University of Groningen Unique features of several microbial α-amylases active on soluble and native starch Sarian, Fean Davisunjaya IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2016 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Sarian, F. D. (2016). Unique features of several microbial α-amylases active on soluble and native starch. University of Groningen. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). 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Download date: 11-10-2021 Unique features of several microbial α‐amylases active on soluble and native starch Fean Davisunjaya Sarian The work described in this thesis was performed in the research group of the Microbial Physiology of the Groningen Biomolecular Sciences and Biotechnology Institute (GBB), the Aquatic Biotechnology and Bioprocessing Engineering of the Engineering and Technology Institute Groningen (ENTEG) of the University of Groningen, The Netherlands, and the Biochemistry Division, Institut Teknologi Banding Indonesia. This work was financially supported by a Bernoulli scholarship and by Asahi Glass Foundation. Cover design and layout of the book: F. D. Sarian Cover front: art of α‐amylase with glucose and maltooligosacharides Cover back: art of glucose and maltooligosaccharides Printed by: Ipskamp Printing ISBN: 978-90-367-8850-2 ISBN: 978-90-367-8849-6 (electronic version) Copyright © 2016 by Fean Davisunjaya Sarian All right reserved. No part of this thesis may be reproduced, stored in a retrieval system or transmitted in any form by any means, without permission from the author. Unique features of several microbial α-amylases active on soluble and native starch PhD thesis to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. E. Sterken and in accordance with the decision by the College of Deans. This thesis will be defended in public on Friday 3 June 2016 at 09.00 hours by Fean Davisunjaya Sarian born on 31 July 1981 in Yogyakarta, Indonesia Supervisors Prof. M.J.E.C. van der Maarel Prof. L. Dijkhuizen Assessment Committee Prof. G.J.W. Euverink Prof. M.W. Fraaije Prof. B. Svensson Table of Contents Chapter 1 Introduction ......................................................................................... 1 Chapter 2 Cold enzymatic hydrolysis of Indonesian roots and tuber starches ........................................................... 81 Chapter 3 Enzymatic degradation of granular starch By Microbacterium aurum strain B8.A ................................ 99 Chapter 4 Degradation of granular starch by the bacterium Microbacterium aurum B8.A involves a novel modular α‐amylase enzyme system with FnIII and CBM25 domains ........................................................................ 125 Chapter 5 Identification of a novel α‐amylase from marine Bacillus megaterium NL3 representing a new GH13 subfamily .......................................................................... 159 Chapter 6 General Discussion ..................................................................... 191 Summary non‐expert ............................................................... 203 Samenvatting ............................................................................. 209 Acknowledgments ........................................................................... vii List of publications .......................................................................... xi Chapter Introduction Introduction What is Starch? Starch is next to cellulose and chitin, the most abundant biopolymer in nature. Plants Page | 2 utilize the light energy absorbed by chlorophyll to produce simple sugars and oxygen from carbon dioxide and water through a process called photosynthesis. These simple sugars may be used directly in cellular respiration, converted into starch, or used to build cell walls. The starch serves as major energy storage for plants, which can later be converted back to glucose and used in respiration when required. Starch is densely packed in microscopic particles called granules and is extremely insoluble in water at room temperature. The composition, size and shape of these granules are often characteristic for the plant species from which they are extracted (1). Structurally, starch is made up of individual units of glucose, linked together by α(14) and occasional α(16)‐glycosidic linkages (2). Amylose, the minor component, is a relatively long and linear molecule with almost exclusively α(14)‐glycosidic bonds with number average degree of polymerization (DPn) of 900‐3300 (3). It has a slight degree of branching of 9‐20 branches per molecule (4, 5). Amylopectin is the major component of most starches and has a DPn of 4800‐ 15900 with a molecular mass that is about 5 times bigger than amylose (3). It consists of short α(14) linked chains while branching by α(16)‐glycosidic bonds occurs every 15‐45 glucosyl units (5, 6). Within the starch granule, amylopectin molecules are ordered radially with the ends of non‐reducing chains pointing toward the outer surface (7). Although the exact molecular architecture is still not clear, its structure is often described by a cluster model (Fig. 1.1) (4, 8). Hizukuri (9) and Kobayashi et al. (10) have shown that an amylopectin molecule contains several types of chains (A, B, and C) which differ in their chain length and are present in almost equal proportions. The A‐chains (unbranched) are the shortest chains, linked to B chains and do not carry any other chains, whereas the B chains (B1‐B4, depending on their length and the number of clusters the chain passes through), connect to one or more A chains and/or B chains. B1 chains are short chains that span only one cluster, while B2, B3, and B4 chains are long chains that connect two to four cluster chains in the amylopectin molecule (9). The C‐chain contains the reducing end group of the molecule (11). Chapter 1 Page | 3 Fig 1.1 Schematic representation of the cluster model of amylopectin. For definition of A, B1‐B2, and C, see text. Straight line, α(14) glycosidic bonds; dashed line, α(16) glycosidic bonds. Adapted from Tester et al. (12), with modifications. The relative content of amylose and amylopectin varies widely, depending on the botanical origin (Table 1.1). The amylose content of regular starch is typically from 10 to 35% of the total starch. High‐amylose starches contain approximately 35‐70% amylose, while ‘waxy’ (high‐amylopectin) starches have almost no amylose (0% to 4%) (13). Functional properties of starch, such as water‐binding capacity, gelatinization and pasting, retrogradation, and susceptibility to enzymatic degradation are influenced by the amylose content and the placement of the amylose in the amylopectin (14). Both amylose and amylopectin are packed together in complex structures consisting of crystalline and amorphous areas and contain a few phosphate groups mainly at the C‐6 of the glucose residues (15). Phosphorus, present as phosphate monoester and phospholipids, at low amounts significantly affects the functional properties of the starch (16). Apart from these main components, smaller amounts of other components, such as proteins and lipids may also be present in amounts that vary depending on the botanical origin and starch isolation procedure (3). Introduction Structure and properties of starch Native starches are semi‐crystalline structures. The degree of crystallinity of native Page | 4 starch varies between 15 – 45% depending on the starch source (6, 17). Observed by light microscopy or scanning electron microscopy (after a slight acid or enzyme treatment), starch granules are formed by the deposition of growth rings that consist of alternating semi‐crystalline and amorphous layers (18–20). Irrespective of the source of the starch, the repeat distance of alternating amorphous and crystalline layers is highly conserved at 9 nm (21–23) (Fig. 1.1). The starch granule organization is very complex and depends strongly on the botanical origin. A schematic representation of the granule’s architecture is given in Fig. 1.2. The outer branches of the amylopectin molecule form double helices that are arranged in crystalline regions. The amorphous part of the starch is believed to consist of amylose that forms single helical structures near the branch points of the amylopectin molecule (24). Jenkins and Donald (21) hypothesized that amylose is predominantly located in the amorphous growth ring based on