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Twenty-four branched maltodextrins were synthesized from eight starches using three thermostable microbial glycogen branching enzymes. The maltodextrins have a degree of branching (DB) ranging from 5 % to 13 %. This range of products allows us to explore the effect of DB on the digestibility, which was quantified under conditions that mimic the digestion process in the small intestine. The rate and extent of digestibility were analyzed using the logarithm of the slope method, revealing that the branched maltodextrins consist of a rapidly and slowly digestible fraction. The amount of slowly digestible maltodextrin increases with an increasing DB. Surprisingly, above 10 % branching the fraction of slowly digestible maltodextrin remains constant. Nevertheless, the rate of digestion of the slowly digestible fraction was found to decline with increasing DB and shorter average internal chain length. These observations increase the understanding of the structural factors important for the digestion rate of branched maltodextrins., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Scope: Resistant starches (RSs) are classically considered to elicit health benefits through fermentation. However, it is recently shown that RSs can also support health by direct immune interactions. Therefore, it has been hypothesized that the structural traits of RSs might impact the health benefits associated with their consumption., Methods and Results: Effects of crystallinity, molecular weight, and chain length distribution of RSs are determined on immune Toll-like receptors (TLRs), dendritic cells (DCs), and T-cell cytokines production. To this end, four type-3 RSs (RS3) are compared, namely Paselli WFR, JD150, debranched Etenia, and Amylose fraction V, which are extracted from potatoes and enzymatically modified. Dextrose equivalent seems to be the most important feature influencing immune signaling via activation of TLRs. TLR2 and TLR4 are most strongly stimulated. Especially Paselli WFR is a potent activator of multiple receptors. Moreover, the presence of amylose, even to residual levels, enhances DC and T-cell cytokine responses. Paselli WFR and Amylose fraction V influence T-cell polarization., Conclusions: It has been shown here that chain length and particularly dextrose equivalent are critical features for immune activation. This knowledge might lead to tailoring and design of immune-active RS formulations., (© 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Dietary fibers are at the forefront of nutritional research because they positively contribute to human health. Much of our processed foods contain, however, only small quantities of dietary fiber, because their addition often negatively affects the taste, texture, and mouth feel. There is thus an urge for novel types of dietary fibers that do not cause unwanted sensory effects when applied as ingredient, while still positively contributing to the health of consumers. Here, we report the generation and characterization of a novel type of soluble dietary fiber with prebiotic properties, derived from starch via enzymatic modification, yielding isomalto/malto-polysaccharides (IMMPs), which consist of linear (α1 → 6)-glucan chains attached to the nonreducing ends of starch fragments. The applied Lactobacillus reuteri 121 GTFB 4,6-α-glucanotransferase enzyme synthesizes these molecules by transferring the nonreducing glucose moiety of an (α1 → 4)-glucan chain to the nonreducing end of another (α1 → 4)-α-glucan chain, forming an (α1 → 6)-glycosidic linkage. Once elongated in this way, the molecule becomes a better acceptor substrate and is then further elongated with (α1 → 6)-linked glucose residues in a linear way. Comparison of 30 starches, maltodextrins, and α-glucans of various botanical sources, demonstrated that substrates with long and linear (α1 → 4)-glucan chains deliver products with the highest percentage of (α1 → 6) linkages, up to 92%. In vitro experiments, serving as model of the digestive power of the gastrointestinal tract, revealed that the IMMPs, or more precisely the IMMP fraction rich in (α1 → 6) linkages, will largely pass the small intestine undigested and therefore end up in the large intestine. IMMPs are a novel type of dietary fiber that may have health promoting activity.

Starch is the major food reserve in plants and forms a large part of the daily calorie intake in the human diet. Industrially, starch has become a major raw material in the production of various products including bio-ethanol, coating and anti-staling agents. The complexity and diversity of these starch based industries and the demand for high quality end products through extensive starch processing, can only be met through the use of a broad range of starch and alpha-glucan modifying enzymes. The economic importance of these enzymes is such that the starch industry has grown to be the largest market for enzymes after the detergent industry. However, as the starch based industries expand and develop the demand for more efficient enzymes leading to lower production cost and higher quality products increases. This in turn stimulates interest in modifying the properties of existing starch and alpha-glucan acting enzymes through a variety of molecular evolution strategies. Within this review we examine and discuss the directed evolution strategies applied in the modulation of specific properties of starch and alpha-glucan acting enzymes and highlight the recent developments in the field of directed evolution techniques which are likely to be implemented in the future engineering of these enzymes.

Cyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch. The Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose. Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts. (i) The loop forming acceptor subsite +3 had a different conformation, providing the G2-amylase with more space at acceptor subsite +3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites -3/-4 (Biochemistry, 38 (1999) 8385). In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase. The loop forming acceptor subsite +3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered. The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme. Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity. Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites.

Cyclodextrin glycosyltransferase (CGTase) preferably catalyzes transglycosylation reactions, whereas many other alpha-amylase family enzymes are hydrolases. Despite the availability of three-dimensional structures of several transglycosylases and hydrolases of this family, the factors that determine the hydrolysis and transglycosylation specificity are far from understood. To identify the amino acid residues that are critical for the transglycosylation reaction specificity, we carried out error-prone PCR mutagenesis and screened for Bacillus circulans strain 251 CGTase mutants with increased hydrolytic activity. After three rounds of mutagenesis the hydrolytic activity had increased 90-fold, reaching the highest hydrolytic activity ever reported for a CGTase. The single mutation with the largest effect (A230V) occurred in a residue not studied before. The structure of this A230V mutant suggests that the larger valine side chain hinders substrate binding at acceptor subsite +1, although not to the extent that catalysis is impossible. The much higher hydrolytic than transglycosylation activity of this mutant indicates that the use of sugar acceptors is hindered especially. This observation is in favor of a proposed induced-fit mechanism, in which sugar acceptor binding at acceptor subsite +1 activates the enzyme in transglycosylation [Uitdehaag et al. (2000) Biochemistry 39, 7772-7780]. As the A230V mutation introduces steric hindrance at subsite +1, this mutation is expected to negatively affect the use of sugar acceptors. Thus, the characteristics of mutant A230V strongly support the existence of the proposed induced-fit mechanism in which sugar acceptor binding activates CGTase in a transglycosylation reaction.

Starch is a major storage product of many economically important crops such as wheat, rice, maize, tapioca, and potato. A large-scale starch processing industry has emerged in the last century. In the past decades, we have seen a shift from the acid hydrolysis of starch to the use of starch-converting enzymes in the production of maltodextrin, modified starches, or glucose and fructose syrups. Currently, these enzymes comprise about 30% of the world's enzyme production. Besides the use in starch hydrolysis, starch-converting enzymes are also used in a number of other industrial applications, such as laundry and porcelain detergents or as anti-staling agents in baking. A number of these starch-converting enzymes belong to a single family: the alpha-amylase family or family13 glycosyl hydrolases. This group of enzymes share a number of common characteristics such as a (beta/alpha)(8) barrel structure, the hydrolysis or formation of glycosidic bonds in the alpha conformation, and a number of conserved amino acid residues in the active site. As many as 21 different reaction and product specificities are found in this family. Currently, 25 three-dimensional (3D) structures of a few members of the alpha-amylase family have been determined using protein crystallization and X-ray crystallography. These data in combination with site-directed mutagenesis studies have helped to better understand the interactions between the substrate or product molecule and the different amino acids found in and around the active site. This review illustrates the reaction and product diversity found within the alpha-amylase family, the mechanistic principles deduced from structure-function relationship structures, and the use of the enzymes of this family in industrial applications.

In most tropical countries, carbohydrate-based agricultural products occur in large quantities. Wider utilization of local starch crops will offer various economic and ecological advantages. Local starch crops can be a catalyst for rural industrial development and eventually open up new markets. The potential glucose yield from the typical Indonesian starches (edible canna, arrowroot, sago, and sweet potato) using lower dosage of cold-starch hydrolyzing enzyme preparation Stargen™ 002 was compared with that of corn and potato. The glucose equivalent yield reached 88.4 g/L and 86.3 g/L after 24 h of hydrolysis when 40% (w/v) raw sago and sweet potato starches were used, respectively, as compared to corn starch (89.6 g/L). While arrowroot and edible canna gave much lower amounts (53.3 g/L and 39.7 g/L, respectively). This study demonstrates that sago and sweet potato starches may provide interesting alternatives to corn starch in a cold hydrolysis conversion process of starch into glucose. [ABSTRACT FROM AUTHOR]

Abstract: Starch is an agricultural raw material used in many food and industrial products. It is present in granules that vary in shape in the form of amylose and amylopectin. Starch-degrading enzymes are used on a large scale in the production of sweeteners (high fructose corn syrup) and concentrated glucose syrups as substrate for the fermentative production of bioethanol and basic chemicals. Over the last two decades α-glucanotransferases (EC 2.4.1.xx), such as branching enzyme (EC 2.4.1.18) and 4-α-glucanotransferase (EC 2.4.1.25), have received considerable attention. These enzymes do not hydrolyze the starch as amylases do. Instead, α-glucanotransferases remodel parts of the amylose and amylopectin molecules by cleaving and reforming α-1,4- and α-1,6-glycosidic bond. Here we review the properties of α-glucanotransferases and discuss the emerging use of these enzymes in the generation of novel starch derivatives. [Copyright &y& Elsevier]

Abstract: Glucansucrases are extracellular enzymes that synthesize a wide variety of α-glucan polymers and oligosaccharides, such as dextran. These carbohydrates have found numerous applications in food and health industries, and can be used as pure compounds or even be produced in situ by generally regarded as safe (GRAS) lactic acid bacteria in food applications. Research in the recent years has resulted in big steps forward in the understanding and exploitation of the biocatalytic potential of glucansucrases. This paper provides an overview of glucansucrase enzymes, their recently elucidated crystal structures, their reaction and product specificity, and the structural analysis and applications of α-glucan polymers. Furthermore, we discuss key developments in the understanding of α-glucan polymer formation based on the recently elucidated three-dimensional structures of glucansucrase proteins. Finally we discuss the (potential) applications of α-glucans produced by lactic acid bacteria in food and health related industries. [Copyright &y& Elsevier]

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering. [ABSTRACT FROM AUTHOR]

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, β- and γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity. [ABSTRACT FROM AUTHOR]

Cyclodextrin glycosyltransferase (CGTase) is a member of the α-amylase family, a large group of enzymes that act on α-glycosidic bonds in starch and related compounds. Over twenty different reaction and product specificities have been found in this family. Although three-dimensional structure elucidation and the biochemical characterization of site-directed mutants have yielded a detailed insight into the mechanism of bond cleavage, the variation in reaction and product specificity is far from understood. This article gives an overview of recent developments in the understanding and engineering of transglycosylation and hydrolysis specificity in CGTase, which is one of the best-studied α-amylase family enzymes. [ABSTRACT FROM AUTHOR]

Food ingredients that can activate and improve immunological defense, against e.g., pathogens, have become a major field of research. Resistant starches (RSs) can resist enzymes in the upper gastrointestinal (GI) tract and induce health benefits. RS-3 physicochemical characteristics such as chain length (DP), A- or B-type crystal, and polydispersity index (PI) might be crucial for immunomodulation by activating human toll-like receptors (hTLRs). We hypothesize that crystal type, DP and PI, alone or in combination, impact the recognition of RS-3 preparations by hTLRs leading to different RS-3 immunomodulatory effects. We studied the activation of hTLR2, hTLR4, and hTLR5 by 0.5, 1 and 2 mg/mL of RS-3. We found strong activation of hTLR2-dependent NF-kB activation with PI <1.25, DP 18 as an A- or B-type crystal. At different doses, NF-kB activation was increased from 6.8 to 7.1 and 10-fold with A-type and 6.2 to 10.2 and 14.4-fold with B-type. This also resulted in higher cytokine production in monocytes. Molecular docking, using amylose-A and B, demonstrated that B-crystals bind hTLR2 promoting hTLR2-1 dimerization, supporting the stronger effects of B-type crystals. Immunomodulatory effects of RS-3 are predominantly hTLR2-dependent, and activation can be tailored by managing crystallinity, chain length, and PI. [Display omitted] [ABSTRACT FROM AUTHOR]