Carbohydrate-Active Enzymes

Structure, Function and Applications

K.-H. Park

• Publisher: Woodhead Publishing
• Year: 2008
• Print ISBN: 9781845695194
• eBook ISBN: 9781845695750
• Topic: Technology & Engineering
• Subtopic: Food Science
• Index: Technology & Engineering

Recent advances in biochemistry and biotechnology have enabled significant progress in basic research on carbohydrate-active enzymes and advances in their effective application. The mechanism of catalytic reaction of carbohydrate-active enzymes is not fully understood, though, as they often show unusual substrate specificity and modes of action. This comprehensive collection summarises some of the most important research in the field of carbohydrate-active enzymes, focusing on the enzymatic reaction mechanism, structure-function relationship and role in the living organismThe book is based on papers presented in the 2008 Agricultural Biotechnology Symposium Carbohydrate-active enzymes: structure, function and applications held on September 26th-27th 2008 in Seoul National University, Korea. This symposium was organized by the Center for Agricultural Biomaterials, Seoul National University, Korea, which has organized symposia on agricultural biotechnology annually since 1990. Many important results on new types of carbohydrate-active enzymes and their applications have been reported at these meetings. Papers in Part one of this collection focus on structure-function relationships of carbohydrate-active enzymes. Papers in Part two discuss functions and applications of carbohydrate-active enzymes, such as enzymes for grain processing and glycosidases and their mutants as useful tools for glycoside synthesis.With its distinguished editor and international team of contributors, Carbohydrate-active enzymes: structure, function and applications is an essential reference for research scientists, post-graduate students and those in the food industry with an interest in enzymes.- Summarises some of the most important research in the field of carbohydrate-active enzymes- Covers topics ranging from enzyme classification and structural elucidation to applications of enzymes in food processing and other industries

Part I

Structure–function relationship of carbohydrate-active enzymes

Outline

Chapter 1: BIOSYNTHESIS OF POLYSACCHARIDES

Chapter 2: α-AMYLASES. INTERACTION WITH POLYSACCHARIDE SUBSTRATES, PROTEINACEOUS INHIBITORS AND REGULATORY PROTEINS

Chapter 3: WHY COULD ISOPULLULANASE, AN ODD PULLULAN-HYDROLYZING ENZYME, BE DISCOVERED?

Chapter 4: SEQUENCE FINGERPRINTS IN THE EVOLUTION OF THE α-AMYLASE FAMILY

Chapter 5: MOLECULAR MECHANISM OF α-GLUCOSIDASE

Chapter 6: STRUCTURE, FUNCTION AND APPLICATIONS OF MICROBIAL β-GALACTOSIDASE (LACTASE)

Chapter 7: STRUCTURAL FEATURE OF THE ARCHEAL GLYCOGEN DEBRANCHING ENZYME FROM SULFOLOBUS SOLFATARICUS

Chapter 8: MOLECULAR CLONING OF THE AMYLOSUCRASE GENE FROM A MODERATE THERMOPHILIC BACTERIUM DEINOCOCCUS GEOTHERMALIS AND ANALYSIS OF ITS DUAL ENZYME ACTIVITY

Chapter 9: SUBSTRATE SPECIFICITY, KINETIC MECHANISM AND OLIGOMERIC STATES OF CYCLOMALTODEXTRINASE FROM ALKALOPHILIC BACILLUS SP. I-5

BIOSYNTHESIS OF POLYSACCHARIDES

John F. Robyt

ABSTRACT

The mechanisms involved in the biosynthesis of six polysaccharides is described in the following order: (1) Introduction to the first purported biosynthesis of polysaccharides, glycogen and starch by phosphorylases; (2) biosynthesis of Salmonella O-antigen polysaccharide; (3) biosynthesis of bacterial cell wall polysaccharide, peptido-murein; (4) biosynthesis of dextran by B-512FMC dextransucrase; (5) biosynthesis of bacterial cellulose and xanthan; (6) biosynthesis of starch in starch granules. The structures of the six polysaccharides are quite diverse. There are four β-linked hetero-polysaccharides (2), (3), and (5), and two α-linked homo-polysaccharides (4) and (6). The first five are biosynthesized by prokaryote bacteria and the sixth polysaccharide (starch) was shown to be biosynthesized by eight different eukaryotic plant sources. All six of the poly-saccharides have been shown to be biosynthesized by a common mechanism in which the monomer or repeating unit is added to the reducing-end of a growing polysaccharide chain in a two catalytic-site insertion mechanism. The β-linked polysaccharides are covalently α-linked to a lipid pyrophosphate, bactoprenol pyrophosphate, at the active-site of the synthesizing enzymes; the α-linked polysaccharides are β-linked directly to the synthesizing enzymes. When the monomer or repeating unit is inserted between the growing polysaccharide and the lipid pyrophosphate or the enzyme, the configuration of the linkage of the polysaccharide is inverted, giving the correct stereochemistry for the specific polysaccharide. Eventually, the poly-saccharides are released from the active-sites by an acceptor reaction with water or with another carbohydrate.

Key words

cellulose synthase

dextransucrase

insertion mechanism

primer mechanism

starch synthase

INTRODUCTION

Polysaccharides were the first biopolymers purported to be biosynthesized in vitro (Cori and Cori 1939) observed that the reaction of liver phosphorylase with α-d-glucose-1-phosphate (α-Glc-1-P) and glycogen added glucose residues to the nonreducing-ends of glycogen chains. Shortly thereafter, Hanes (1940) reported a similar reaction for potato phosphorylase in which α-Glc-1-P and starch also added glucose residues to the nonreducing-ends of the starch chains. Up to this time, the reaction catalyzed by phosphorylases was with inorganic phosphate (P_i) and glycogen or starch chains to give α-Glc-1-P and a partially degraded polysaccharide. It was found that phosphorylases catalyzed these two reactions with equilibrium constants close to one (Swanson and Cori, 1948). The equilibrium, however, seemed to favor the degradation reaction than the synthetic reaction. The reactions were formulated for glycogen and starch chains, as the following:

The reactions show that the degradation involves inorganic phosphate that removes glucose residues from the nonreducing-end of the polysaccharide chains to remove glucose residues and form α-Glc-1-P and a partially degraded polysaccharide chain. The reverse, synthetic reaction, involves the transfer of glucose from α-Glc-1-P to α-1→4 glucan chains or to the nonreducing-ends of an α-1→4 linked glucose oligosaccharide. The addition of just α-Glc-1-P to the phosphorylases, however, gave no reaction. It was, thus, recognized that a preformed polysaccharide or oligosaccharide chain was absolutely required to have synthesis by these reactions and the concept of a required primer was established.

As the reaction was studied more carefully, it was found that starting with α-Glc-1-P and a starch or glycogen chain, the reaction rapidly slowed down and stopped, as the concentration of P_i increased. It was further found that the synthetic reaction did not occur in vivo at all, as the concentration of P_i in animal and plant tissue was 20- to 40-times the concentration of α-Glc-1-P (Trevelyan et al., 1952; Ewart et al,. 1954; Liu and Shannon, 1981) and the in vivo conditions greatly favored degradation, rather than synthesis. Further, the addition of phosphorylases to just α-Glc-1-P gave no reaction. It, thus, appeared that phosphorylases only catalyzed the degradation of glycogen and starch and not the synthesis.

The studies of (Cori and Cori, 1939; Hanes, 1940; and Swanson and Cori, 1948), however, led to the development of the hypothesis for a required primer chain for the biosynthesis of polysaccharides. With essentially no evidence this concept has stuck in the minds of many people since then and relatively recently, it has been postulated for the mechanism of biosynthesis of polysaccharides, even with a paucity of experimental evidence (Bocca et al., 1997; Ball et al. 1998; Ball and Morell, 2003; and Tomlinson and Denyer, 2003).

Some 20 years after the phosphorylase experiments, (De Fekete et al., 1960; Recondo and Leloir, 1961; Leloir et al., 1961) found that the high-energy donor of glucose for starch biosynthesis was uridine diphospho glucose (UDPGlc) and adenosine diphospho glucose (ADPGlc) and that when ADPGlc was incubated with starch granules, starch chains were biosynthesized. ADPGlc was the better of the two donors. The biosynthetic enzymes, starch synthase and starch branching enzyme were apparently entrapped in the granules during their synthesis. Many years later, (Robyt and Mukerjea, 2000) found that starch granules that h...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• CONTRIBUTORS
• PREFACE
• Part I: Structure–function relationship of carbohydrate-active enzymes
• Part II: Functions and applications of carbohydrate-active enzymes