in greater changes in the structure of the cell wall and in the permeability of the cell membrane. Sudharshan et al. [1992] also demonstrated that the chitooligosaccharide could alter permeability and further prevent the entry of materials or cause leakage of cell constituents, leading to the death of bacteria. Another suggested mechanism for antibacterial activity of chitooligosaccharide was the blockade of RNA transcription by adsorption of penetrated chitosan to bacterial DNA [Kim et al., 2003]. The antibacterial activity of oligosaccharides is related to the hydrophilicity of cell wall, whereas the inhibition capacity does not fit well with the hydrophilicity of cell wall for Gramnegative bacteria [Chung et al., 2004]. So the hydrophilicity of the cell wall could not fully explain the difference in the antibacterial activity for Gram-negative bacteria. It was found that the distribution of negative charges on their cell surfaces was quite different although cell wall hydrophilicity was similar among Gram-negative bacteria. Hence, positively charged chitooligosaccharide had higher antibacterial activity or inhibition activity in Staphylococcus aureus than Streptococcus faecalis [Chen et al., 2002]. This clearly explained why most Gram-negative bacteria were sensitive to chitooligosaccharide, and negative charge density on the cell surface apparently determined whether the bacteria were easily inhibited by chitooligosaccharide or not. Because the amount of adsorbed chitooligosaccharide to the different bacterial cells is exactly the same order determined for the antibacterial activity of chitooligosaccharide on these bacteria, the antibacterial activity of chitooligosaccharide and the surface characteristics of the cell wall are closely related [Wang, 1992]. More negatively charged cell surfaces had a greater interaction with oligosaccharides. The number of amino groups in chitooligosaccharides has been proved to play a major role in antibacterial activity, and several mechanisms have been proposed to describe this activity [Chen et al., 2002]. In general, when the positively charged group of chitooligomers interacts with the negatively charged carboxylic acid group of macromolecules on bacterial cell surface, the polyelectrolyte complexes will be formed on the cell surface of pathogen

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Xianzhen Li and Xiaoyi Chen

[Choi et al., 2001; Kim et al., 2003]. This complex acts as an impermeable layer around the cells and suppresses the metabolic activity of the bacteria by blocking of nutrient permeation through the cell wall, and lastly results in the cell death. With respect to the formation of polyelectrolyte complexes, higher number of primary amino groups presented in chitooligosaccharide can make stronger interactions with bacterial cells [Tsai et al., 2002]. Many pathogens use carbohydrate-binding proteins to attach to cells and initiate plant disease. The first line of defense against these infectious diseases consists of decoy oligosaccharides in the mucous layer. When decoy oligosaccharides bind to the microbe’s carbohydrate-binding proteins competitively, the attached pathogens are released from the host cells and cleared by the physiological mechanism (Figure 3). So the soluble oligosaccharides can both prevent bacterial attachment and separate bacteria already attached [Zopf and Roth, 1996]. The decoy is a homologue, regardless of size, provided it is a numerically and isomerically identical to the corresponding fragment of the native carbohydrate. The pathogen proteins (adhesins, lectins) have strict requirements for their oligosaccharide ligands. Usually the specific sugar sequence required by an adhesin is at the terminal, non-reducing end of the oligosaccharide chain, although many adhesins also recognize internal sugars [Zopf and Roth, 1996].

a

Pathogen

Plant

b

Plant

Pathogen

Plant cell Figure 3. Binding of cell surface carbohydrate on microbial adhesion (a) and carbohydrate-binding proteins on bacterial cell surface occupied by oligosaccharides preventing their interaction with plant cell surface (b). Adapted and modified from Zopf and Roth [1996].

A lock-and-key hypothesis was proposed for explaining the interrelation between the plant and pathogen [Zopf and Roth 1996]. The cell surface carbohydrates are named as "keys", and carbohydrate binding proteins on cell membrane of pathogen are asserted as the

Biodegradation of Polysaccharide Sourced from Virulence Factor…

9

"locks" in such lock-and-key interactions. The role of oligosaccharides is to make themselves to excellently competitive inhibit the target interaction. The optimum size of the competing oligosaccharide depends on the number of monosaccharide subunits recognized by the complementary protein. If the carbohydrate-binding domain of a protein has a cleft fitting four terminal residues of a specific octasaccharide, for example, the terminal tetrasaccharide alone will bind to the protein with an avidity being the same as that of the native octasaccharide. Whereas the terminal trisaccharide will bind less well than the native compound, and the terminal disaccharide worse still. A non-carbohydrate analogue may bind efficiently to a microbial adhesin [Ofek et al., 1990], but it is more likely to be toxic and immunogenic than that of the carbohydrate homologue. When cell surface carbohydrates are linked to a flexible polymer backbone to create a macromolecule, they can simultaneously engage many adhesin molecules at a bacterial cell surface, forming a stable complex (Figure 3a). Whereas the oligosaccharides may effectively competitive inhibit the combination between plant and pathogen. The pathogen attached on plant cells can be removed on binding of oligosaccharide to a few sites of microbial adhesin (figure 3b) [Spaltenstein and Whitesides, 1991].

4. BIODEGRADATION OF POLYSACCHARIDES 1) Biodegradation of glucan Certain plant defense reactions are elicited by compounds referred to as elicitors, such as oligosaccharides released from fungal and plant cell walls [Yoshikawa et al., 1993]. Oligo-βglucan elicitor, released from the cell wall of the phytopathogenic fungus Phytophthora megasperma by soybean glucanases, caused defense reactions in soybean cells [Umemoto et al., 1997]. The first event between the soybean and fungus is an attack of the fungal cell wall by soybean β-1,3-glucanase, resulting in the release of active oligo-β-glucan elicitors. The released oligo-β-glucan then initiated phytoalexin accumulation in plants [Yoshikawa et al., 1981]. β-Glucans were recognized to be actively involved in plant-pathogen interactions in the mid-1970s, the ability of which to induce phytoalexin accumulation in soybean cells was first detected in the culture filtrates of Phytophthora sojae [Ayers et al., 1976a]. These elicitors are composed of 3-, 6-, and 3,6-linked β-glucosyl residues, a composition very similar to glucans that are major constituents of various mycelial walls [Ayers et al., 1976b; Bartnicki-Garcia, 1968]. The elicitor-active glucans can be released from the mycelial walls of Phytophthora spp. by either partial chemical degradation or enzymatic hydrolysis [Ayers et al., 1976b; Keen and Yoshikawa, 1983; Sharp et al., 1984a; Yoshikawa et al., 1981]. Lichenase cleaved the β-(1,4)-linkages adjacent to α-(1,3)-linkage. The oligosaccharides thus obtained were the (1,4)-linked building blocks of β-glucan with (1,3)-linked end group [Johansson et al., 2000; Wood et al., 1994]. Other enzymes used for depolymerization of βglucan samples were cellulase, which cleaved only β-(1,4)-glycosidic linkages [Roubroeks et al., 2001], and β-glucosidase together with lichenase were used for quantitative measurement of β-glucan in cereal products [Boyac et al., 2002; McCleary and Codd, 1991].

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Xianzhen Li and Xiaoyi Chen

Eight hexa (β-D-glucopyranosyl)-D-glucitols, two of which were not separated from each other, were purified to homogeneity from the mixture of oligoglucosides generated by partial hydrolyzed mycelial walls of Phytophthora megaaperma f. sp. glyeinea [Sharp et al., 1984b]. Only one of these hexa (β-D-glucosyl)-D-glucitols was shown to have elicitor activity. The smallest elicitor-active oligoglucoside is a branched hepta-β-glucoside with a backbone of five (1,6)-linked β-glucosyl residues and have two terminal β-glucosyl residues attached at C3 of the second and fourth backbone glucose rings [Sharp et al., 1984a]. The structure of this hexa (β-D-glucopyranosyl)-D-glucitol was shown as below: β D Glcp (1

6) β D Glcp (1 3 1 β D Glcp

6) β D Glcp (1

6) β D Glcp (1 3

6) Glucitol

1 β D Glcp

The glycosyl-linkage compositions of the hexa (β-D-glucosy1)-D-glucitols indicated that only small structural differences existed between the elicitor-active and the elicitor-inactive molecules. These results indicated that the similarity in structure of six elicitor-inactive oligosaccharides to a highly defined structure of the elicitor-active molecules established was required for elicitor activity. The elicitor-active hexa (β-D-glucopyranosy1)-D-glucitol was the first example of complex carbohydrate acting as a regulatory molecule in plants [Sharp et al., 1984a]. Evidences of glucan as an elicitor were obtained from the crude or only partially purified fungal cell wall fractions, such as the Phytophthora sojae cell wall hydrolysate, a heterogeneous β-1,3-1,6 glucan extracted from the mycelial walls of Phytophthora sojae f. sp. glycinea [Sharp et al., 1984a]. The elicitor activity of this glucan was mainly studied in leguminous plants but it was also reported to induce a Gly-rich protein [Brady et al., 1993] and antiviral protection in tobacco cells [Kopp et al., 1989]. A pure glucan heptasaccharide [Sharp et al., 1984a] prepared from Phytophthora sojae glucan induced the synthesis of phytoalexins in soybean (Glycine max) [Sharp et al., 1984a]. The minimal structural requirements for the elicitation of phytoalexin synthesis in soybean by this glucan were established as a succession of five β-1,6-linked glucosyl residues with two side branches of β1,3-glucose [Cheong et al., 1991]. Specific binding sites for the β-1,6-1,3 heptaglucan from Phytophthora sojae have been described in soybean [Cheong et al., 1991], alfalfa (Medicago sativa), bean (Phaesoleus vulgarus), lupine (Lupinus albus), and pea (Pisum sativum) [Cosio et al., 1996; Côté et al., 2000]. The smallest elicitor-active β-glucan has been determined (hepta-β-glucoside), and the chemically synthesized elicitor was reported not only to induce phytoalexin accumulation but also to have a high affinity for the plasma membrane fraction [Cheong et al., 1991; Sharp et al., 1984a]. These reports strongly suggested that these oligo-glucan elicitors bind to a receptor on the plasma membrane of soybean, resulting in the accumulation of phytoalexins.

Biodegradation of Polysaccharide Sourced from Virulence Factor…

11

2) Biodegradation of chitin and chitosan Chitin is a natural polymer, being a linear polysaccharide of β-(1,4)-linked N-acetyl glucosamine (GlcNAc) residues, found in the exoskeletons of crustacea and insects and in the cell walls of certain fungi. It is the second most abundant polymer in nature next to cellulose [Rudrapatnam et al., 2003]. However the insolubility of chitin in most solvents seriously restricts its biological activities. Chitosan is derived from chitin by deacetylation in the presence of alkali, which is a copolymer consisting of β-1,4-linked glucosamine (GlcN) with various degrees of N-acetylated glucosamine residues [Arvanitoyannis et al., 1998]. Chitosan has been demonstrated to have different biological activities, such as antitumor effect , antibacterial effect, and antifungal effect [Choi et al., 2001; Jeon et al., 2001; Roller et al., 1999; Seo et al., 2000]. Like the difficult situation chitin is facing, however, the application of polymeric chitosan was also limited due to its solubility only in weakly acidified water and its high viscosity. Recently the studies on chitin and chitosan have been paid more interest in biodegradation of chitin and chitosan to form chitooligosaccharides, especially the hydrolysis of chitosan into oligosaccharides. Because the chitosan oligomers not only are water-soluble and have low viscosity due to their shorter chain lengths and free amino groups in D-glucosamine units, but also possess versatile functional properties such as antitumor activity, immuno-enhancing effects, enhancement of protective effects against infection with some pathogens, antifungal activity, and antibacterial activity [Jeon & Kim, 2001; Jeon et al., 2000; Jeon et al., 2001; Hirano & Nagao, 1989; Kendra et al., 1989; Suzuki, 1996; Suzuki et al., 1986; Tokoro et al., 1989; Tsukada et al., 1990]. With respect to antimicrobial activity, it has been known that chitosan oligomers are superior to chitin oligomers because the partially degraded chitosan possesses a lot of polycationic amines, which can interact with the negatively charged residues of macromolecules on the cell surface of microorganisms and subsequently inhibit the cell growth of microorganisms [Young & Kauss, 1983]. The antimicrobial effect of chitooligosaccharides has also been shown to be greatly dependent on their degree of polymerization (DP) or molecular weight and requires glucosamine polymers with DP 6 or greater [Kendra and Hadwiger, 1984]. Chitosan can be depolymerized by partial hydrolysis with concentrated HCl [Horowitz et al., 1957]. However, acidic hydrolysis produced low yields of oligosaccharides and a large amount of its constituent units D-glucosamine. Also, the oligosaccharides prepared by the acidic hydrolysis might be toxic because of chemical changes during conversion. Therefore, the preferred method for producing chitooligosaccharides with specific lengths and sequences should be depolymerization of chitin/chitosan by enzymatic hydrolysis [Sikorski et al., 2005]. Up to now, there have been many different enzymes being isolated and studied for this purpose. The hydrolysis of chitin/chitosan to monomer N-acetyl glucosamine and glucosamine oligosaccharides has been studied for decades, in which the main representative chitolytic enzyme was chitinase catalyzing the hydrolysis of chitin. However, it was found that the chitin degradation was difficult due to its insolubility. The better suitable substrate for chitinase should be partially deacetylated chitin. Chitin/chitosan is generally susceptible to a number of different enzymes indicating its broad substrate specificity [Aiba, 1994a, b]. Up to date, a range of chitino/chitosanolytic enzymes have been found in the most living organisms including bacteria [Lee et al., 1996; Ohtakara et al., 1990; Varum et al., 1996; Yang et al.,

12

Xianzhen Li and Xiaoyi Chen

2005], actinomycetes [Hoell et al., 2006; Ohtakara et al., 1990], fungi [Kim et al., 1998; Muzarelli et al., 1994a], plant [Eilenberg et al., 2006; Lakhtin et al., 1995], and mammals or humans [Boot et al., 2001; Escott and Adams, 1995; Renkema et al., 1995]. Chitinolytic bacteria produced multiple chitinases, but there was comparatively little information available about the properties and roles of the individual chitinases in a chitinolytic system. Alteromonas sp. strain O-7 secretes four chitinases (ChiA, ChiB, ChiC, and ChiD) in the presence of chitin, in which ChiA plays a central role in chitin degradation [Orikoshi et al., 2005]. Amycolatopsis orientalis (Nocardia orientalis) was known to secrete various chitinolytic enzymes such as chitinase, β-N-acetyl hexosaminidase, and endo-βglucosaminidase [Nanjo et al., 1989, 1990]. These enzymes have been shown to catalyze efficient transglycosylation activities [Usui et al., 1987, 1990]. In addition, some other common carbohydrases and proteases have also been proved their hydrolytic ability on chitosan to produce chitooligosaccharides with different molecular weights [Aiba, 1994a; Zhang et al., 1999]. Lysozyme from hens’ eggs has been investigated and shown to be the most efficacious when the chitosan is only partially deacetylated [Nordtveit et al., 1994, 1996]. Enzymes from a variety of sources have been used for examining chitosan degradation. Uchida et al. [1989] reported that chitosanase from Bacillus sp. produced mainly the oligosaccharides with DP 2–6 and a small amount of D-glucosamine after prolonged incubation, suggesting its endo-action on chitosan and its ability to degrade partially chitosan molecules. The susceptibility of chitosan to a number of different enzymes has been investigated. Aiba [1994a, 1994b] carried out the hydrolysis of partially N-acetylated chitosan with chitinase and lysozyme because these enzymes can recognize N-acetyl glucosamine residues in chitosan. Pantaleone et al. [1992] reported the hydrolytic susceptibility of chitosan to a wide range of enzymes, including 10 kinds of glycanases, 21 kinds of protease, five lipases and a tannase, which were derived from different bacterial, fungal, mammalian and plant sources. Among them papain from Carica papaya and hemicellulase and lipase from Aspergillus niger were reported as effective enzymes to hydrolyze chitosan. Muzzarelli et al. [1994b, 1995] examined the action of papain and lipase in depolymerizing chitosan. From these results, a lot of commercial enzymes have been developed for efficient hydrolysis of chitosan. These enzymes, however, were added at relatively high concentrations, while chitosanase showed substantial activities at low concentrations. Further, it has been revealed that the structure of glycosidic bonds in chitosan affects enzymatic hydrolysis process. Differentially deacetylated chitosan have four different types of randomly distributed glycosidic bonds in their structures. These include linkages GlcNGlcN, GlcNAc-GlcN, GlcN-GlcNAc and GlcNAc-GlcNAc. Egg white lysozyme was found to be almost exclusive towards the cleavage of glycosidic linkage of GlcNAc-GlcNAc, while Bacillus chitosanase was found to be highly specific towards GlcN-GlcN linkages [Varum et al., 1996]. In addition, chitinase could act on partially N-acetylated chitosan by recognizing GlcNAc residues in the chitosan sequence [Aiba, 1994b]. Chitosanases from different organisms also differ in their catalytic action and that is mainly dependent on deacetylated degree of chitosan [Kurita, 1998]. However, it has been generally observed that chitosanases obtained from microbes produce relatively a higher yield of chitooligosaccharides compared to chitosanases from the other sources. Although microbial chitosanases have shown to have excellent performances in chitooligosaccharides production, they are too expensive to be utilized in large-scale

Biodegradation of Polysaccharide Sourced from Virulence Factor…

13

industrial applications. Therefore, other commercial enzymes were utilized under specific conditions to produce chitooligosaccharides with a relatively low cost [Zhang et al., 1999]. Several plants, insects and microorganisms have chitinolytic enzyme systems that are capable of degrading chitin/chitosan [Azarkan et al., 1997; Patil et al., 2002]. For example, Serratia marcescens produced at least three chitinases with complementary activities [Suzuki et al., 2002]. Degradation of chitin results in a range of chitooligosaccharides with different degree of polymerization. In the past decades, several biotechnological approaches have been taken to prepare industrially chitooligosaccharides. Biodegradation of chitin/chitosan was carried out in batch reactors in the early period of enzymatic production of chitooligosaccharides [Izume and Ohtakara, 1987; Jeon and Kim, 2000; Varum et al. 1996]. However this batch method has some disadvantages: (1) the high cost associated with large quantities of expensive enzymes; (2) low yields due to the limited ability to control the degree of polymer; (3) mixture of chitooligosaccharide with a broad molecular weight range; (4) large quantities of expensive chitosanase used. Especially, the high cost associated with hydrolytic enzymes demotes the application of enzymatic methods. To reduce production cost, it is recommended to reuse hydrolytic enzymes instead of a single use in batch reactors. Enzyme immobilization method was introduced to degrade chitin/chitosan for overcoming the high process cost problems caused by the single used enzyme in batch reactors [Jeon et al. 1998]. In this system the immobilized chitosanase exhibited the highest enzymatic activity. It was found that the effective production of target chitooligosacchrides depended greatly on surface enzyme density, supported particle size, aggregation speed, and initial substrate concentration [Kuroiwa et al., 2002, 2003]. However the poor affinity of immobilized enzymes to chitin/chitosan substrates than that of free enzymes limited this method for hydrolysis of chitin/chitosan effectively. Such problems that the immobilization column reactors faced can be overcome by using ultrafiltration (UF) membrane enzymatic reactor system [Jeon and Kim, 2000]. The enzymatic production of chitooligosaccharides with relatively a high degree of polymerization was processed in this system. The chitin/chitosan-hydrolyzing enzyme added in the reaction vessel was recycled, and the produced oligosaccharides were separated from the substrates and the enzymes in the process of chitosan degradation. The production of reducing sugar progressively increased with arising permeation rate, and the composition of the oligosaccharides was dependent on the permeation rate. Thus permeation rate was a key factor for the control of oligosaccharide production. The molecular weight distribution of the hydrolysate could be controlled within limits by the appropriate membrane used. Large quantities of pentamers and hexamers but no monomers were obtained with the UF membrane reactor system, suggesting this system enable effective production of relatively larger oligosaccharides [Jeon et al. 2001]. Oligosaccharides obtained using this reactor system showed antibacterial activity [Jeon and Kim, 2000; Kittur et al., 2003]. This reactor system could hydrolyze at least 11 batches of substrates for the same amount of enzymes used in the batch reactor. However, UF membrane method did not allow continuous production of chitooligosaccharides because of the increased transmembrane pressure during the reaction. It seems to occur due to the high viscosity of chitosan. Therefore reducing chitosan viscosity prior to treatment in the membrane system may allow membrane fouling to be all eviated.

14

Xianzhen Li and Xiaoyi Chen

Considering the membrane fouling occurred due to the high viscosity of chitosan, researchers have been performed to develop new strategies to degrade chitin/chitosan. As schematically shown in Figure 4, a dual reactor system composed of an UF membrane reactor and a column reactor packed with an immobilized enzyme was developed. With this combination of an immobilized enzyme reactor and the UF membrane reactor, continuous production of chitooligosaccharides was feasible [Jeon & Kim, 2000]. Firstly, chitosan was partially degraded by the immobilized enzyme prepacked in the column reactor, and the hydrolysates with low viscosity were immediately supplied to a substrate feed tank of an UF membrane reactor. Secondly, the partial fragments of chitin/chitosan from immobilization reactor were continuously added to the UF membrane reactor system for the enzymatic degradation to produce chitooligosaccharide. The different molecular weight cutoffs of UF membranes can be used in the system to obtain different molecular weight distribution. This method ensures a greater productivity per unit of enzyme, ability to control molecular weight distribution, and more efficient continuous production process. Kuroiwa et al. [2003] has determined the optimum conditions for continuous production of pentamers and hexamers of chitooligosaccharides using a dual reactor. Under the optimum conditions, continuous production of pentamers and hexamers was achieved for a month without significant decrease in products.

Figure 4. Schematic diagram of the dual reactor system developed for continuous production of chitooligosaccharides. Adapted from Jeon and Kim [2000].

In the enzymatic hydrolysis of chitin/chitosan, the molecular size of the final products is very important because the functional properties of chitin/chitosan and their oligosaccharides are greatly dependent on their molecular weights [Hirano and Nagao 1989]. In enzymatic hydrolysis of chitosan using a batch reactor, chitosan may be cut randomly by endo-type enzymes and then higher oligosaccharides produced may be immediately hydrolyzed by the enzyme in the solution, leading to the preparation of lower oligosaccharides. In a continuous

Biodegradation of Polysaccharide Sourced from Virulence Factor…

15

UF membrane reactor, however, smaller oligosaccharides than the cutoff of the used membrane were permeated and separated from the enzyme during reaction. This probably prevented the oligosaccharides proceeding to further hydrolysis [Izume and Ohtakara, 1987]. For these reasons, the dual reactor system has been mainly used in the enzymatic process for hydrolysis of polysaccharide [Jeon & Kim, 2000].

3) Biodegradation of xyloglucan Xyloglucan is a major structural carbohydrate of hemicellulose occurred in the primary walls of many land plants [Somerville et al., 2004]. It has a cellulose-like backbone of β-1,4linked D-Glcp residues, which is substituted in a regular pattern with xylosyl residues plus other sugars that vary depending on the plant species. Xyloglucan forms a structural network by interaction with cellulose microfibrils via hydrogen bonds, which plays a key role in cell wall integrity [Fry et al., 1993b; Pauly et al., 1999]. Xyloglucan being substituted mainly occurs at C-6 with α-1,6 D-xylosyl (Xylp) residues. Other saccharides, frequently β-Dgalactose (D-Galp) and α-1,2 L-fucose (l-Fucp) residues, may be attached to it [Carpita and McCann, 2000]. In order to conventionally write [Fry et al., 1993b], the unsubstituted D-Glcp residue is named G, α-D-Xylp-(1,6)-β-D-Glcp segment is named X, β-D-Galp-(1,2)-α-D-Xylp-(1,6)-βd-Glcp is L and a-L-Fucp-(1,2)-β-d-Galp(1,2)-α-D-Xylp-(1,6)- β-D-Glcp is F (shown in Figure 5). Two general types of xyloglucans are XXXG-type having a repeating unit with one unsubstituted D-Glcp residue and XXGG-type having two unsubstituted D-Glcp residues [Vincken et al., 1997b]. HO HO OH

OH O

O O

HO OH x OH HO H

O

O

O

O O

O

HO

OH

OH

HO O

O

OH

O HO

O

OH O

O O

OH OH

RO O

O OH OH

OH HO

OH y

OH OH

Figure 5. General structure of xyloglucan including XXXG (x=0, y=0, R=H), XLXG (x=1, y=0, R=H), XXLG (x=0, y=1, R=H), and XLLG (x=1, y=1, R=H). XXFG (x=0, y=1, R=a-1,2 L-Fuc.

n

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Xianzhen Li and Xiaoyi Chen

Xyloglucan-degrading enzymes have attracted the attention of researchers during the last decade. Some cellulases (endo-1,4-β-glucanases) are able to hydrolyze the xyloglucan backbone at unsubstituted D-Glcp [Vincken et al., 1997a]. Two of seven cellulases from Chrysosporium lucknowense possess a notable activity against xyloglucan [Bukhtojarov et al., 2004]. For a long time, three xyloglucan-degrading endoglucanases from plants have been the only examples of specific xyloglucanases acting on the polymer backbone [Edwards et al., 1986; Matsumoto et al., 1997]. A few specific xyloglucanases from fungi have been discovered only recently [Hasper et al., 2002; Yaoi and Mitsuishi, 2004]. Three xyloglucanases isolated from Aspergillus japonicus, Chrysosporium lucknowense and Trichoderma reesei have high specific activity toward tamarind xyloglucan, whereas the activity against carboxylmethyl cellulose and barley β-glucan is absent or very low. All enzymes produced XXXG, XXLG/XLXG and XLLG oligosaccharides as the end products of xyloglucan hydrolysis. Aspergillus japonicus xyloglucanase displayed an endo-type of attack on the polymeric substrate, while the action mode of two other xyloglucanases was similar to the exo-type [Grishutin et al., 2004]. The recent enzymatic characterization of the endoxyloglucanase, Xgh74A, showed that the enzyme hydrolyzed the glycosidic bond of the unbranched glucosyl residues in xyloglucan, to yield XXXG, XLXG, and XLLG oligosaccharides [Zverlov et al., 2005]. Two xyloglucan-specific endo-β-1,4-glucanases were isolated from the Gram-positive bacterium Paenibacillus sp. strain KM21, one of which is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while the other of which has dual endo- and exo-mode activities or processive endo-mode activity [Yaoi et al., 2005].

4) Biodegradation of pectin Pectin is a family of complex and highly heterogeneous polysaccharides that contributes to the structure of plant tissues as a component of the middle lamella and primary cell walls [Daas et al., 1999，2001]. Pectin is built of a backbone mainly consisting of β-1,4-linked αD-galacturonic acid (GalUA) residues, with various degrees of methyl esterification of the carboxyl groups on α-D-galacturonic acid residues. The main chain of the polymer also contains rhamnose residues that can be highly substituted by arabinose and galactose side chains. Phytopathogenic fungi produce extracellular enzymes, which can degrade the cell wall components of plants. These fungi not only degrade cell wall polymers to use their sugars as an important nutrient source, but also digest the cell walls to aid in penetrating cells and spreading through the plant tissue. In this rotting process oligomers can be produced by degradation of pectic carbohydrates in cell walls, for example, pectin derived oligosaccharides were generated when cell wall pectins were digested in fruit as they were ripen or they were degraded by pathogen enzymes during tissue colonization [Melotto et al., 1994; Tonukari et al., 2000; Wanjiru et al., 2002]. It is thought that these oligosaccharides are generally important factors for regulating fruit responses to infection by pathogens [OlanoMartin et al., 2002; Ridley et al., 2001]. Degradation of the pectin polymer occurs via a set of pectinolytic enzymes, which can roughly be divided into two groups. One is pectin esterase removing methoxyl groups from

Biodegradation of Polysaccharide Sourced from Virulence Factor…

17

pectin. The other is depolymerase being classified further as lyases (β-elimination) and hydrolases, which degrade the backbone chains (Figure 6) [Sakai et al., 1993]. All hydrolases involved in the degradation of pectin are classified as members of family 28 of the glycoside hydrolases, including the endopolygalacturonases, exopolygalacturonases and rhamnogalacturonases [Markovič and Janeček, 2001]. Polygalacturonate (pectate) lyases are specific for unmethylated pectate, although they can be active on pectin with a low degree of methyl esterification [Tardy et al., 1997]. For example, Pectate lyase C from Bacillus substilis can depolymerize polygalacturonate and pectin of methyl esterification degree from 22% to 89%, but exhibits maximum activity on 22% esterified citrus pectin [Soriano et al., 2006]. Pectate lyases are widely distributed among plant pathogens, where they play an important role as virulence factors [Herron et al., 2000]. They have also been found in saprophytic microorganisms, including members of the genus Bacillus and in some thermophilic bacteria [Berensmeier et al., 2004; Hatada et al., 2000; Kluskens et al., 2003]. Pectin lyases cleave the 1,4-α-linkages in pectin molecules and are mainly synthesized by fungal species, whereas few bacteria produce this type of enzyme. The pectin lyases have a specificity on pectin molecules, for example, an exo-acting pectin lyase from an Aspergillus sp. acted on highly esterified pectin but not on polygalacturonic acid [Delgado et al., 1992]. In the culture of anaerobe Bacteroides thetaiotamicron, there were both polygalacturonate hydrolysase and polygalacturonate lyase activities to be detected, which have been identified as membrane-associated enzymes [McCarthy et al., 1985]. Phytopathogenic Pseudomonas spp. causing soft rots of plant secreted a constitutive pectate lyase when grown in calcium-containing media [Liao, 1991]. R1 O

R1

R1 COX

HO

OH

COX

COX OH

O

HO

OH

OH COX HO

OH

Pectin lyase Exo-polygalacturonate lyase Pectate lyase

O

Pectinase Exo-polygalacturonase

O

O

O

O

COX

COX

OH

O

HO

O

R2

R2

Pectin methyl esterase

R1

OH OH

O

HO

O

HO

O OH

O

R2

O -

COO HO

O OH

O HO

COO

-

O OH

O R2

Figure 6. Pectin degradation by pectiolytic enzymes (X is predominantly O- for pectate and O-Me for pectin, R1 and R2 are polygalacturonate).

In studying the pectin-degrading enzymes produced by plant pathogens, such as Erwinia chrysanthemi, the hydrolytic action and production of pectinases and polygalacturonate lyase

18

Xianzhen Li and Xiaoyi Chen

have provided a mechanism for the bacteria to degrade the plant cell wall and effect on pathogenicity, and also bacteria utilize such breakdown products for bacterial growth. It was found that pectate lyases produced by soft-rotting bacterium Erwinia spp. play a significant role in pathogenic infection [Reverchon and Baudouy, 1987]. The enzymes are induced by breakdown products of polygalacturonic acid such as 2-keto-3-deoxygluconate, and their production is repressed by glucose. What is perhaps surprising is that the bacteria should synthesize five pectate lyases, apparently in addition to an oligogalacturonate lyase active against the unsaturated digalacturonate. As well as playing a very important role in the plant infection process, the lyases enable the bacteria to grow on pectin as the sole carbon and energy source. Oligogalacturonides with degrees of polymerization between 10 and 20 have been shown to have important regulatory activity for plant defense mechanisms [Cervone et al., 1989].

5) Biodegradation of xylan Xylan, a β -1,4 linked polymer of D-xylose with D-glucuronic acid or L-arabinose substituents, occurs in almost all parts of plant cell walls, which is the one main component of plant hemicelluloses. Production of xylooligosaccharides from plant hemicelluloses generally includes extraction of xylans and subsequent enzymatic hydrolysis of the extracted xylans [Yuan et al., 2004]. Xylan can be depolymerized by the enzymatic degradation of xylanases. The enzymatic degradation of plant cell wall xylan requires the concerted action of a dedicated enzymatic consortium (diverse enzymatic syndicate) due to the constituent groups in xylan acting as a limited factor in efficient hydrolysis. Among these enzymes there are endo-1,4-β-xylanses and β-xylosidases that cleave the backbone chain, and β-Larabinofuranosidase, xylan esterases, and β-D-glucuronidase that cleave the side chain of xylan (Figure 7) [Coughlan and Hazlewood, 1993]. Esterases hydrolyze the O-acetyl substituents, primarily at the O-2 position of the xylan backbone, which include two distinct ferulae and acetylxylan esterases catalyzing to remove the ferulate groups linked via arabinosides to the xylan backbone and to deacetylate the O-3 and (primarily) O-2 positions of the xylan backbone in acetylxylan [Taylor et al., 2006]. Several bacterial and fungal species produce the full complement of enzymes necessary to utilize xylan as a carbon source [Uffen, 1997]. Xylanase activity has been detected in some members of the genus Paenibacillus, Bacillus, Xylanibacter, Streptomyces [Jiang et al., 1994; John et al., 2006; Lee et al., 2000; Morales et al., 1995; Ueki et al., 2006; Velázquez et al., 2004].

Biodegradation of Polysaccharide Sourced from Virulence Factor… -

O

O

α-Glucuronidase

O

MeO HO

Acetyl xylan esterase H3C Xylanase

OH O

HO O

O

O

O MeO HO

O

O

O

19

OH O

OH

HO O

O O O O

n

α-L-Arabinofuranosidase OH

Ferulate esterase

Figure 7. Schematic diagram of xylan degradation.

Endoxylanases are classified into two groups, family 10 or 11, based on hydrophobic cluster analysis and amino acid sequence homologies [Henrissat, 1991]. Compared to endoxylanases of the family 11, those belonging to the family 10 show better capability of cleaving glycosidic linkages in the xylan main chain closer to the substituents, such as MeGlcA and acetic acid. The thermophilic fungi, Thermoascus aurantiacus and Sporotrichum thermophile, produce high activities of highly thermostable xylanases under solid state and submerged culture respectively. A high molecular xylanase (XYL I) with catalytic properties similar to those belonging to the family 10 was purified and characterized by the culture filtrates of Thermoascus aurantiacus, and a low molecular weight xylanase (XYL A) with catalytic properties similar to those belonging to the family 11 was isolated from the culture filtrates of Sporotrichum thermophil [Kalogeris et al., 2001]. When treatment with a Thermoascus aurantiacus family 10 or a Sporotrichum thermophile family 11 endoxylanases, acidic oligosaccharides can be degraded from birch wood xylan. The main difference between the products liberated by xylanases of the family 10 and that from the family 11 concerned the length of the products containing 4-O-methyl-D-glucuronic acid. The xylanase from Thermoascus aurantiacus liberated an aldotetrauronic acid as the shortest acidic fragment from glucuronoxylan in contrast with the enzyme from Sporotrichum thermophile, which liberated an aldopentauronic acid. It was found that xylooligosaccharides could be typically produced by enzymatic degradation of lignocellulosic materials, having xylan as major hemicellulose component [Parajó et al., 2004]. However, such degradation was concerned with the substituent in polysaccharides. The substituted xylooligosaccharides, arabinoxylooligosaccharides, can be formed from wheat flour arabinoxylans. Arabinoxylans consist a backbone of (1,4)- β-linked D-xylopyranosyl units with substitutions of α-L-arabinofuranosyl units at position C-(O)-2 and/or C-(O)-3 [Courtin and Delcour, 2001; Gruppen et al., 1992]. Endoxylanases attacked the arabinoxylans backbone internally, depending on the arabinose substitution pattern [Swennen et al., 2005]. They degraded arabinoxylans yielding series of oligomeric and polymeric arabinoxylans with different DP [Courtin and Delcour, 2001; Maes et al., 2004].

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Xianzhen Li and Xiaoyi Chen

Debeire et al [1990] purified an extracellular xylanase from a hydrolytic thermophilic anaerobe, Clostridium thermolacticum. They observed that the xylanase was an endo-1,4xylanase and the main hydrolyzed end products of the larchwood xylan were xylobiose and xylotriose. Pellerin et al [1991] employed this xylanase for production of xylooligosaccharides from xylan derived from corncob meal by alkaline extraction. However, a long hydrolysis time of 48 h was required with 100 000 U of the xylanase. The substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina (Trichoderma reesei) was examined using several polysaccharides and oligosaccharides. The results revealed that xyloglucan chains were hydrolyzed at substituted glucose residues, in contrast to the action of all known xyloglucan endoglucanases [Desmet et al., 2007]. It has been proved that the xylooligosaccharides with DP less than 5 failed to induce the xylanase activity for plant defense [Miyazaki et al., 2005].

6) Biodegradation of carrageenan Carrageenans are sulfated galactans occurred in the cell wall of marine red seaweeds. They are major components of the matrix involved in the building up of the cell-wall architecture and mediate cell-cell recognition in host-pathogen interactions [Kloareg et al., 2001]. Carrageenan is a generic name for a family of natural, water-soluble, sulfated galactans [De Ruiter and Rudolph, 1997]. This large family of anionic polymers share the same backbone structure, which consists of a linear chain of alternating α-(1,3)-linked β-Dgalactopyranose (Galp) and β-(1,4)-linked α-D-galactopyranose. In these units, various hydroxyl groups may be substituted by ester sulfate [Knutsen et al., 1994, 1995]. Usually a 3,6-anhydro-α-D-galactopyranose (AnGalp) in place of α-D-galactopyranose may contains in linear chain. Carrageenan can be classified according to the number and the position of sulfated ester. For example, the three most industrially exploited carrageenans, namely kappa(κ, A-G4S), iota- (ι, A2S-G4S) and lambda- (λ, D2S6S-G2S) carrageenans, are distinguished by the presence of one, two and three ester sulfate groups each repeating disaccharidic unit respectively (Figure 8). However, carrageenans have very heterogeneous chemical structures, depending on the algal sources, the life stages and the extraction procedures of the polysaccharides. It was reported that carrageenan could be degraded by using an active oxygen species or organic acid solution [Yamada et al. 2000; Yu et al. 2002]. However, the degradation degree of carrageenan with these chemical methods was controlled difficultly, and usually very small molecules even its constituent monomer was produced, in which the products were a complex mixture. Therefore it was thought that the most popular technical method should be the enzymatic hydrolysis of carrageenan. Several genera of marine bacteria have been found to produce carrageenase, such as Vibrio, Alteromonas, Pseudomonas carrageenovora and Cytophaga [Araki et al. 1999; Michel et al. 2000, 2001; Potin et al. 1991].

Biodegradation of Polysaccharide Sourced from Virulence Factor…

a

OSO3CH2OH O

O O

O

O

OH

n

OH

b

OSO3CH2OH O

O O

O OH

c

OH

O OSO3-

OSO3-

CH2OH O

n

O O OSO3-

O OSO3-

21

HO

n

Figure 8. Disaccharide repeating units of carrageenans. a, k-Carrageenan (β-D-galactopyranosyl-4sulfate(1,4)-O-3,6-anhydro-α-D-galactopyranosyl); b, ι-Carrageenan (β-D-galactopyranosyl-4-sulfate(1,4)O-3,6-anhydro-α-D-galactopyranosyl-2-sulfate); c, λ-Carrageenan (β-D-galactopyranosyl-2-sulfate(1,4)-O-aD-galactopyranosyl-2,6-disulfate).

To prepare sulfated oligosaccharides with special biological activities, a marine bacterium Cytophaga strain MCA-2 was isolated, which produced a special extracellular carrageenase in the presence of crude κ-carrageenan [Mou et al. 2002]. This κ-carrageenase can cleave specifically the internal α-(1,4) linkages of κ-carrageenans to yield oligosaccharides of the neocarrabiose series [Barbeyron et al., 2000; Michel et al., 2001]. Enzymatic products of κ-carrageenans have been shown to elicit biological activities in plants [Bouarab et al., 1999]. Depolymerization of κ-carrageenan was performed also using other carrageen-degrading enzymes for oligosaccharides production [Ekeberg et al., 2001; Knutsen et al., 2001]. In order to prepare the oligosaccharides with different molecular weights and sulfur contents, carrageenan was first fragmented by enzymatic hydrolysis, and then followed by sulfonation with formamide-chlorosulfonic acid. Semi-preparative chromatography based on a strong

22

Xianzhen Li and Xiaoyi Chen

anion-exchange (SAX)-HPLC column has been used to separate such oligosaccharides [Yu et al., 2002]. Carrageenan derived oligosaccharides have been shown to have biological activities such as being an disintegrant and anti-viral [Shi et al. 2000]. In general, the biological activities of sulfated oligosaccharides are close related with the molecular weight, the carbohydrate structure and the content and linking position of sulfur groups [Liu et al., 2000].

7) Biodegradation of xanthan Xanthan is an extracellular heteropolysaccharide produced by the phytopathogenic bacterium Xanthomonas campestris pv. campestris. Xanthan molecule consists of a main cellulosic backbone with linear trisaccharide side chains, each of which is composed of a mannosyl (β-1,4)-glucuronyl-(β-1,2)-mannose sequence attached at the C-3 position on alternate glucosyl residues through α-1,3 linkages (Figure 9) [García-Ochoa et al. 2000]. The molecular weight distribution ranges from 2 × 106 to 20 × 106 Da. The internal mannosyl residues of the side chain are mostly acetylated at O-6 position, and approximately 50% of the terminal mannosyl residues may be substituted with a pyruvate ketal at C-4 and C-6 positions. The levels of pyruvate and acetyl substitution are varied depending on growth conditions and bacterial strains [García-Ochoa et al. 2000]. It has been proved that exopolysaccharide xanthan is the virulence factor of Xanthomonas campestris pv. campestris causing block rot process in cruciferous plants, which leads to a significant loss of crop in the worldwide [Chou et al., 1997; Ishikawa et al., 2004]. It has been believed that the defense response in plant cells mediated by elicitor molecules could help plant resist disease [Fry et al. 1993b]. Like some elicitor-active oligosaccharides produced artificially by the enzymatic fragmentation of glucan, chitosan and pectin [Boudart et al. 1998; Fry et al. 1993b; Küpper et al. 2001], xanthan molecule has the similar structural features to these polysaccharides such as the side chain and charged residues [García-Ochoa et al. 2000], presumably xanthan degradation products have elicitor activity. In fact, some xanthooligosaccharides derived from xanthan are elicitor-active to induce the accumulation of phytoalexin in the soybean cotyledon [Liu et al., 2005]. The xanthan degradation products have also been shown to directly inhibit Xanthomonas. campestris pv. campestris, but none of the other organisms was tested in vitro [Qian et al., 2006]. It is interesting that xanthan produced by Xanthomonas campestris pv. campestris was involved in the pathogenicity [Ishikawa et al., 2004], but its partial hydrolysates could inhibit the cell growth of Xanthomonas campestris pv. campestris [Qian et al., 2006]. Therefore the xanthan degradation products were potential protecting agent for the management of diseases caused by Xanthomonas campestris pv. campestris [Liu et al., 2005; Qian et al., 2006].

Biodegradation of Polysaccharide Sourced from Virulence Factor… CH2OH O HO

OH CH2OAc

HO

O Man OH

Glc

CH2OH O Glc HO OH

CH3

HO

O HO

O Man OH

CH2OH O O

OH CH2OAc HO Man HO

OH

COOH HO

OH

β -D-Glucanase

Glc

O

OH

CH2OAc O HO Man OH HO

CH2OH O COOH

n

O O

β -D-Glucanase

O

O OH

O

COOH O GlcA O HO OH

O

O

O

HO Man HO

COOH CH3

CH2OH O

O

Glc

Xanthal lyase

23

O

Glc

O OH

n

O O

CH2OH O Glc OH

α -D-Manosidase

CH2OAc O O HO Man OH HO

O

GlcA

OH COOH

HO

O OH

GlcA OH

OH

β -D-Glucosidase CH2OH O HO Glc HO

O OH CH2OAc HO Man HO

COOH HO

O

CH2OH O Glc OH

CH2OH O OH

Glc

O O

CH2OAc HO Man HO

O

COOH

GlcA OH

OH OH

HO

O

GlcA OH

O O

CH2OH O HO Glc HO

O

OH

Unsaturated glucuronyl hydrolase

Figure 9. Xanthan depolymerization pathway in Bacillus sp. strain GL1 [Nankai et al., 1999].

Xanthan is a highly stable polysaccharide that can be completely degraded into its smallest constituents by a few microorganisms [Ahlgren, 1993; Cadmus et al., 1982; Cheetham and Mashimba, 1991; Christensen et al. 1996; Nankai et al., 1999; Sutherland, 1984], although in the unordered conformation it can be degraded partially by cellulase [Rinaudo and Milas, 1980]. Cadmus et al. [1982] described the isolation of a salt-tolerant bacterium Bacillus sp. K11 capable of eliminating side chains from xanthan. The xanthandegrading bacterium Paenibacillus alginolyticus degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact [Ruijssenaars et al. 1999b]. Bacillus sp. GL1 was found to utilize xanthan for its growth and thereby produced xanthan lyase [Hashimoto et al. 1998a]. Although some microbial mixed cultures have been found to assimilate xanthan, an enzymatic route for the complete depolymerization of a xanthan in Bacillus sp. strain GL1 was elucidated in 1999 by analyzing the structures of xanthan depolymerization products [Nankai et al., 1999]. As shown in Figure 9, the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains was first attacked by extracellular

24

Xianzhen Li and Xiaoyi Chen

xanthan lyase to remove the pyruvylated mannose. And then, this modified xanthan was depolymerized to a tetrasaccharide by extracellular β-D-glucanase, without the terminal mannosyl residue of the side chain in a repeating unit of xanthan. After that, the tetrasaccharide was incorporated into cells and one glucose residue was cleaved from nonreducing end by β-D-glucosidase. The produced trisaccharide (unsaturated glucuronylacetylated mannosyl-glucose) was degraded successively to unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. At last, the disaccharide was hydrolyzed to the constituent monosaccharides (mannose and glucose) by α-D-mannosidase. According to the enzymatic route of xanthan, there are five xanthan-degrading enzymes involved in xanthan degradation. Xanthanase (β-D-glucanase) catalyses the hydrolysis of the xanthan backbone. A few xanthanases have been identified, some of which were categorized as cellulase family members [Sutherland, 1987]. It was found that some cellulase showed partially hydrolytic activity on xanthan [Rinaudo and Milas, 1980]. The endoglucanases probably acted in conjunction with xanthan lyase and showed a higher activity on xanthanderived oligosaccharides than on intact xanthan [Ahlgren, 1991; Hashimoto et al. 1998b; Sutherland, 1987]. However, the enzyme from Paenibacillus alginolyticus XL-1 was active only on intact xanthan and was not found to be associated with endoglucanases [Ruijssenaars et al., 1999]. Xanthan lyase is an enzyme first attacking xanthan, which removes the terminal mannosyl residue via β-elimination and yields a free mannose and an unsaturated glucuronyl terminal in the side chain of xanthan [Ahlgren, 1991; Sutherland, 1987]. Xanthan lyase is usually used as a xanthan-modifying enzyme for studying structure-function relationships and producing modified xanthan. There are two types found in xanthan lyase, non-specific and pyruvated mannose-specific xanthan lyases [Ahlgren, 1991; Hashimoto et al., 1998b; Ruijssenaars et al., 1999; Sutherland, 1987]. It was clear that the acetyl group was not required for lyase activity, since the enzyme was active on xanthans that were originally low in acetyl substituents as well as on chemically deacetylated xanthan [Ruijssenaars et al., 1999]. The cloning and sequencing of xalA gene was first described in Paenibacillus alginolyticus XL-1, encoding pyruvated mannose-specific xanthan lyase. The mature enzyme could be expressed functionally in Escherichia coli showing no activity on depyruvated xanthan like the native enzyme [Ruijssenaars et al., 2000]. Soon after, another gene for the xanthan lyase was cloned from Bacillus sp. strain GL1 in which the lyase was induced by xanthan [Hashimoto et al., 2001]. Pyruvated mannose-specific xanthan lyases have been purified from a salt-tolerant mixed culture and Bacillus sp. [Ahlgren, 1991; Hashimoto et al., 1998b]. Mixed or pure bacterial cultures grown on xanthan generally produced a mix of xanthandegrading enzymes [Hashimoto et al., 1998b; Ruijssenaars et al., 1999]. Cadmus et al. [1982] described the isolation of a xanthan-degrading bacterium. The xanthanase occurred in culture was a mixture of the enzymes attacking all of the side chain linkages in the xanthan molecules, including the xanthan lyase, α-D-mannosidase, and unsaturated glucuronyl hydrolase. They found no depolymerase activity in their cultures because the β-1,4-linked glucan backbone remained intact. Also the enzymes excreted by Paenibacillus alginolyticus XL-1 only removed residues from the xanthan side chains, whereas long stretches of the β1,4-glucan remained intact [Ruijssenaars et al., 1999].

Biodegradation of Polysaccharide Sourced from Virulence Factor…

25

Recently a xanthooligosaccharide has been produced by degradation of a virulence factor of xanthan [Liu et al., 2005], which in turn inhibited the cell growth of the xanthan-producing phytopathogen Xanthomonas campestris pv. campestris [Qian et al., 2006]. Such inhibitory activity was greatly dependent on their hydrolytic degree, which was critical for microorganism inhibition [Jeon et al., 2001]. It was suggested that the degraded xanthan was potentially valuable biological control agent, because Xanthomonas campestris pv. campestris is one of the most important phytopathogen causing crucifer black rot, and the xanthan is its important virulence determinant.

8) Biodegradation of alginate Alginate is a linear anionic copolymer composed of (1,4) linked β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). As shown in Figure 10, they consist of the alternation of homopolymeric blocks of poly-β-1,4-D-mannuronic acid (referenced to MM blocks, a), of homopolymeric blocks of poly-α-1,4-L-guluronic acid (GG blocks, b), and of heteropolymeric blocks with random arrangements of both monomers (MG blocks, c). The proportion and sequence of the block structures vary greatly in alginate molecules, depending on alginates derived from the seaweed sources [Gacesa, 1988]. Like other polysaccharide derived from fungi and plant [Sharp et al., 1984a; Shibuya and Minami, 2001], alginate is also the cell wall constituent of brown seaweeds and as an exopolysaccharide produced by pathogen during infection process [Gacesa, 1988]. One of the critic feature of alginate is having many negative charges occurred in alginate molecules. It has been proved that alginate produced by brown seaweeds is not acetylated [Onsøyen, 1996]. The acetylated form of alginate is synthesized by certain bacteria, such as mucoid cells of Pseudomonas aeruginosa and Azotobacter vinelandii [Cote and Krull 1988; Pier, 1998]. Alginate has also been found to function as a major virulence factor of several pathogenic bacteria during the infectious process such as Pseudomonas aeruginosa, Pseudomonas syringae [Keith et al., 2003; Peñaloza-Vázquez et al., 2004; Pier, 1998]. Marine alga alginate is widely used in food, cosmetics and pharmaceutical industries owning to its gelling ability, stabilizing properties and high viscosity [Ci et al., 1999]. However, the increasing attention has been paid to alginate oligomers produced by alginate lyases, because it has been revealed that alginate hydrolysates and their derivatives exhibit many important bioactivities, such as stimulating the growth of plant root or Bifidobacterium [Akiyama et al., 1992], and causing cytotoxic cytokine production in human mononuclear cells [Iwamoto et al., 2003]. Alginate lyase also has attracted public attention recently because the enzymatic degradation of alginate expands the potential application of this polysaccharide [Hu et al., 2004a, 2004b]. Depolymerized alginates with low molecular weight act like oligosaccharins in their ability to regulate physiological process in plants [Hu et al., 2004a].

26

Xianzhen Li and Xiaoyi Chen

a

-

COO

O HO

M

b

HO

O

OH

O

COOO

OH - O COO

COO-

OH

G

OH

O

O

O

OH

GO

O HO

COO- O OH

M

OH COO-

O

OH

GO O

O

O

G

OH COO-

COO-

OH

c

O

OH

OH

O

G

O HO

M

COO- O M OH

O HO

M

O OH

O

OH COO-

Figure 10. Chemical structure of alginates from brown algae.

Alginates can be degraded enzymatically by alginase, however there are no other alginate hydrolyase being reported except alginate lyase, which catalyze the β-elimination of the 4-Olinked glycosidic bond to form a unsaturated double bond between C-4 and C-5. In this lytic action molecule containing 4-deoxy-L-erythro-hex-4-enepyranosyluronate at the nonreducing end was generated [Gacesa et al., 1989]. As already indicated, alginate lyases are of widespread occurrence, being found in marine gastropods, bacteriophage, various marine microorganisms and some soil. Up to date, alginate lyase has been found to occur in many microorganism, such as Alginovibrio aquatilis, Bacillus circulans, Dendryphiella salina, Enterobacter cloacae, Klebsiella aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas alginovora, etc. [Boyd and Turvey, 1977; Boyen et al., 1990; Caswell et al., 1989; Hansen et al., 1984; Linker and Evans, 1984; Nibu et al., 1995; Shimokawa et al., 1997; Stevens and Levin, 1977]. Not all of the reported alginate lyases have been extensively studied, but those which have been characterized seem to be capable of degrading both bacterial and/or algal alginates and partially hydrolyzed polyuronic acid substrates [Davidson et al., 1976; Dunne and Buckmire, 1985; Hansen et al., 1984]. We recently isolated a bacterium of Flavobacterium sp. LXA as a high alginate-degrading enzyme producer from decayed seaweed. Alginate could be decomposed controllably by alginase from the strain LXA to form alginooligosaccharide. It has been found that some of those oligosaccharides are elicitor-active on plants, inducing the accumulation of phytoalexin in the soybean cotyledon (unpublicated results). The alginate lyases have an absolute specificity for either a D-mannuronate or an Lguluronate residue at the non-reducing side of the bond to be cleaved, although usually there is no restriction on the uronic acids present at the reducing side [Boyd and Turvey, 1977]. Moreover, most of the alginate lyases are randomly endolytic enzymes, whereas some with

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exo-activity have also been reported [Doubet and Quantrano 1984]. To date, three types of alginate lyases have been identified [Østgaard, 1993], the specificities of which are defined in terms of the preference for polymannorate block or polyguluronate block. It was difficult to determine which linkage bond of the M-G, the G-M or mixed random PolyMG being degraded. The alginate lyases found in Photobacterium sp. and Pseudomonas aeruginosa are representatives of poly M lyase [Linker and Evans, 1984; Romeo and Preston, 1986], and those found in Klebsiella aerogenes are representatives of poly G lyase. [Lange et al., 1989] The alginate lyase purified from Azotobacter vinelandii, Alteromonas sp. strain H-4 and Vibrio sp. 510-64 was shown to be capable of degrading both poly M and poly G, which was similar to an enzyme from Bacillus circulans [Ertesvåg et al., 1998; Hu et al., 2006; Sawabe et al., 1997]. The efficiency of degradation depends on the block type [Larsen et al., 1993]. The polymannuronate specific lyases from Dolabella yielded incomplete degradation of substrates, the main products being di-and tri-uronides but no monomeric material [Nisizawa et al., 1968]. It was found that Sphingomonas sp. strain A1 depolymerized alginate by three types of cytoplasmic alginate-depolymerizing enzymes (alginate lyases A1-I, A1-II, and A1-III) [Hashimoto et al., 2000]. Briefly, A1-I was active on acetylated and non-acetylated alginates. A1-II preferred polyG and non-acetylated alginate produced by brown seaweeds. A1-III efficiently liquefied polyM and acetylated alginates produced by mucoid cells of Pseudomonas aeruginosa [Yoon et al., 2000]. These three alginate lyases produced di- and trisaccharides from alginate as major final products [Hashimoto et al., 1998b; Yoon et al., 2000], which implied that the cells of Sphingomonas sp. strain A1 had an additional enzyme responsible for the degradation of alginate oligosaccharides to the constituent monosaccharides. Although several bacterial and algal alginate can be degraded by the enzymes, the specificity of the alginate lyase was not identical to different source of alginate. When compared to other polysaccharide lyases, the mode of action of enzymes degrading alginate is further complicated by the differences in distribution of monosaccharide residues in different substrates. Thus, alginates with the same or very similar mannuronate:guluronate ratios can have very great differences in monosaccharide sequence and in the frequency of adjacent residues. It is true that the choice of enzyme sources and reaction conditions affect the end products. Therefore, the elucidation of the substrate specificities of alginate lyases toward different kinds of oligouronic acids is important for the preparation of desired oligouronic acids by enzymatic degradation of alginate.

5. WELL-KNOWN OLIGOSACCHARIDES AND THEIR STRUCTURAL EFFECT ON BIOLOGICAL ACTIVITIES Higher plants have the ability to initiate various defense reactions, such as hypersensitive responses, production of phytoalexins and antimicrobial proteins, and reinforcement of cell walls when they are infected by various pathogens [Dangl and Jones, 2001]. They can distinguish self and non-self, or detect specific pathogens through the perception of signal molecule elicitors [Kaku et al., 2006]. Fragments of cell surface macromolecules typical of microorganisms such as cell wall polysaccharides often serve as a potential elicitor to induce

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defense reactions. Oligosaccharides as elicitor molecules have been shown to induce defense responses in plant cells [Hahn, 1996]. Some oligosaccharides produced artificially by the enzymatic fragmentation of the constituent of plant or fungi cell wall, as well as the virulence factor of extracellular polysaccharides produced by pathogen, have been demonstrated to be elicitor- or antimicrobe-active [Boudart et al., 1998; Fry et al., 1993b; Liu et al., 2005; Qian et al., 2006]. Their size, polyanionic or polycationic characteristics, and molecular shape are important structural features for the biological activity of oligosaccharides [Aldington et al., 1991].

1) β-glucan-derived oligosaccharides Oligosaccharide elicitors derived from the β-glucans of pathogenic mycetes have been very well characterized, in which a branched hepta-β-glucoside generated from Pseudomonas sojae glucan by partial hydrolysis is the most active elicitor in soybean cells [Sharp et al., 1984a]. Plant cells can recognize the specific features of the hepta-β-glucoside structure, including all three non-reducing terminal glucosyl residues and their spacing along the backbone of the molecule [Cheong et al., 1991]. However, the hepta-β-glucoside did not act as an elicitor in tobacco cells, but a linear β-1,3-linked glucooligosaccharide (laminari oligosaccharides) was elicitor-active on tobacco cells [Klarzynski et al., 2000]. Moreover, it has not been demonstrated any β-glucan fragment to be elicitor-active on monocots up to now. Inui et al. [1997] reported the induction of chitinase and PAL activity in cultured rice cells by laminari hexaose. Recently, Yamaguchi et al. [2000] reported that a reduced pentasaccharide (glucopentaose) derived from the β-glucan of Magnaporthe grisea (Pyricularia oryzae) can induce phytoalexin biosynthesis as elicitor in rice cells at 10 nM concentrations. Whereas its structure is quite different compared with hepta-β-glucoside. Hepta-β-glucoside is a 1,6-linked β-glucooligosaccharide with branches at the C-3 of two 6linked glucosyl residues, but glucopentaose is a 1,3-linked β-glucooligosaccharide with branch at the C-6 of a 3-linked glucosyl residue. Kobayashi et al. [1993] examined the influence of the terminal group of glucan oligosacxcharides on the induction of phytoalexin biosynthesis in alfalfa by comparing a pyridylaminated hepta-β-1,3–1,6 linear glucan with the non-modified hepta-β-glucoside, and found the latter being far less active than the former. The similar results were obtained in the bean cotyledon and the essential minimal structure for biological activity was shown to be a β-1,3–1,6 triglucoside [Tai et al., 1996a, b]. An elicitor active β-1,3-, 1,6-oligoglucans with DP of between 8 and 17 on tobacco was isolated from Alternaria alternata, in which the 1,6linked and non-reducing terminal residues are essential for the elicitor activity. Its activity was about 1000 times more potent than that of laminarin [Shinya et al., 2006]. Degree of polymerization also is an important attribute for biological activities of oligosaccharides. In general, oligosaccharides able to induce a biological response have a DP higher than 4 [Darvill et al., 1992]. An α-(1,3)-linked D-glucan with an average DP of 23 glucose units inhibited completely local lesion development of potato virus Y on Nicotiana tabacum [Singh et al., 1970]. β-1,3-Linked glucan oligomers with a DP over 4 stimulate the release of chitinase, and DP 6 trigger the expression of PAL activity in rice cells [Inui et al., 1997].

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Three non-reducing terminal glucosyl residues, as well as the distribution of the sidechains along the backbone of the molecule, are essential for the elicitor activity [Cheong et al., 1993, 1991] when using a family of chemically synthesized oligo-β-glucosides ranging in size from hexamer to decamer for elucidation elicitor activity. In contrast, the reducing terminal glucosyl residue of the hepta-β-glucoside elicitor is not essential for activity [Cheong et al., 1991].

2) Chitin and chitosan oligosaccharides Chitin/chitosan oligosaccharides, chitooligosaccharide, can inhibit the growth of some strains of bacteria, fungi, and yeasts [Kendra and Hadwiger, 1984; Roller and Covill, 1999; Sudarshan et al., 1992; Wang, 1992]. The reported microorganisms inhibited by chitooligosaccharides include Bacillus subtilis, Candida sp., Enterobacter sakazakii, Escherichia coli, Lactobacillus sp., Listeria monocytogenes, Micrococcus luteus, Pseudomonas aerginosa, Rhodotorula sp., Salmonella typhimurium, Staphylococcus aureus, Staphylococus epidermidis, Streptococus mutans, Streptococcus faecalis, etc. [Jeon et al., 2001; No et al., 2002; Rhoades & Roller, 2000; Tsai et al., 2000]. The antimicrobial activity of chitooligosaccharides is regulated by the subtle difference in oligosaccharide structure such as degrade of polymerization, charge number and distribution, nature of chemical modification to the molecule, and type of microorganism [Chung et al., 2004; Gerasimenko et al., 2004; Jeon et al., 2001; Muzzarelli, 1996; Park et al., 2004a, 2004b; Tsai et al., 2002]. Uchida et al. [1989] observed that chitooligosaccharides mainly with a DP of 4-6 possessed maximal antimicrobial and antifungal effects, while those mainly with a DP of 3-4 showed no activity. It was found that chitooligosaccharide with average molecular weight of less than 2200 Da was hard to suppress microbial growth, but those with molecular weight of around 5500 Da suppressed the cell growth. In another study, Kendra and Hadwiger [1984] examined the extent to which DP can be reduced before antifungal activity was adversely affected using phytopathogen Fusarium solani. The shortest chitooligosaccharide exhibiting the maximum antifungal activity was the heptamer and then the antimicrobial activity decreased with chain length, where the dimer and trimer were inactive. Antibacterial activity of chitosan oligomer is generally superior to chitin oligomer because chitosan possesses a lot of polycationic amines, facilitating their binding on the bacterial cell surface with the negatively charged residues of macromolecules, and subsequently inhibiting bacterial growth [Young and Kauss, 1983]. The death rate of bacterial cells tends to increase upon the increase in degree of deacetylation of chitooligosaccharides [Tsai et al., 2002]. In most cases, 85-95% deacetylation has shown to be responsible for performing the highest antibacterial activity [Chung et al., 2004]. Also the increase in antibacterial activity of chitooligosaccharides is correlated with the arising molecular weight in the same deacetylation case [Park, et al., 2004b]. The charge distribution of chitooligosaccharides is in conjunction with its antifungal activity [Hirano & Nagao, 1989]. Structural modification of chitooligosaccharides by introducing positively charged groups can improve the antibacterial activity.

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In addition, charge distribution of bacterial cell wall plays a considerable role in antibacterial activities. Chung et al. [2004] studied the cell surface characteristics and revealed a close relationship between hydrophilicity and negative charge distribution of bacterial cell surface. The negative charge distribution on cell surface of Gram-negative bacteria was higher than that of Gram-positive bacteria, leading to a higher hydrophobicity on former cell surface. Moreover, inhibition degree of chitooligosaccharides against tested bacteria fits very well the order of high negatively charged Gram-negative bacteria to less negatively charged Gram-positive bacteria [Wang, 1992].

3) Pectin oligosaccharide Pectin oligosaccharide, oligogalacturonides, derived from pectic polysaccharides of plant cell walls have also been known to act as elicitors to induce biosynthesis of phytoalexins, proteinase inhibitors, and lignification [Bruce and West, 1989; Dixon et al., 1989; Farmer et al., 1991]. In general, oligogalacturonides is the most active only if the number of galacturonic acid residues is not lower than 10 [Cervone et al., 1989]. Whereas Reymond et al. [1995] showed that the ability of oligogalacturonides to induce protein phosphorylation increased with the size of the oligogalacturonides even to a DP more than 20. Oligogalacturonides usually require higher concentrations to show elicitor activity compared to chitin- or glucan-oligomers. Compared with the underived oligogalacturonides, the modified oligogalacturonides on the terminal residues reduce the biological activity. The introduction of a 4,5-unsaturated bond in the non-reducing terminal residues by pectate lyase can lower the most active size of oligogalacturonides from DP 10 to 12 [Spiro et al., 1998]. Although some oligogalacturonides able to stimulate defense response have been characterized, a little is known about signal transduction in these systems. Some evidences supported that these molecules activated defense genes in plants via the octadecanoic pathway, which increases the jasmonic acid level in leaves of tomato plants through an intermediary formation of linolenic acid hydroperoxidase [Doares et al., 1995]. The increase of jasmonic acid levels leads to the transcriptional activation of defense genes [Doares et al., 1995]. Up to date, there has been no reports of elicitor activity of oligogalacturonides in monocot plants, which is identical to the much lower content of pectic polysaccharides occurred in monocot cell walls.

4) Alginate-derived oligosaccharide Most known bioactive oligosaccharides are sourced from the structural components of pathogen or plant cell walls [Fry et al., 1993b]. And an active oligosaccharide has also been degraded from a virulence factor of extracellular polysaccharide produced by phytopathogen [Liu et al., 2005; Qian et al., 2006]. Alginate is both the component of algal cell walls and the virulence factor of some pathogen [Gacesa, 1988; Peñaloza-Vázquez et al., 2004], therefore it should be also elicitor-active on host plants or have inhibitory effect on pathogen. In fact, alginooligosaccharide derived from algal cell walls was found to be potential signals for the induction of chitinase expression in plant cells [Fry et al., 1993b]. After being stimulated with alginooligosaccharide, the phytoalexin accumulation in soybean cotyledon was observed, and

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such elicitor activity was related to the DP of alginooligosaccharides. The maximal production of phytoalexin was obtained when the alginooligosaccharide with DP of 6 was applied on the wound cotyledon (unpublicated data). The suppression of PAL gene expression has been proved to lead the increased fungal disease susceptibility in plants [Maher et al., 1994]. Thus the increase in PAL activity was a direct response of host plants to attempted penetration by the pathogens, which was associated with the resistance of plant to fungal diseases [Hahlbrock and Scheel, 1989; Shiraishi et al., 1995]. When alginooligosaccharide was applied on the surface of the wound soybean cotyledon, the increase in PAL could be detected instantly, and the peak value of PAL activity occurred at 1.5-2 h. After that there was another peak PAL activity appeared at 4-5 h.

6. CONCLUSION Polysaccharides can be degraded by different enzymes sourced from various living organisms, including microorganism, plant and animal. The carbohydrate-degrading enzymes can be classified as hydrolyase and lyase, which catalyses the degradation of polysaccharides in endo- or exo-type respectively. The enzymes degrading polysaccharides showed the specificity on their substrates. Some oligosaccharides, produced by enzymatic degradation of polysaccharides sourced from main constituents of pathogen or plant cell walls and virulence factors of exopolysaccharides of pathogen, have been shown biological activity, such as elicitor activity and antimicrobial activity. Such bioactivity on plant cells greatly depends on the molecular weight or degree of polymerization, the charge distribution, the arrangement of branch side chain, and terminal groups, etc.. The biodegradation of polysaccharides and their products, bioactive oligosaccharides, play a significant role in protect phytopathogen infection.

7. REFERENCES Ahlgren JA (1991) Purification and characterization of a pyruvated-mannose-specific xanthan lyase from heat-stable, salt-tolerant bacteria. Appl Environ Microbiol, 57, 2523-2528. Ahlgren JA (1993) Purification and properties of a xanthan depolymerase from a heat-stable salt-tolerant bacterial consortium. J Ind Microbiol, 12, 87-92. Aiba S (1994a) Preparation of N-acetylchitooligosaccharides by lysozymic hydrolysates of partially N-acetylated chitosans. Carbohyd Res, 261, 297-306. Aiba S (1994b) Preparation of N-acetylchitooligosaccharides by hydrolysis of chitosan with chitinase followed by N-acetylation. Carbohyd Res, 256, 323-328. Akiyama H, Endo T, Nakakita R, Murata K, Yonemoto Y & Okayama K (1992) Effect of depolymerized alginates on the growth of Bifidobacteria. Biosci Biotechnol Biochem, 56, 355-356. Aldington S, McDougall GJ & Fry SC (1991) Structure-activity relationships of biologically active oligosaccharides. Plant Cell Environ, 14, 625-636

32

Xianzhen Li and Xiaoyi Chen

Alvarez ME, Pennell RI, Meijer PJ, Ishikawa A, Dixon RA & Lamb C (1998) Reactive oxygen intermediates mediate systemic signal network in the establishment of plant immunity. Cell, 92, 773-784 Annon (1996) Pesticide statistics. Pestic Res J, 8, 106-113. Araki T, Higashimoto Y & Morishita T (1999) Purification and characterization of kappacarrageenase from a marine bacterium Vibrio sp. CA-1004. Fish Sci, 65, 937-942. Arvanitoyannis IS, Nakayama A & Aiba S (1998) Chitosan and gelatin based edible films: state diagrams, mechanical and permeation properties. Carbohyd Polym, 37, 371-382. Ayers AR, Ebel J, Valent B & Albersheim P (1976a) Host-pathogen interactions: X. Fractionation and biological activity of an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae. Plant Physiol, 57, 760-765. Ayers AR, Valent B, Ebel J & Albersheim P (1976b) Host-pathogen interactions: XI. Composition and structure of wall-released elicitor fractions. Plant Physiol, 57, 766-774. Azarkan M, Amrani A, Nijs M, Vandermeers A, Zerhouni S, Smoulders N & Looze Y (1997) Carica papaya latex is a rich source of class II chitinase. Phytochemistry, 46,1319-1325. Barnard M, Padgitt M & Uri ND (1997) Pesticide use and its measurement. Int Pest Control, 39, 161-164. Barbeyron T, Michel G, Potin P, Henrissat B & Kloareg B (2000) Iota-carrageenases constitute a novel family of glycoside hydrolases, unrelated to that of kappacarrageenases J Biol Chem, 275, 35499-35505. Bartnicki-Garcia S (1968) Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annu Rev Microbiol, 22, 87-108. Bell AA (1981) Biochemical mechanisms of disease resistance. Annu Rev Plant Physiol, 32, 21-81 Benhamou N (1996) Elicitor-induced plant defense pathways. Trends plant Sci Rev, 1, 233– 240. Benhamou N, Bélanger RR & Paulitz TC (1996) Induction of differential host responses by Pseudomonas fluorescens in Ri T-DNA-transformed pea roots after challenge with Fusarium oxysporum f. sp. pisi and Pythium ultimum. Phytopathology, 86, 1174-1185. Berensmeier S, Singh SA, Meens J & Buchholz K (2004) Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high alkaline pectate lyase. Appl Microbiol Biotechnol, 64, 560-567. Bishop PD, Pearce G, Bryant JE & Ryan CA (1984) Isolation and characteization of the proteinase inhibitor-inducing factor from tomato leaves. J Biol Chem, 259, 13172-13177. Blumwald E, Aharon GS & Lam BCH (1998) Early signal transduction pathways in plantpathogen interactions. Trends Plant Sci, 3, 342-346 Boller T (1995) Chemoperception of microbial signals in plant cells. Annu Rev Plant Physiol Mol Biol, 46, 189-214. Boot RG, Blommaart EFC, Swart E, Ghauharali-van der Vlugt K, Bijl N, Moe C, Place A & Aerts JMG (2001) Identification of a nivel acidic mammalian chitinase distinct from chitotriosidase. J Biol Chem, 276, 6770-6778. Boudart G, Lafitte C, Barthe JP, Frasez D & Esquerré-Tugayé MT (1998) Differential elicitation of defense responses by pectic fragments in bean seedlings. Planta, 206, 8694.

Biodegradation of Polysaccharide Sourced from Virulence Factor…

33

Bouarab K, Potin P, Correa J, Kloareg B (1999) Sulfated oligosaccharides mediate the interaction between a marine red alga and its green algal pathogenic endophyte. Plant Cell, 11, 1635-1650. Boyac IH, Seker UÖS & Mutlu M (2002) Determination of β-glucan content of cereals with an amperometric glucose electrode. Eur Food Res Technol, 215, 538-541. Boyd J & Turvey JR (1977) Isolation of a poly-α-L-guluronate lyase from Klebsiella aerogenes. Carbohydr Res, 57, 163-171 Boyen C, Kloarag B, Polne-Fuller M & Gibor A (1990) Preparation of alginate lyases from marine mollusks for protoplast isolation in brown algae. Phycologia, 29, 173-181. Bradley DJ, Kjellbom P & Lamb CJ (1992) Elicitor- and wound induced oxidative crosslinking of a proline-rich plant cell wall protein: a novel, rapid defense response. Cell, 70, 21-30 Brady KP, Darvill AG & Albersheim P (1993) Activation of a tobacco glycine-rich protein gene by a fungal glucan preparation. Plant J, 4, 517-524. Bruce RJ & West CA (1989) Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension-cultures of castor bean. Plant Physiol, 91, 889-897. Bukhtojarov FE, Ustinov BB, Salanovich TN, Antonov AI, Gusakov AV, Okunev ON & Sinitsyn AP (2004) Cellulase complex of the fungus Chrysosporium lucknowense: isolation and characterization of endoglucanases and cellobiohydrolases. Biochemistry (Mosc.), 69, 542-551. Cadmus MC, Jackson LK, Burton KA, Plattner RD & Slodki ME (1982) Biodegradation of xanthan gum by Bacillus sp.. Appl Environ Microb, 44, 5-11. Carpita N & McCann M (2000) The cell wall. In: Buchanan B, Gruissem W & Jones R (Eds), Biochemistry and Molecular Biology of Plants, pp. 52-108, Somerset: John Wiley & Sons, Inc. Cartwright D, Langcake P, Pryce RJ Leworthy DP & Ride JP (1977) Chemical activation of host defense mechanisms as a basis for crop protection. Nature, 267, 511-513. Caswell RC, Gacesa P, Lutrell KE & Weightman AJ (1989) Molecular cloning and heterologous expression of a Kiebsiella pneumoniae gene encoding alginate lyase. Gene, 75, 127-134. Cervone F, Hahn MG, De LorenzoG, Darvill A & Albersheim P (1989) Host pathogen interactions. XXXIII. A plant protein converts a fungal pathogenesis factor into an elicitor of plant defense responses. Plant Physiol, 90,542-548. Cheetham NWH & Mashimba ENM (1991) Characterisation of some enzymic hydrolysis products of xanthan. Carbohydr Polym, 15, 195-206. Chen C, Bélanger RR, Benhamou N & Paulitz T (2000) Defense enzymes induced in cucumber roots by treatment with plant growth promoting rhizobacteria (PGPR) and Pythium aphanidermatum. Physiol Mol Plant Pathol, 56, 13-23. Chen YM, Chung YC, Wang LW, Chen KT & Li SY (2002) Antibacterial properties of chitosan in waterborne pathogen. J Environ Sci Health A, 37, 1379-1390. Cheong J-J, Alba R, Cǒté F, Enkerli J & Hahn MG (1993) Solubilization of unctional plasma membrane-localized hepta-β-glucoside elicitor binding proteins rom soybean. Plant Physiol,103, 1173-1182.

34

Xianzhen Li and Xiaoyi Chen

Cheong J-J, Birberg W, Fugedi P, Pilotti A, Garegg PJ, Hong N, Ogawa T & Hahn MG (1991) Structure-activity relationships of oligo-β-glucoside elicitors of phytoalexin accumulation in soybean. Plant Cell, 3, 127-136. Cheong JJ & Hahn MG (1991) A specific high-affnity binding site for the hepta-β-glucoside elicitor exists in soybean membranes. Plant Cell, 3, 137-147. Choi BK, Kim KY, Yoo YJ, Oh SJ, Choi JH & Kim CY (2001) In vitro antimicrobial activity of chitooligosaccharide mixture against Actinobacillus actinomycetemcomitans and Streptococcus mutans. Int J Antimicrob Agents, 18, 553-557. Chou F-L, Chou H-C, Lin Y-S, Yang B-Y, Lin N-T, Weng S-F & Tseng Y-H (1997) The Xanthomonas campestris gumD gene required for synthesis of xanthan gum is involved in normal pigmentation and virulence in causing black rot. Biochem Biophys Res Commun, 233, 265-269. Christensen BE, Myhr MH & Smidsrod O (1996) Degradation of double-stranded xanthan by hydrogen peroxide in the presence of ferrous ions: comparison to acid hydrolysis. Carbohydr Res, 280, 85-99. Chung Y-C, Su Y-P, Chen C-C, Jia G, Wang H-L, Wu JCG & Lin J-G (2004) Relationship between antibacterial activity of chitosan and surface characteristics of cell wall. Acta Pharmacol Sin, 25, 932-936. Ci SX, Huynh TH, Louie LW, Yong A, Beals BJ, Ron N Tsang WG, Soon-Shiang P & Desai NPJ (1999) Molecular mass distribution of sodium alginate by high-performance sizeexclusion chromatography. Chromatogr A, 864, 199-210. Cosio EG, Feger M, Miller CJ, Antelo L & Ebel J (1996) High-affinity binding of fungal βglucan elicitors to cell membranes of species of the plant family Fabaceae. Planta, 200, 92-99. Côté F & Hahn MG (1994) Oligosaccharins: structure and signal transduction. Plant Mol Biol, 26, 1397-1411. Côté F, Roberts K & Hahn M (2000) Identification of high affinity binding sites for the heptaβ-glucoside elicitor in membranes of the model legumes Medicago truncatula and Lotus japonicus. Planta, 211, 596-605. Cote GL & Krull LH (1988) Characterization of the extracellular polysaccharides from Azotobacter chroococcum. Carbohydr Res, 181, 143-152 Coughlan MP & Hazlewood GP (1993) β-1,4-D-Xylan-degrading enzyme system: biochemistry, molecular biology and applications. Biotechnol Appl Biochem, 17, 259289. Courtin CM & Delcour JA (2001) Relative activity of endoxylanses towards waterextractable and water-unextractable arabinoxylan. J Cereal Sci, 33, 301-312. Daas PJH, Boxma B, Hopman AMCP, Voragen AGJ & Schols HA (2001) None sterified galacturonic acid sequence homology of pectins. Biopolymers, 58, 1-8. Daas PJH, Meyer-Hansen K, Schols HA, De Ruiter GA & Voragen AGJ (1999) Investigation of the non-esteri Wed galacturonic acid distribution in pectin with endopolygalacturonase, Carbohydr Res, 318, 135-145. Daayf F, Bel–Rhlid R & Bélanger RR (1997) Methyl ester of pcoumaric acid: a phytoalexinlike compound from long English cucumber leaves. J Chem Ecol, 23, 1517-1526. Dangl JL & Jones JDG (2001) Plant pathogens and integrated defense responses to infection. Nature 411, 826-833.

Biodegradation of Polysaccharide Sourced from Virulence Factor…

35

Daniels MJ, Collinge DB, Dow JM, Osbourn AE & Roberts IN (1987) Molecular biology of the interaction of Xanthomonas campestris with plants. Plant Physiol Biochem, 25, 353359. Darvill AG, Augur C, Bergmann C, Carlson RW, Cheong JJ, Eberhard S, Hahn MG, Lo VM, Marfá V, Meyer B, Mohnen D, O’Neill MA, Spiro MD, van Halbeek H, Zork WS & Albersheim P (1992) Oligosaccharins: oligosaccharides that regulate growth, development and defense responses in plants. Glycobiology, 2, 181-198. Datta K, Velazhahan R, Oliva N, Ona I, Mew T, Khush GS, Muthukrishnan S & Datta SK (1999) Over expression of cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. Theor Appl Genet, 98, 1138-1145. Davidson IW, Sutherland IW & Lawson CJ (1976) Purification and properties of an alginate lyase from a marine bacterium. Biochem J, 159, 707-713. De Ruiter GA & Rudolph B (1997) Carrageenan biotechnology. Trends Food Sci Tech, 12, 389-395. Debeire P, Priem B, Strecker G and Vignon M (1990) Purification and properties of an endo1,4-xylanase excreted by a hydrolytic thermophilic anaerobe Clostridium thermolacticum: A proposal for its action mechanism on larchwood 4-Omethylglucuronoxylan. Eur J Biochem, 187, 573-580. Delgado L, Trejo BA, Huitron C & Aguilar G (1992) Pectin lyase from Aspergillus sp. CHY-1043. Appl Microbiol Biotechnol, 39, 515-519. Desmet T, Cantaert T, Gualfetti P, Nerinckx W, Gross L, Mitchinson C & Piens K (2007) An investigation of the substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina. FEBS J, 274, 356-363. Dixon RA, Harrison MJ & Lamb CJ (1994) Early events in the activation of plant defense responses. Annu Rev Phytopathol, 32: 479-501. Dixon RA, Jennings AC, Davies IA, Gerrish C & Murphy DL (1989) Elicitor-active components from French bean hypocotyls. Physiol Mol Plant Pathol, 3, 99-115. Doares SH, Syrovets L, Weiler EW & Ryan CA (1995) Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoic pathway. Proc Nat/Acad Sci USA, 92, 4095-4098. Dunne WM & Buckmire FLA (1985) Partial purification and characterization of a polymannuronic acid depolymerase produced by a mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis. Appl Environ Microbiol, 50, 562569. Edwards M, Dea ICM, Bulpin PV & Reid JSC (1986) Purification and properties of a novel xyloglucan-specific endo-1-4-β-D-glucanase from germinated nasturtium seeds (Tropaeolum majus L.). J Biol Chem, 261, 9489-9494. Eilenberg H, Pnini-Cohen S, Schuster S, Movtchan A & Zilberstein A (2006) Isolation and characterization of chitinase genes from pitchers of the carnivorous plant Nepenthes khasiana. J Exp Bot, 57, 2775-2784. EI-Ghaouth A, Arul J, Asselin A & Benhamou N (1992) Antifungal activity of chitosan on two post-harvest pathogen of straw berry fruits. Phytooath, 82, 398-402. Ekeberg D, Knutsen SH, Sletmoen M (2001) Negative-ion electrospray ionization mass spectrometry (ESI-MS) as a tool for analyzing structural heterogeneity in kappacarrageenan oligosaccharides. Carbohydr Res, 334, 49-59.

36

Xianzhen Li and Xiaoyi Chen

Ertesvåg H, Erlien F, Skjåk-Bræk G, Rehm BHA & Valla S (1998) Biochemical properties and substrate specificities of a recombinantly produced Azotobacter vinelandii alginate lyase. J Bacteriol, 180, 3779-3784 Escott GM & Adams DJ (1995) Chitinase activity in human serum and leukocyte. Infect Immun, 63, 4770-4773. Farmer EE, Moloshok TD, Saxton MJ & Ryan CA (1991) Oligosaccharide signaling in plants. Specificity of oligouronide-enhanced plasma membrane protein phosphorylation. J Biol Chem, 266, 3140-3145. Flach J, Pilet P-E & Jollefs P (1992) What's new in chitinase research? Experientia, 48, 701716. Frindlender M, Inbar J & Chet I (1993) Biological control of soilborne plant pathogens by a

β -1,3-glucanase producing Pseudomonas cepacia. Soil Biol Biochem, 25, 1211-1221. Fry SC, Aldington S, Hetherington PR & Aitken J (1993a) Oligosaccharides as signals and substrates in the plant cell wall. Plant Physiol, 103, 1-5. Fry SC, York WS, Albersheim P, Darvill A, Hayashi T, Joseleau J-P, Kato Y, Lorences EP, Maclachlan GA, McNeil M, Mort AJ, Reid JSG, Seitz HU, Selvendran RR, Voragen AGJ & White AR (1993b) An unambiguous nomenclature for xyloglucan-derived oligosaccharides, Physiol Plant, 89, 1-3. Gacesa P. (1988) Alginates. Carbohydr Polymer 8, 161-182. Gacesa P, Caswell RC & Kille P (1989) Bacterial alginases. Antibiot Chemother, 42, 67-71. García-Ochoa F, Santos VE, Casas JA & Gómez E (2000) Xanthan gum: production, recovery, and properties. Biotechnol Adv, 18, 549-579 Gatz A (1997) Chemical control of gene expression. Annu Rev Plant Physiol Plant Mol Biol, 48, 89-108. Gerasimenko DV, Avdienko ID, Bannikova GE, Zueva OY & Varlamov VP (2004) Antibacterial effects of water-soluble low molecular-weight chitosans on different microorganisms. Appl Biochem Microb, 40, 253-257. Greenberg JT (1997) Programmed cell death in plant-pathogen interactions. Annu Rev Plant Physiol Plant Mol Biol, 48, 525-545. Grishutin SG, Gusakov AV, Markov AV, Ustinov BB, Semenova MV & Sinitsyn AP (2004) Specific xyloglucanases as a new class of polysaccharide-degrading enzymes. Biochimica et Biophysica Acta, 1674, 268-281. Gruppen H, Hamer RJ & Voragen AGJ (1992) Water unextractable cell wall material from wheat flour. II. Fractionation of alkali-extracted polymers and comparison with waterextractable arabinoxylans. J Cereal Sci, 16, 53-67. Hahlbrock K. & Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol, 40, 347-369. Hahn MG (1996) Microbial elicitors and their receptors in plants. Annu Rev Phytopathol, 34, 387-412. Hansen JB, Doubet RS & Ram J (1984) Alginase production by Bacillus circulans. Appl Environ Microbiol, 47, 704-709. Hashimoto W, Miki H, Tsuchiya N, Nankai H & Murata K (1998a) Xanthan lyase of Bacillus sp. strain GL1 liberates pyruvylated mannose from xanthan side chains. Appl Environ Microb, 64, 3765-3768.

Biodegradation of Polysaccharide Sourced from Virulence Factor…

37

Hashimoto W, Miki H, Tsuchiya N, Nankai H & Murata K (2001) Polysaccharide lyase: molecular cloning, sequencing, and over expression of the xanthan lyase gene of Bacillus sp. strain GL1. Appl Environ Microbiol, 67, 713-720 Hashimoto W, Miyake O, Momma K, Kawai S & Murata K (2000) Molecular identification of oligoalginate lyase of Sphingomonas sp. strain A1 as one of the enzymes required for complete depolymerization of alginate. J Bacteriol, 182, 4572-4577 Hashimoto W, Okamoto M, Hisano T, Momma K & Murata K (1998b) Sphingomonas sp. A1 lyase active on both poly-β-D-mannuronate and heteropolymeric regions in alginate. J Ferment Bioeng, 86, 236-238. Hasper AA, Dekkers E, van Mil M, van de Vondervoort PJI & de Graaff LH (2002) EglC, a new endoglucanase from Aspergillus niger with major activity towards xyloglucan, Appl Environ Microbiol, 68, 1556-1560. Hatada Y, Saito K, Koike K, Yoshimatsu T, Ozawa T, Kobayashi T & Ito S (2000) Deduced amino-acid sequence and possible catalytic residues of a novel pectate lyase from an alkalophilic strain of Bacillus. Eur J Biochem, 267, 2268-2275. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J, 280, 309-316. Herron SR, Benen JAE, Scavetta RD, Visser J & Jurnak F (2000) Structure and function of pectic enzymes: virulence factors of plant pathogens. Proc Natl Acad Sci USA, 97, 87628769. Hirano S & Nagao N (1989) Effects of chitosan, pectic acid, lysozyme and chitinase on the growth of several phytopathogens. Agric Biol Chem, 53, 3065-3066. Hoell IA, Dalhus B, Heggset EB, Aspmo SI & Eijsink VGH (2006) Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes. FEBS J, 273 4889-4900 Horowitz ST, Roseman S & Blumenthal HJ (1957) The preparation of glucosamine oligosaccharides. I separation. J Am Chem Soc, 79, 5046-5049. Hu XK, Jiang X & Hwang H (2006) Purification and characterization of an alginate lyase from marine bacterium Vibrio sp. mutant strain 510-64. Curr Microb, 53, 135-140 Hu XK, Jiang XL, Hwang HM, Liu SL & Guan HS (2004a) Promotive effects of alginatederived oligosaccharide on maize seed germination. J Appl Phycol, 16, 73-76 Hu XK, Jiang XL, Hwang HM, Liu SL & Guan HS (2004b) Antitumour activities of alginatederived oligosaccharides and their substitution derivatives. Eur J Phycol, 39, 67-71 Hunt MD & Ryals JA (1996) Systemic acquired resistance signal transduction. Critical Rev Plant Sci, 15, 583-606. Inui H, Yamaguchi Y & Hirano S (1997) Elicitor actions of N-acetylchitooligosaccharides and laminari oligosaccharides for chitinase and L-phenylalanine ammonia-lyase induction in rice suspension culture. Biosci Biotech Biochem, 61, 975-978. Ishikawa R, Nishimito MS, Fukuchi A & Matsuura K (2004) Effective control of cabbage black rot by valid amycin A and its effect on extracellular polysaccharide production of Xanthomonas campestris pv. campestris. J Pestic Sci, 29, 209-213. Iwamoto Y, Xu X, Tamura T, Oda T & Muramatsu T (2003) Enzymatically depolymerized alginate oligomers that cause cytotoxic cytokine. Biosci Biotechnol Biochem, 67, 258263. Izume M & Ohtakara A (1987) Preparation of D-glucosamine oligosaccharides by the enzymatic hydrolysis of chitosan. Agric Biol Chem, 51, 1189-1191.

38

Xianzhen Li and Xiaoyi Chen

Jabs T, Tschöpe M, Colling C, Hahlbrock K & Scheel D (1997) Elicitor-stimulated ion fluxes and O2 from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. Proc Natl Acad Sci USA, 94, 4800-4805. Jeon YJ & Kim SK (2000) Continuous production of chitooligosaccharides using a dual reactor system. Process Biochem, 35,623-632. Jeon YJ & Kim SK (2001) Effect of antimicrobial activity by chitosan oligosaccharides Nconjugated with asparagines. J Microbiol Biotechnol, 11, 281-286. Jeon Y-J, Park P-J, Byun H-G, Song B-K & Kim S-K (1998) Production of chitosan oligosaccharides using chitin-immobilized enzyme. Korean J Biotechnol Bioeng, 13,147154. Jeon YJ, Park PJ & Kim S (2001) Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohyd Polym, 44, 71-76. Jeon YJ, Shahidi F & Kim SK (2000) Preparation of chitin and chitosan oligomers and their applications in physiological functional foods. Food Rev Int, 61, 159-176. Ji C & Kuc J (1996) Antifungal activity of cucumber β-1,3-glucanase and chitinase. Physiol Mol Plant Pathol, 49, 257-265. Jiang Z-Q, Deng W, Li L-T, Ding C-H, Kusakabe I & Tan S-S (1994) A novel, ultra-large xylanolytic complex (xylanosome) secreted by Streptomyces olivaceoviridis. Biotechnol Lett, 26, 431-436. Johansson L, Virkki L, Maunu S, Lehto M, Ekholm P & Varo P (2000) Structural characterization of water-soluble β-glucan of oat bran. Carbohyd Polym, 42, 143-148. John FSJ, Rice JD & Preston JF (2006) Paenibacillus sp. strain JDR-2 and xynA1: a novel system for methylglucuronoxylan utilization. Appl Environ Microb, 72, 1496-1506. Kaku H, Nishizawa Y, Ishii-Minami N, Akimoto-Tomiyama C, Dohmae N, Takio K, Minami E, & Shibuya N (2006) Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc Nat/ Acad Sci USA, 103, 11086-11091. Kalogeris E, Christakopoulos P, Vrsanska M, Kekos D, Biely P & Macris BJ (2001) Catalytic properties of the endoxylanase I from Thermoascus aurantiacus. J Mol Catal B: Enzym, 11, 491-501. Keen NT & Yoshikawa M (1983) β-1,3-Endoglucanase from soybean releases elicitor-active carbohydrates from fungus cell walls. Plant Physiol, 71, 460-465. Keen NT (2000) A Century of plant pathology: a retrospective view on understanding hostparasite interactions. Annu Rev Phytopathol, 38, 31-48. Keith RC, Keith LMW, Hernández-Guzmán G, Uppalapati SR & Bender CL (2003) Alginate gene expression by Pseudomonas syringae pv. tomato DC3000 in host and non-host plants. Microbiology, 149, 1127-1138. Kendra DF, Christian D & Hadwiger LA (1989) Chitosan oligomers from Fusarium solani pea interactions, chitinase β-glucanase digestion of spore lings and from fungal wall chitin actively inhibit fungal growth and enhance disease resistance. Physiol Mol Plant Pathol, 35, 215-230. Kendra DF & Hadwiger LA (1984) Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Experimental Mycology, 8, 276-281.

Biodegradation of Polysaccharide Sourced from Virulence Factor…

39

Kim JY, Lee JK, Lee TS & Park WH (2003) Synthesis of chitooligosaccharide derivative with quaternary ammonium group and its antimicrobial activity against Streptococcus mutans. Int J Biol Macromol, 32, 23-27. Kim SY, Shon DH & Lee KH (1998) Purification and characteristics of two types of chitosanases from Aspergillus fumigatus. J Microbiol Biotechnol, 8, 568-574. Kirsch C, Hahlbrock K & Kombrink E (1993) Purification and characterization of extracellular acidic chitinase isoenzymes from elicitor-stimulated parsley cells. Eur J Biochem, 213, 419-425. Kittur FS, Kumar ABV & Tharanathan RN (2003) Low molecular weight chitosanspreparation by depolymerization with Aspergillus niger pectinase, and characterization. Carbohyd Res, 338, 1283-1290 Klarzynski O, Plesse B, Joubert JM, Yvin JC, Kopp M, Kloareg B & Fritig B (2000) Linear β-1,3 glucans are elicitors of defense responses in tobacco. Plant Physiol, 124, 10271037. Kloareg K, Potin P, Weinberger F, Correa J & Kloareg B (2001) The Condrus crispus Acrochaete operculata host-pathogen association, a novel model in glycobiology and applied phycopathology. J Appl Phycol, 13, 185-193. Kluskens LD, Van Alebeek GJWM, Voragen AGJ, De Vos WM & Van Der Oost J (2003) Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima. Biochem J, 370, 651-659. Knutsen SH, Myslabodski DE, Larsen B & Usov AI (1994) A modified system of nomenclature for red algal galactans. Bot Mar, 37, 163-169. Knutsen SH, Sletmoen M, Kristensen T, Barbeyron T, Kloareg B & Potin P (2001) A rapid method for the separation and analysis of carrageenan oligosaccharide released by iotaand kappa-carrageenase. Carbohydr Res, 331, 101-106. Kobayashi A, Tai A, Kanzaki H & Kawazu K (1993) Elicitor active oligosaccharides from algal laminaran stimulate the production of antifungal compounds in alfalfa. Natur sch, 48c: 575-579. Kombrink E & Hahlbrock K (1986) Responses of cultured parsley cells to elicitors from phytopathogenic fungi. Plant Physiol, 81, 216-21. Kopp M, Rouster J, Fritig B, Darvill A & Albersheim P (1989) Host-pathogen interactions: XXXII. A fungal glucan preparation protects Nicotianae against infection by viruses. Plant Physiol, 90, 208–216. Kurita K (1998). Chemistry and application of chitin and chitosan. Polym Degrad Stabil, 59, 117-120. Küpper FC, Kloareg B, Guern J & Potin P (2001) Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata. Plant Physiol, 125, 278-291. Kuroiwa T, Ichikawa S, Sato S, Hiruta O, Sato S & Mukataka S (2002) Factors affecting the composition of oligosaccharides produce din chitosan hydrolysis using immobilized chitosanases. Biotechnol Progr, 18, 969-974. Kuroiwa T, Ichikawa S, Sato S & Mukataka S (2003) Improvement of the yield of physiologically active oligosaccharides in continuous hydrolysis of chitosan using immobilized chitosanases. Biotechnol Bioeng, 84, 121-127. Lakhti VM, Kostanova EA & Arbatski NP (1995) Isolation and characterization of multiple forms of papaya latex lysozyme. Appl Biochem Microbiol, 31, 214-220.

40

Xianzhen Li and Xiaoyi Chen

Lange B, Wingender J & Winkler UK (1989) Isolation and characterization of an alginate from Klebsiella aerogenes. Arch Microbiol, 152, 302-308. Larsen B, Hooen K & Ostgaard K (1993) Kinetics and specificity of alginate lyases. Hydrobiologia, 260/261, 557-56l. Lawton MA & Lamb C (1987) Transcriptional activation of plant defense genes by fungal elicitors, wounding and infection. Mol Cell Biol, 7, 335-341. Lee HJ, Shin DJ, Cho NC, Kim HO, Shin SY, Im SY, Lee HB, Chum SB & Bai S (2000) Cloning, expression and nucleotide sequences of two xylanase genes from Paenibacillus sp.. Biotechnol Lett, 22, 387-392. Lee HW, Choi JW, Han DP, Park MJ, Lee NW & Yi DH (1996) Purification and characteristics of chitosanase from Bacillus sp. HW-002. J Microbiol Biotechnol, 6, 1925. Lee HW, Parkb Y-S, Jungb J-S & Shinb W-S (2002) Chitosan oligosaccharides, dp 2-8, have prebiotic effect on the Bifidobacterium bifidium and Lactobacillus sp.. Anaerobe, 8, 319324. Lee S, Choi H, Suh SJ, Doo I-S, Oh K-Y, Choi EJ, Taylor ATS, Low PS & Lee Y (1999) Oligogalacturonic acid and chitosan reduce stomatal aperture by the evolution of reactive oxygen species from guard cells of tomato and commelina communis. Plant Physiol, 121, 147-152. Levine A, Tenhaken R, Dixon R & Lamb C (1994) H2O2 from the oxidative burst or chestrates the plant hypersensitive disease resistance response. Cell, 79, 583-593. Liao C-H (1991) Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida. J Bacteriol, 173, 4386-4393. Linker A & Evans LR (1984) Isolation and characterization of an alginate lyase from mucoid strains of Pseudomonas aeruginosa. J Bacteriol, 159, 958-964. Liu H, Huang C, Dong W, Du Y, Bai X & Li X (2005) Biodegradation of xanthan by newly isolated Cellulomonas sp. LX, releasing elicitor-active xantho-oligosaccharides induced phytoalexin synthesis in soybean cotyledons. Proc Biochem, 40, 3701-3706. Liu JM, Haroun-Bouhedja F & Boisson-Vidal C (2000) Analysis of the in vitro inhibition of mammary adenocarcinoma cell adhesion by sulphated polysaccharides. Anticancer Res, 20, 3265-3271. Maes C, Vangeneugden B & Delcour JA (2004) Relative activity of two endoxylanases towards water-unextractable arabinoxylans in wheat bran. J Cereal Sci, 39, 181-186. Maher EA, Bate NJ, Ni W, Elkind Y, Dixon RA & Lamb CJ (1994) Increased disease susceptibility of transgenic tobacco plants with suppressed levels of preformed phenylpropanoid products. Proc Natl Acad Sci USA, 91, 7802-7806. Markovič O & Janeček S (2001) Pectin degrading glycoside hydrolases of family 28: sequence-structural features, specificities and evolution. Protein Eng, 14, 615-631. Matsumoto T, Sakai F & Hayashi T (1997) A xyloglucan-specific endo-1,4-β-glucanase isolated from auxin-treated pea stems. Plant Physiol, 114, 661-667. Mauch F, Hadwiger LA & Boller T (1984) Ethylene: symptom, not signal for the induction of chitinase and β-1,3-glucanasein pea pods by pathogens and elicitors. Plant Physiol, 76, 607-611.

Biodegradation of Polysaccharide Sourced from Virulence Factor…

41

Maurhofer M, Hase C, Meuwly P, Metraux JP & Defago G (1994) Induction of systemic resistance of tobacco to tobacco necrosis virus by the root-colonizing Pseudomonas fluorescens strain CHAO: influence of the gacA gene and of pyoverdine production. Phytopathology, 84, 139-146. May RM (1985) Evolution of pesticide resistance. Nature, 15, 12-13. McCarthy RE, Kotarski SF & Salyers AA (1985) Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotamicron. J Bacteriol, 161, 493-499. McCleary BV & Codd R (1991) Measurement of (1,3), (1,4)- β-D-glucan in barley and oats: a stream lined enzymic procedure. J Sci Food Agric, 55, 303-312. Mehdy MC (1994) Active oxygen species in plant defense against pathogens. Plant Physiol, 105: 467-472 Melotto E, Greve LC & Labavitch JM (1994) Cell-wall metabolism in ripening fruit. 7. Biologically-active pectin oligomers in ripening tomato (Lycopersicon-Esculentum mill) fruits. Plant Physiol, 106, 575-581. Michel G, Chantalat L, Duee E, Barbeyron T, Henrissat B, Kloareg B & Dideberg O (2001) The κ-carrageenase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of clan-B glycoside hydrolases. Structure, 9, 513-525. Michel G, Flament D, Barbeyron T, Vernet T, Kloareg B & Dideberg O (2000) Expression, purification, crystallization and preliminary X-ray analysis of the iota-carrageenase from Alteromonas fortis. Acta Crystallogr D, 56, 766-768. Miyazaki K, Hirase T, Kojima Y and Flint HJ (2005) Medium- to large-sized xylooligosaccharides are responsible for xylanase induction in Prevotella bryantii B. Microbiology, 151, 4121-4125. Mohan R, Vijayan P & Kolattukudy PE (1993) Developmental and tissue specific expression of a tomato anionic peroxidase (tap 1) gene by a minimal promoter with wound and pathogen induction by an additional 5’-flanking region. Plant Mol Biol, 22, 475-490. Morales P, Madarro A, Flors A, Sendra JM & Pérez-González JA (1995) Purification and characterization of a xylanase and an arabinofuranosidase from Bacillus polymyxa. Enzyme Microb Technol, 17, 424-429. Mou HJ, Jiang XL, Jiang X & Guan HS (2002) Isolation and properties of a carrageenandegrading bacterium. J Fish Sci China, 9, 251-254. Muzzarelli RAA (1996) Chitosan-based dietary foods. Carbohyd Polym, 29, 309–316. Muzarelli RAA, Ilari P, Tarsi R, Dubini B & Xia W (1994a) Chitosan from Absidia coerulea. Carbohyd Polym, 25, 45-50. Muzzarelli RAA, Tomasetti M & Ilari P (1994b) Depolymerization of chitosan with the aid of papain. Enzyme Microbial Technology, 16, 110-114. Muzzarelli RAA, Xia W, Tomasetti M & Ilari P (1995) Depolymerization of chitosan and substituted chitosans with the aid of a wheat germ lipase preparation. Enzyme Microb Technol, 17, 541-545. Nanjo F, Katsumi R & Sakai K (1990) Purification and characterization of exo-β-Dglucosaminidase, a novel type of enzyme, from Nocardia orientalis. J Biol Chem, 256, 10088-10094. Nanjo F, Sakai K, Ishikawa M, Isobe K & Usui T (1989) Properties and transglycosylation reaction of a chitinase from Nocardia orientalis. Agric Biol Chem, 53, 2189-2195.

42

Xianzhen Li and Xiaoyi Chen

Nankai H, Hashimoto W, Miki H, Kawai S & Murata K (1999) Microbial system for polysaccharide depolymerization: enzymatic route for xanthan depolymerization by Bacillus sp. strain GL1. Appl Environ Microbiol, 65, 2520-2526. Nibu Y, Satoh T, Nishi Y, Takeuchi T, Maruta K & Kusakabe I (1995) Purification and characterization of extracellular alginate lyase from Enterobacter cloacae M-1. Biosci Biotechnol Biochem 59: 632-637. Nisizawa K, Fujibayashi S & Kashiwabara Y (1968) Alginate lyases in the hepatopancre as of a marine mollusc, Dolabella auricula Solander. J Biochem, 64, 25-37. No HK, Park NY, Lee SH, Hwang HJ & Meyers SP (2002) Antibacterial activities of chitosans and chitosan oligomers with different molecular weights on spoilage bacteria isolated from tofu. J Food Sci, 67, 1511-1514. Nordtveit RJ, Vårum KM & Smidsrød O (1994) Degradation of fully water-soluble, partially N-acetylated chitosans with lysozyme. Carbohydr Polym, 23,253-260. Nordtveit RJ, Vårum KM & Smidsrød O (1996) Degradation of partially N-acetylated chitosans with hen egg white and human lysozyme. Carbohydr Polym, 29, 163-167. Ofek I & Sharon N (1990) Adhesins as lectins: specificity and role in infection. Curr Top Microbiol Immunol, 151, 91-114. Ohtakara A, Matsunaga H & Mitsutomi M (1990) Action pattern of Streptomyces griseus chitinase on partially N-acetylated chitosan. Agric Biol Chem, 54:3191-3199. Olano-Martin E, Gibson GR & Rastall RA (2002) Comparison of the in vitro bifidogenic properties of pectins and pectic-oligosaccharides. J Appl Microbiol, 93, 505-511. Onsøyen E (1996) Commercial applications of alginates. Carbohydr Eur, 14, 26-31. Orikoshi H, Nakayama S, Miyamoto K, Hanato C, Yasuda M, Inamori Y & Tsujibo H (2005) Roles of four chitinases (ChiA, ChiB, ChiC, and ChiD) in the chitin degradation stem of marine bacterium Alteromonas sp. strain O-7. Appl Environ Microbiol, 71, 1811-1815. Pantaleone D, Yalpani M & Scollar M (1992) Unusual susceptibility of chitosan to enzymatic hydrolysis. Carbohyd Res, 237, 325-332. Parajó JC, Garrote G, Cruz JM & Domýnguez H (2004) Production of xylooligosaccharides by autohydrolysis of lignocellulosic materials. Trends Food Sci Technol, 15, 115-120. Park PJ, Je JY, Byun HG, Moon SH & Kim SK (2004a) Antimicrobial activity of heterochitosans and their oligosaccharides with different molecular weights. J Microbiol Biotechnol, 14, 317-323. Park PJ, Lee HK & Kim SK (2004b) Preparation of hetero chitooligosaccharides and their antimicrobial activity on Vibrio parahaemolyticus. J Microbiol Biotechnol, 14, 41-47. Patil RS, Ghormade V & Deshpande MV (2002) Chitinolytic enzymes: an exploration. Enzyme Microb Technol, 26, 473-483. Pauly M, Albersheim P, Darvill A & York WS (1999) Molecular domains of the cellulose/xyloglucan network in the cell walls of higher plants. Plant J, 20, 629-639. Pellerin P, Goselin M, Lepoutre J, Samain E & Debeire P (1991) Enzymic production of oligosaccharides from corncob xylan. Enzyme Microb Technol, 13:617-621. Peñaloza-Vázquez A, Fakhr MK, Bailey AM & Bender CL (2004) AlgR functions in algC expression and virulence in Pseudomonas syringae pv. syringae. Microbiology, 150, 2727-2737. Pier GB (1998) Pseudomonas aeruginosa: a key problem in cystic fibrosis. Am Soc Microbiol News, 64, 339-347.

Biodegradation of Polysaccharide Sourced from Virulence Factor…

43

Potin P, Sanseau A, Le Gall Y, Rochas C & Kloareg B (1991) Purification and characterization of a new κ-carrageenase from a marine Cytophaga-like bacterium. Eur J Biochem, 201, 241-247. Qian F, An L, He X, Han Q & Li X (2006) Antibacterial activity of xantho-oligosaccharide cleaved from xanthan against phytopathogenic Xanthomonas campestris pv. campestris. Proc Biochem, 41, 1582-1588. Ramanathan A, Samiyappan R & Vidhyasekaran P (2000) Induction of defense mechanisms in greengram leaves and suspension cultured cells by Macrophomina phaseolina and its elicitors. J Plant Dis Protect, 107, 245-257. Reimers PJ, Guo A & Leach JE (1992) Increased activity of acationic peroxidase associated with an incompatible interaction between Xanthomonas oryzae pv. oryzae and rice (Oryza sativa). Plant Physiol, 99, 1044-1050. Renkema GH, Boot RG, Muijsers AO, Donker-Koopman WE, Aerts JMFG (1995) Purification and characterization of human chitotriosidase, a novel member of the chitinase family of proteins. J Biol Chem, 270 2198-2202. Reverchon S & Baudouy J (1987) Regulation of expression of pectate lyase genes pelA, pelD and pelE in Erwinia chrysanthemi. J Bacteriol, 169, 2417-2423. Reymond P, Grûnberger S, Paul K, Mûller M & Farmer EE (1995) Oligogalacturonide defense signals in plants: large fragments interact with the plasma membrane in vitro. Proc Natl Acad Sci USA, 92, 4145-4149. Rhoades J & Roller S (2000) Antimicrobial actions of degraded and native chitosan against spoilage organisms in laboratory media and foods. Appl Environ Microbiol, 66, 80-86. Ridley BL, O’Neill MA & Mohnen DA (2001) Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry, 57, 929-967. Rinaudo M & Milas M (1980) Enzymic hydrolysis of the bacterial polysaccharide xanthan by cellulase. Int J Biol Macromol, 2, 45-48. Rogovin SP, Anderson RF & Cadmus MC (1961) Production of polysaccharide with Xanthomonas campestris. J Biochem Microbiol Technol Eng, 3, 51-63. Roller S & Covill N (1999) The antifungal properties of chitosan in laboratory media and apple juice. Int J Food Microbiol, 47, 67-77. Romeo T & Preston JF (1986) Purification and structural properties of an extracellular (1-4)β-D-mannuronan-specific alginate lyase from a marine bacterium. J Biochem, 25, 83858391. Roubroeks JP, Andersson R, Mastromauro DI, Christensen BE & Axman P (2001) Molecular weight, structure and shape of oat (1,3), (1,4)-β-D-glucan fractions obtained by enzymatic degradation with (1,4)-β-D-glucan 4-glucanohydrolase from Trichoderma reesei. Carbohyd Polym, 46, 275-285. Rudrapatnam N, Tharanathan RN & Kittur FS (2003) Chitin: the undisputed biomolecule of great potential. Critical Rev Food Sci Nutr, 43, 61-87. Ruijssenaars HJ, de Bont JAM & Hartmans S (1999) A pyruvated mannose-specific xanthan lyase involved in xanthan degradation by Paenibacillus alginolyticus XL-1. Appl Environ Microbiol, 65, 2446-2452. Ruijssenaars HJ, Hartmans S & Verdoes JC (2000) A novel gene encoding xanthan lyase of Paenibacillus alginolyticus strain XL-1. Appl Environ Microbiol, 66, 3945-3950.

44

Xianzhen Li and Xiaoyi Chen

Ryan CA (1988) Oligosaccharides as recognition signals for the expression of defensive genes in plants. Biochemistry, 27, 8879-8883. Sakai T, Sakamoto T, Hallaert J & Vandamme EJ (1993) Pectin, pectinase and protopectinase: production, properties, and applications. Adv Appl Microbiol, 39, 213294. Sawabe T, Ohtsuka M & Ezura Y (1997) Novel alginate lyases from marine bacterium Alteromonas sp. strain H-4. Carbohydr Res, 304, 69-76 Seo WG, Pae HO, Kim NY, Oh GS, Park IS, Kim YH, Kim YM, Lee YH, Jun CD & Chung HT (2000) Synergistic cooperation between water soluble chitosan oligomers and interferong for induction of nitric oxide synthesis and tumoricidal activity in murine peritoneal macrophages. Cancer Lett, 159, 189-195. Sharp JK, McNeil M & Albersheim P (1984a) The primary structures of one elicitor-active and seven elicitor in active hexa (β-D-glucopyranosyl)-D-glucitols isolated from the mycelial walls of Phytophthora megasperma f. sp. glycinea. J Biol Chem, 259, 1132111336 Sharp JK, Valent B & Albersheim P (1984b) Purification and partial characerization of a βglucan fragment that elicits phytoalexin accumulation in soybean. J Biol Chem, 259, 11312-11320. Shi RX, Xu ZH & Li ZE (2000) Antitumor activity of degraded Chondrus ocellatus polysaccharide. Oceanol Limnol Sinica, 31, 653-656. Shibuya N & Minami E (2001) Oligosaccharide signaling for defense responses in plant. Physiol Mol Plant Pathol, 59, 223-233. Shimokawa T, Yoshida S, Takeuchi T, Murata K, Kobayashi H & Kusakabe I (1997) Purification and characterization of extracellular poly(β-D-1,4-mannuronide) lyase from Dendryphiella salina IFO 32139. Biosci Biotech Biochem, 61, 636-640. Shinya T, Ménard R, Kozone I, Matsuoka H, Shibuya N, Kauffmann S, Matsuoka K & Saito M (2006) Novel β-1,3-, 1,6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco. FEBS J, 273, 2421-2431 Shiraishi T, Yamada T, Nicholson RL & Kunoh H (1995) Phenylalanine ammonia-lyase in barley: activity enhancement in response to Erysiphe graminis sp. hordei (race 1) a pathogen, and Erysiphe pisi, a non pathogen. Physiol Mol Plant Pathol, 46, 153-162. Sikorski P, Stokke BT, Sørbotten A, Våum KM, Horn SJ & Eijsink VGH (2005) Development and application of a model for chitosan hydrolysis by a family 18 chitinase. Biopolymers, 77, 273-285. Singh RP, Wood FA & Hodgson WA (1970) The nature of virus inhibition by a polysaccharide from Phytophthora infestans. Phytopathology, 60, 1566-1569 Slováková L, Subiková V & Farka V (1994) Influence of xyloglucan oligosaccharides on some enzymes involved in the hypersensitive reaction to TNV (tobacco necrosis virus) of cucumber cotyledons. Zeitschrift flur Pflanzenkrankheiten und Pflanzenschutz, 101, 278285 Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J, Osborne E, Paredez A, Persson S, Raab T, Vorwerk S & Youngs H (2004) Toward a systems approach to understanding plant cell walls. Science, 306:2206-2211. Somssich IE & Hahlbrock K (1998) Pathogen defense in plants: a paradigm of biological complexity. Trends Plant Sci, 3, 86-90.

Biodegradation of Polysaccharide Sourced from Virulence Factor…

45

Soriano M, Diaz P &Pastor FIJ (2006) Pectate lyase C from Bacillus subtilis: a novel endocleaving enzyme with activity on highly methylated pectin. Microbiology, 152, 617-625 Spaltenstein A & Whitesides GM (1991) Polyacrylamides bearing pendant agrisialoside groups strongly inhibit agglutination of erythrocytes by influenza virus. J Am Chem Soc, 113, 686-687. Spiro MD, Ridley BL, Eberhard S, Kates KA, Mathieu Y, O’Neill MA, Mohnen D, Guern J, Darvill A & Albersheim P (1998) Biological activity of reducing-end-derivatized oligogalacturonides in tobacco tissue cultures. Plant Physiol, 116: 1289-1298. Stevens RA & Levin RE (1977) Purification and characteristics of an alginase from Alginovibrio aquatilis. Appl Environ Microbiol, 33, 1156-1161. Sudharshan NR, Hoover DG & Knorr D (1992) Antibacterial action of chitosan. Food Biotechnol, 6, 257-272. Sutherland IW (1984) Hydrolysis of unordered xanthan in solution by fungal cellulases. Carbohydr Res, 131, 93-104. Sutherland IW (1987) Xanthan lyases: novel enzymes found in various bacterial species. J Gen Microbiol, 133, 3129-3134. Suzuki K, Mikami T, Okawa Y, Tokoro A, Suzuki S & Suzuki M (1986) Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose. Carbohyd Res, 151, 403-408. Suzuki K, Sugawara N, Suzuki M, Uchiyama T, Katouno F, Nikaidou N, Watanabe T & Chitinases A (2002) B and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation. Bioscie Biotechnol Biochem, 66, 1075-1083. Suzuki S (1996) Studies on biological effects of water-soluble lower homologous oligosaccharides of chitin and chitosan. Fragrance J, 15, 61-68. Swennen K, Courtin CM, van der Bruggen B, Vandecasteele C & Delcour JA (2005) Ultrafiltration and ethanol precipitation for isolation of arabinoxylooligosaccharides with different structures. Carbohyd Polym 165, 1-10 Tai A, Kawazu K & Kobayashi A (1996a) Species-specificity of an elicitor-active oligosaccharide, LN-3, to leguminous plants. Z Naturforsch, 51: 371-378. Tai A, Ohsawa E, Kawazu K & Kobayashi A (1996b) Aminimum essential structure of LN-3 elicitor activity in bean cotyledons. Z Naturforsch, 51: 15-19. Tardy F, Nasser W, Robert-Baudouy J & Hugouvieux-Cotte-Pattat N (1997) Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors. J Bacteriol, 179, 2503-2511. Taylor EJ, Gloster TM, Turkenburg JP, Vincent F, Brzozowski AM, Dupont C, Shareck F, Centeno MSJ, Prates JM, Puchart V, Ferreira LMA, Fontes CMGA, Biely P & Davies GJ (2006) Structure and activity of two metal ion-dependent acetylxylan esterases involved in plant cell wall degradation reveals a close similarity to peptidoglycan deacetylases. J Biol Chem, 281, 10968-10975 Tokoro A, Kobayashi M, Tatekawa N, Suzuki S & Suzuki M (1989) Protective effect of Nacetyl chitohexaose on Listeria monocytogens infection in mice. Microb Immun, 33, 357367. Tonukari NJ, Scott-Craig JS & Walton JD (2000) The Cochliobolus carbonum SNF1 gene is required for cell wall-degrading enzyme expression and virulence on maize. Plant Cell, 12, 237-247.

46

Xianzhen Li and Xiaoyi Chen

Tsai GJ, Su WH, Chen HC & Pan CL (2002) Antimicrobial activity of shrimp chitin and chitosan from different treatments and applications of fish preservation. Fish Sci, 68, 170-177. Tsai GJ, Wu Z & Su W-H (2000) Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. J Food Preser, 63, 747-752. Tsukada K, Matsumoto T, Aizawa K, Tokoro A, Naruse R, Suzuki S & Suzuki M (1990) Antimetastatic and growth-inhibitory effects of N-acetylchitohexaose in mice bearing Lewis lung carcinoma. Japan J Cancer Res, 81, 259-265. Ueki A, Akasaka H, Suzuki D, Hattori S & Ueki K (2006) Xylanibacter oryzae gen. nov., sp. nov., a novel strictly anaerobic, Gram-negative, xylanolytic bacterium isolated from riceplant residue in flooded rice-field soil in Japan. Int J Syst Evol Microbiol, 56, 2215-2221. Uffen RL (1997) Xylan degradation, a glimpse at microbial diversity. J Ind Microbiol Biotechnol, 19, 1-6. Umemoto N, Kakitani M, Iwamatsu A, Yoshikawa M, Yamaoka N & Ishida I (1997) The structure and function of a soybean β-glucan-elicitor-binding protein. Proc Natl Acad Sci USA, 94, 1029-1034. Usui T, Hayashi Y, Nanjo F, Sakai K & Ishido Y (1987) Transglycosylation reaction of a chitinase purified from Nocardia orientalis. Biochim Biophys Acta, 923, 302-309. Usui T, Matsui H & Isobe K (1990) Enzymatic synthesis of useful chitooligosaccharides utilizing transglycosylation by chitinolytic enzymes in a buffer containing ammonium sulfate. Carbohydr Res, 203, 65-77. Van Loon LC 1997 Induced resistance in plants and the role of pathogenesis related proteins. Eur J Plant Pathol, 103, 753-765. Varum KM, Holme HK, Izume M, Stokke BT & Smidsrod O (1996) Determination of enzymatic hydrolysis specificity of partially N-acetylated chitosans. Biochemica et Biophysica Acta, 1291, 5-15. Velázquez E, de Miguel T, Poza M, Rivas R, Rosselló-Mora R & Villa TG (2004) Paenibacillus favisporus sp. nov., a xylanolytic bacterium isolated from cow faeces. Int J Syst Evol Microbiol, 54, 59-64. Vincken J-P, Beldman G & Voragen AGJ (1997a) Substrate specificity of endoglucanases: what determines xyloglucanase activity? Carbohydr Res, 298, 299-310. Vincken J-P, York WS, Beldman G & Voragen AGJ (1997b) Two general branching patterns of xyloglucan, XXXG and XXGG, Plant Physiol, 114, 9-13. Wang GH (1992) Inhibition and inactivation of five species of foodborne pathogens by chitosan. J Food Protec, 55, 916-925. Wanjiru WM, Kang ZS & Buchenauer H (2002) Importance of cell wall degrading enzymes produced by Fusarium graminearum during infection of wheat heads. Eur J Plant Pathol, 108, 803-810. Williams RJ & Heymann DL (1998) Containment of antibiotic resistance. Science, 279, 1153-1154. Wood PJ, Weisz J & Blackwell BA (1994) Structural studies of (1,3), (1,4)-β-D-glucans by 13 C-nuclear magnetic resonance spectroscopy and by rapid analysis of cellulose-like regions using high-performance anion-exchange chromatography of oligosaccharides released by lichenase. Cereal Chem, 71, 301-307.

Biodegradation of Polysaccharide Sourced from Virulence Factor…

47

Yamada T, Ogamo A, Saito T, Uchiyama H & Nakagawa Y (2000) Preparation of O-acylated low-molecular-weight carrageenans with potent anti-HIV activity and low anticoagulant effect. Carbohydr Polym, 41, 115-120. Yamaguchi T, Yamada A, Hong N, Ogawa T, Ishii T & Shibuya N (2000) Differences in the recognition of glucan elicitor signals between rice and soybean: β-glucan fragments from the rice blast disease fungus Pyricularia oryzae that elicit phytoalexin biosynthesis in suspension-cultured rice cells. Plant Cell, 12, 817-826. Yang H-C, Im W-T, An D-S, Park W-S, Kim IS & Lee S-T (2005) Silvimonas terrae gen. nov., sp. nov., a novel chitin-degrading facultative anaerobe belonging to the ‘Betaproteobacteria’. Int J Syst Evol Microbiol, 55, 2329-2332 Yaoi K & Mitsuishi Y (2004) Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128, FEBS Lett, 560, 45-50. Yaoi K, Nakai T, Kameda Y, Hiyoshi A & Mitsuishi Y (2005) Cloning and characterization of two xyloglucanases from Paenibacillus sp. strain KM21. Appl Environ Microbiol, 71, 7670-7678. Yoon H-J, Hashimoto W, Miyake O, Okamoto M, Mikami B & Murata K (2000) Over expression in Escherichia coli, purification, and characterization of Sphingomonas sp. A1 alginate lyases. Protein Expr Purif, 19, 84-90. Yoshikawa M, Keen NT & Wang MC (1983) A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation. Plant Physiol, 73, 497-506. Yoshikawa M, Matama M & Masago H (1981) Release of a soluble phytoalexin elicitor from mycelial walls of Phytophthora megasperma var. sojae by soybean tissues. Plant Physiol, 67, 1032-1035. Yoshikawa M, Yamaoka N & Takeuchi Y (1993) Elicitors: their significance and primary modes of action in the induction of plant defence reactions. Plant Cell Physiol, 34, 11631173. Young D & Kauss H (1983) Release of calcium from suspension cultured glycine max cells by chitosan, other polycations, and polyamines in relation to effects on membrane permeability. Plant Physiology, 73, 698-702. Yu G, Guan H, Ioanoviciu AS, Sikkander SA, Thanawiroon C, Tobacman JK, Toida T & Linhardt RJ (2002) Structural studies on κ-carrageenan derived oligosaccharides. Carbohyd Res, 337 433-440 Yuan QP, Zhang H, Qian ZM & Yang XJ (2004) Pilot-plant production of xylooligosaccharides from corncob by steaming, enzymatic hydrolysis and nanofiltration. J Chem Technol Biotechnol, 79, 1073-1079 Zhang H, Du Y, Yu X, Mitsutomi M & Aiba S (1999) Preparation of chitooligosaccharides from chitosan by a complex enzyme. Carbohyd Res, 320, 257-260. Zopf D & Roth S (1996) Oligosaccharide anti-infective agents. Lancet, 347, 1017-1021 Zverlov VV, Schantz N, Schmitt-Kopplin P & Schwarz WH (2005) Two new major subunits in the cellulosome of Clostridium thermocellum: xyloglucanase Xgh74A and endoxylanase Xyn10D. Microbiology, 151, 3395-3401.

In: Environmental Biodegradation Research Focus Editor: B. Y. Wang, pp. 49-76

ISBN: 978-1-60021-904-7 © 2007 Nova Science Publishers, Inc.

Chapter 2

BIODEGRADATION OR METABOLISM OF BISPHENOL A IN THE ENVIRONMENT Jeong-Hun Kang∗ and Yoshiki Katayama Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-Ku, Fukuoka City, Japan

ABSTRACT Recently, there has been increasing interest in the effects of endocrine disruptors on organisms. Bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane; CAS Registry No. 8005-7) is an endocrine disruptor with estrogenic activity and acute toxicity to aquatic organisms. BPA is made by combining acetone and phenol and is used mainly as a material for the production of epoxy resins and polycarbonate plastics. Due to intensified usage of these products, exposure of organisms to BPA via several routes, such as the environment and the food chain, has increased. BPA contamination in the environment occurs through several routes, such as migration from human wastes and effluent from wastewater treatment plants. BPA exposed to the environment can be biodegraded or metabolized by microorganisms (bacteria, fungi and plankton), plants, invertebrates and vertebrates (fish, amphibians and mammals). Biodegradation or metabolism is a very important step for removing or detoxifying BPA in the environment or organisms. Although some metabolites of BPA may exhibit enhanced estrogenicity or toxicity, in general, BPA biodegradation or metabolism by organisms leads to detoxication of BPA. However, excessive BPA doses cause bioaccumulation if detoxification pathways are saturated. In this chapter we describe 1) contamination routes of BPA, 2) biodegradation or metabolism of BPA by organisms, and 3) bioaccumulation of BPA in organisms, with the main subject of this chapter being the biodegradation or metabolism of BPA by organisms.

∗

Corresponding author: Jeong-Hun Kang; Telephone number: 81-92-802-2849; Fax: 81-92-802-2849; E–mail address: [email protected]

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Key words: bisphenol A, biodegradation, metabolism, environment, bioaccumulation.

1. INTRODUCTION Bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane；CAS Registry No. 80-05-7) is a known endocrine disruptors (Krishnan et al., 1993) that is acutely toxic to aquatic organisms in the 1000–10,000 μg/l range for both freshwater and marine species (Alexander et al., 1988). BPA is an organic compound composed of two phenol rings connected by a methyl bridge, with two methyl functional groups attached to the bridge (Figure 1). BPA is used as a material for the production of phenol resins, polyacrylates and polyesters, but mainly for the production of epoxy resins and polycarbonate plastics. The epoxy resins are used as foodcontact surface lacquer coatings for cans, metal jar lids, protective coatings and finishes, automobile parts, adhesives, aerospace applications, and as a coating for PVC pipes. The polycarbonate plastics are used in compact disk manufacturing, automotive lenses, household appliances, food packaging, and plastic bottles (Staples et al., 1998). Due to increased use of products based on epoxy resins and polycarbonates, exposure of organisms to BPA via several routes, such as the environment and the food chain, has increased (Kang et al., 2006b). Thus, it is difficult for organisms (microorganisms, plants, invertebrates, and vertebrates) to escape from the toxic and endocrine disrupting effects of BPA. The transport potential of BPA to air is much lower (