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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Bioorganic Chemistry".

Deadline for manuscript submissions: closed (31 March 2023) | Viewed by 23195

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Special Issue Editor

Prof. Dr. Stefan Janecek

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Laboratory of Protein Evolution, Institute of Molecular Biology, Slovak Academy of Sciences, SK-84551 Bratislava, Slovakia
Interests: amylolytic enzymes; starch/glycogen-binding domains; glycoside hydrolases; in-silico protein structure analysis; protein bioinformatics
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Special Issue Information

Dear Colleagues,

The international symposia on the ALpha-AMYlase family (ALAMYs— http://imb.savba.sk/~janecek/Alamys/) was established in 2001. From the beginning, being organized once every three years, each ALAMY has been held at Smolenice Castle in Slovakia, the Congress Centre of the Slovak Academy of Sciences. The castle is situated on a hill above the small city of Smolenice, which lies at the foot of Small Carpathian Mountains, 60 km north-east from Bratislava. The beautiful natural surroundings and attractive interiors of the castle create a special atmosphere for scientific discussions, relaxation and meeting friends.

This Special Issue of Molecules (https://www.mdpi.com/journal/molecules/special_issues/G7C8D1F97U), entitled “Advances in Amylases”, has been arranged to collect contributions presented during the 8^th edition of this series of conferences, The Eighth Symposium on the Alpha-Amylase Family (ALAMY_8—http://imb.savba.sk/~janecek/Alamys/Alamy_8/), due to be held October 9-13, 2022.

This Special Issue welcomes manuscripts dealing with the cloning, sequencing, expression, biochemical characterization, tertiary structure determination, structure/function relationships, and protein design and evolution of starch hydrolases and related alpha-glucan active enzymes. Topics of interest include, but are not necessarily limited to, various aspects of the main alpha-amylase enzyme clan GH-H (i.e., families GH13, GH70 and GH77), as well as of the smaller alpha-amylase families—mainly GH57, but also GH119 and even GH126. Starch-active LPMOs from the family AA13 are equally welcome. Last but not least, any aspects of starch and glycogen (in general, an alpha-glucan) binding, representing distinct CBMs and/or surface-binding sites, are also within the scope of this Special Issue. In addition to basic research-oriented studies, emphasis may also be given to potential applications, especially in biotechnology and medicine.

Three types of contributions will be considered: (i) articles; (ii) reviews; and (iii) perspectives.

Finally, this Special Issue is open not only for participants of the ALAMY_8 Symposium; all “amylase-positive” people are welcome to contribute!

Prof. Dr. Stefan Janecek
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Published Papers (14 papers)

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Editorial

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4 pages, 231 KiB

Open AccessEditorial

Advances in Amylases—What’s Going on?

by Štefan Janeček

Molecules 2023, 28(21), 7268; https://doi.org/10.3390/molecules28217268 - 25 Oct 2023

Viewed by 748

Abstract

With regard to the CAZy database ( [...] Full article

(This article belongs to the Special Issue Advances in Amylases)

Research

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19 pages, 4976 KiB

Open AccessArticle

Binding Specificity of a Novel Cyclo/Maltodextrin-Binding Protein and Its Role in the Cyclodextrin ABC Importer System from Thermoanaerobacterales

by Jorge Aranda-Caraballo, Roberto A. Saenz, Alonso A. López-Zavala, Beatriz Velazquez-Cruz, Laura Espinosa-Barrera, Yair Cárdenas-Conejo, Andrés Zárate-Romero, Oscar Linares-Vergara, Juan A. Osuna-Castro, Edgar Bonales-Alatorre, Sara Centeno-Leija and Hugo Serrano-Posada

Molecules 2023, 28(16), 6080; https://doi.org/10.3390/molecules28166080 - 16 Aug 2023

Viewed by 1219

Abstract

Extracellular synthesis of functional cyclodextrins (CDs) as intermediates of starch assimilation is a convenient microbial adaptation to sequester substrates, increase the half-life of the carbon source, carry bioactive compounds, and alleviate chemical toxicity through the formation of CD-guest complexes. Bacteria encoding the four steps of the carbohydrate metabolism pathway via cyclodextrins (CM-CD) actively internalize CDs across the microbial membrane via a putative type I ATP-dependent ABC sugar importer system, MdxEFG-(X/MsmX). While the first step of the CM-CD pathway encompasses extracellular starch-active cyclomaltodextrin glucanotransferases (CGTases) to synthesize linear dextrins and CDs, it is the ABC importer system in the second step that is the critical factor in determining which molecules from the CGTase activity will be internalized by the cell. Here, structure-function relationship studies of the cyclo⁄maltodextrin-binding protein MdxE of the MdxEFG-MsmX importer system from Thermoanaerobacter mathranii subsp. mathranii A3 are presented. Calorimetric and fluorescence studies of recombinant MdxE using linear dextrins and CDs showed that although MdxE binds linear dextrins and CDs with high affinity, the open-to-closed conformational change is solely observed after α- and β-CD binding, suggesting that the CM-CD pathway from Thermoanaerobacterales is exclusive for cellular internalization of these molecules. Structural analysis of MdxE coupled with docking simulations showed an overall architecture typically found in sugar-binding proteins (SBPs) that comprised two N- and C-domains linked by three small hinge regions, including the conserved aromatic triad Tyr193/Trp269/Trp378 in the C-domain and Phe87 in the N-domain involved in CD recognition and stabilization. Structural bioinformatic analysis of the entire MdxFG-MsmX importer system provided further insights into the binding, internalization, and delivery mechanisms of CDs. Hence, while the MdxE-CD complex couples to the permease subunits MdxFG to deliver the CD into the transmembrane channel, the dimerization of the cytoplasmatic promiscuous ATPase MsmX triggers active transport into the cytoplasm. This research provides the first results on a novel thermofunctional SBP and its role in the internalization of CDs in extremely thermophilic bacteria. Full article

(This article belongs to the Special Issue Advances in Amylases)

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22 pages, 5404 KiB

Open AccessArticle

Structural and Functional Characterization of Drosophila melanogaster α-Amylase

by Moez Rhimi, Jean-Luc Da Lage, Richard Haser, Georges Feller and Nushin Aghajari

Molecules 2023, 28(14), 5327; https://doi.org/10.3390/molecules28145327 - 11 Jul 2023

Cited by 2 | Viewed by 1317

Abstract

Insects rely on carbohydrates such as starch and glycogen as an energy supply for growth of larvae and for longevity. In this sense α-amylases have essential roles under extreme conditions, e.g., during nutritional or temperature stress, thereby contributing to survival of the insect. This makes them interesting targets for combating insect pests. Drosophila melanogaster α-amylase, DMA, which belongs to the glycoside hydrolase family 13, sub family 15, has been studied from an evolutionary, biochemical, and structural point of view. Our studies revealed that the DMA enzyme is active over a broad temperature and pH range, which is in agreement with the fluctuating environmental changes with which the insect is confronted. Crystal structures disclosed a new nearly fully solvated metal ion, only coordinated to the protein via Gln263. This residue is only conserved in the subgroup of D. melanogaster and may thus contribute to the enzyme adaptive response to large temperature variations. Studies of the effect of plant inhibitors and the pseudo-tetrasaccharide inhibitor acarbose on DMA activity, allowed us to underline the important role of the so-called flexible loop on activity/inhibition, but also to suggest that the inhibition modes of the wheat inhibitors WI-1 and WI-3 on DMA, are likely different. Full article

(This article belongs to the Special Issue Advances in Amylases)

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18 pages, 3879 KiB

Open AccessArticle

Novel Design of an α-Amylase with an N-Terminal CBM20 in Aspergillus niger Improves Binding and Processing of a Broad Range of Starches

by Andika Sidar, Gerben P. Voshol, Erik Vijgenboom and Peter J. Punt

Molecules 2023, 28(13), 5033; https://doi.org/10.3390/molecules28135033 - 27 Jun 2023

Cited by 3 | Viewed by 1354

Abstract

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes. Full article

(This article belongs to the Special Issue Advances in Amylases)

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12 pages, 4027 KiB

Open AccessArticle

The Structure of Maltooctaose-Bound Escherichia coli Branching Enzyme Suggests a Mechanism for Donor Chain Specificity

by Remie Fawaz, Courtney Bingham, Hadi Nayebi, Janice Chiou, Lindsey Gilbert, Sung Hoon Park and James H. Geiger

Molecules 2023, 28(11), 4377; https://doi.org/10.3390/molecules28114377 - 27 May 2023

Cited by 1 | Viewed by 1067

Abstract

Glycogen is the primary storage polysaccharide in bacteria and animals. It is a glucose polymer linked by α-1,4 glucose linkages and branched via α-1,6-linkages, with the latter reaction catalyzed by branching enzymes. Both the length and dispensation of these branches are critical in defining the structure, density, and relative bioavailability of the storage polysaccharide. Key to this is the specificity of branching enzymes because they define branch length. Herein, we report the crystal structure of the maltooctaose-bound branching enzyme from the enterobacteria E. coli. The structure identifies three new malto-oligosaccharide binding sites and confirms oligosaccharide binding in seven others, bringing the total number of oligosaccharide binding sites to twelve. In addition, the structure shows distinctly different binding in previously identified site I, with a substantially longer glucan chain ordered in the binding site. Using the donor oligosaccharide chain-bound Cyanothece branching enzyme structure as a guide, binding site I was identified as the likely binding surface for the extended donor chains that the E. coli branching enzyme is known to transfer. Furthermore, the structure suggests that analogous loops in branching enzymes from a diversity of organisms are responsible for branch chain length specificity. Together, these results suggest a possible mechanism for transfer chain specificity involving some of these surface binding sites. Full article

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13 pages, 2987 KiB

Open AccessArticle

Exploration of the Transglycosylation Activity of Barley Limit Dextrinase for Production of Novel Glycoconjugates

by Malene Bech Vester-Christensen, Jesper Holck, Martin Rejzek, Léa Perrin, Morten Tovborg, Birte Svensson, Robert A. Field and Marie Sofie Møller

Molecules 2023, 28(10), 4111; https://doi.org/10.3390/molecules28104111 - 16 May 2023

Cited by 1 | Viewed by 1508

Abstract

A few α-glucan debranching enzymes (DBEs) of the large glycoside hydrolase family 13 (GH13), also known as the α-amylase family, have been shown to catalyze transglycosylation as well as hydrolysis. However, little is known about their acceptor and donor preferences. Here, a DBE from barley, limit dextrinase (HvLD), is used as a case study. Its transglycosylation activity is studied using two approaches; (i) natural substrates as donors and different p-nitrophenyl (pNP) sugars as well as different small glycosides as acceptors, and (ii) α-maltosyl and α-maltotriosyl fluorides as donors with linear maltooligosaccharides, cyclodextrins, and GH inhibitors as acceptors. HvLD showed a clear preference for pNP maltoside both as acceptor/donor and acceptor with the natural substrate pullulan or a pullulan fragment as donor. Maltose was the best acceptor with α-maltosyl fluoride as donor. The findings highlight the importance of the subsite +2 of HvLD for activity and selectivity when maltooligosaccharides function as acceptors. However, remarkably, HvLD is not very selective when it comes to aglycone moiety; different aromatic ring-containing molecules besides pNP could function as acceptors. The transglycosylation activity of HvLD can provide glycoconjugate compounds with novel glycosylation patterns from natural donors such as pullulan, although the reaction would benefit from optimization. Full article

(This article belongs to the Special Issue Advances in Amylases)

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15 pages, 8073 KiB

Open AccessArticle

The Distinctive Permutated Domain Structure of Periplasmic α-Amylase (MalS) from Glycoside Hydrolase Family 13 Subfamily 19

by Yan An, Phuong Lan Tran, Min-Jee Yoo, Hyung-Nam Song, Kwang-Hyun Park, Tae-Jip Kim, Jong-Tae Park and Eui-Jeon Woo

Molecules 2023, 28(10), 3972; https://doi.org/10.3390/molecules28103972 - 09 May 2023

Cited by 1 | Viewed by 1945

Abstract

Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120–180 (N-terminal) and 646–676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, β-CD binds more weakly than the linear substrate, possibly due to the positioning of A402. MalS has two Ca²⁺ binding sites that contribute significantly to the thermostability of the enzyme. Intriguingly, the study found that MalS exhibits a high binding affinity for polysaccharides such as glycogen and amylopectin. The N domain, of which the electron density map was not observed, was predicted to be CBM69 by AlphaFold2 and might have a binding site for the polysaccharides. Structural analysis of MalS provides new insight into the structure–evolution relationship in GH13 subfamily 19 enzymes and a molecular basis for understanding the details of catalytic function and substrate binding of MalS. Full article

(This article belongs to the Special Issue Advances in Amylases)

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18 pages, 3340 KiB

Open AccessArticle

Increasing Protein Content of Rice Flour with Maintained Processability by Using Granular Starch Hydrolyzing Enzyme

by Jinxing Zhai, Xiaoxiao Li, Birte Svensson, Zhengyu Jin and Yuxiang Bai

Molecules 2023, 28(8), 3522; https://doi.org/10.3390/molecules28083522 - 17 Apr 2023

Viewed by 1780

Abstract

Rice flour (RF) has become a promising food material. In the present study, RF with higher protein content was prepared using a granular starch hydrolyzing enzyme (GSHE). Particle size, morphology, crystallinity, and molecular structures of RF and rice starch (RS) were characterized to establish a hydrolytic mechanism; thermal, pasting, and rheological properties were determined to evaluate processability using differential scanning calorimetry (DSC), rapid viscosity analysis (RVA), and rheometer, respectively. The GSHE treatment resulted in pinholes, pits, and surface erosion through sequential hydrolysis of crystalline and amorphous areas on the starch granule surface. The amylose content decreased with hydrolysis time, while the very short chains (DP < 6) increased rapidly at 3 h but decreased slightly later. After hydrolysis for 24 h, the protein content in RF increased from 8.52% to 13.17%. However, the processability of RF was properly maintained. Specifically, the data from DSC showed that the conclusion temperature and endothermic enthalpy of RS barely changed. The result of rapid RVA and rheological measurement indicated that RF paste viscosity and viscoelastic properties dropped rapidly after 1 h hydrolysis and thereafter recovered slightly. This study provided a new RF raw material useful for improving and developing RF-based foods. Full article

(This article belongs to the Special Issue Advances in Amylases)

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16 pages, 2490 KiB

Open AccessArticle

Alteration of Substrate Specificity and Transglucosylation Activity of GH13_31 α-Glucosidase from Bacillus sp. AHU2216 through Site-Directed Mutagenesis of Asn258 on β→α Loop 5

by Waraporn Auiewiriyanukul, Wataru Saburi, Tomoya Ota, Jian Yu, Koji Kato, Min Yao and Haruhide Mori

Molecules 2023, 28(7), 3109; https://doi.org/10.3390/molecules28073109 - 30 Mar 2023

Viewed by 1587

Abstract

α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on β→α loop 5 of the catalytic (β/α)₈-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation. Full article

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11 pages, 1011 KiB

Open AccessArticle

Importance of Inactivation Methodology in Enzymatic Processing of Raw Potato Starch: NaOCl as Efficient α-Amylase Inactivation Agent

by Signe Schram Zinck, Stefan Jarl Christensen, Ole Bandsholm Sørensen, Birte Svensson and Anne S. Meyer

Molecules 2023, 28(7), 2947; https://doi.org/10.3390/molecules28072947 - 25 Mar 2023

Cited by 3 | Viewed by 1706

Abstract

Efficient inactivation of microbial α-amylases (EC 3.2.1.1) can be a challenge in starch systems as the presence of starch has been shown to enhance the stability of the enzymes. In this study, commonly used inactivation methods, including multistep washing and pH adjustment, were assessed for their efficiency in inactivating different α-amylases in presence of raw potato starch. Furthermore, an effective approach for irreversible α-amylase inactivation using sodium hypochlorite (NaOCl) is demonstrated. Regarding inactivation by extreme pH, the activity of five different α-amylases was either eliminated or significantly reduced at pH 1.5 and 12. However, treatment at extreme pH for 5 min, followed by incubation at pH 6.5, resulted in hydrolysis yields of 42–816% relative to controls that had not been subjected to extreme pH. “Inactivation” by multistep washing with water, ethanol, and acetone followed by gelatinization as preparation for analysis gave significant starch hydrolysis compared to samples inactivated with NaOCl before the wash. This indicates that the further starch degradation observed in samples subjected to washing only took place during the subsequent gelatinization. The current study demonstrates the importance of inactivation methodology in α-amylase-mediated raw starch depolymerization and provides a method for efficient α-amylase inactivation in starch systems. Full article

(This article belongs to the Special Issue Advances in Amylases)

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13 pages, 2716 KiB

Open AccessArticle

Characterization of the Glucan-Branching Enzyme GlgB Gene from Swine Intestinal Bacteria

by Yuqi Shao, Weilan Wang, Ying Hu and Michael G. Gänzle

Molecules 2023, 28(4), 1881; https://doi.org/10.3390/molecules28041881 - 16 Feb 2023

Viewed by 1651

Abstract

Starch hydrolysis by gut microbiota involves a diverse range of different enzymatic activities. Glucan-branching enzyme GlgB was identified as the most abundant glycosidase in Firmicutes in the swine intestine. GlgB converts α-(1→4)-linked amylose to form α-(1→4,6) branching points. This study aimed to characterize GlgB cloned from a swine intestinal metagenome and to investigate its potential role in formation of α-(1→4,6)-branched α-glucans from starch. The branching activity of purified GlgB was determined with six different starches and pure amylose by quantification of amylose after treatment. GlgB reduced the amylose content of all 6 starches and amylose by more than 85% and displayed a higher preference towards amylose. The observed activity on raw starch indicated a potential role in the primary starch degradation in the large intestine as an enzyme that solubilizes amylose. The oligosaccharide profile showed an increased concentration of oligosaccharide introduced by GlgB that is not hydrolyzed by intestinal enzymes. This corresponded to a reduced in vitro starch digestibility when compared to untreated starch. The study improves our understanding of colonic starch fermentation and may allow starch conversion to produce food products with reduced digestibility and improved quality. Full article

(This article belongs to the Special Issue Advances in Amylases)

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17 pages, 2636 KiB

Open AccessArticle

Impact of Starch Binding Domain Fusion on Activities and Starch Product Structure of 4-α-Glucanotransferase

by Yu Wang, Yazhen Wu, Stefan Jarl Christensen, Štefan Janeček, Yuxiang Bai, Marie Sofie Møller and Birte Svensson

Molecules 2023, 28(3), 1320; https://doi.org/10.3390/molecules28031320 - 30 Jan 2023

Cited by 5 | Viewed by 1934

Abstract

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBD_St1 and SBD_St2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBD_GA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBD_GA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2–2.4-fold on amylose and decreased 3–9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3–1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBD_GA-TuαGT and-SBD_St2-TuαGT showed K_d of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBD_St1-TuαGT had 3–5-fold lower affinity. SBD_St2 contributed more than SBD_St1 to activity, substrate binding, and the stability of TuαGT fusions. Full article

(This article belongs to the Special Issue Advances in Amylases)

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17 pages, 8544 KiB

Open AccessArticle

A Novel Subfamily GH13_46 of the α-Amylase Family GH13 Represented by the Cyclomaltodextrinase from Flavobacterium sp. No. 92

by Filip Mareček and Štefan Janeček

Molecules 2022, 27(24), 8735; https://doi.org/10.3390/molecules27248735 - 09 Dec 2022

Cited by 8 | Viewed by 1385

Abstract

In the CAZy database, the α-amylase family GH13 has already been divided into 45 subfamilies, with additional subfamilies still emerging. The presented in silico study was undertaken in an effort to propose a novel GH13 subfamily represented by the experimentally characterized cyclomaltodxtrinase from Flavobacterium sp. No. 92. Although most cyclomaltodextrinases have been classified in the subfamily GH13_20. This one has not been assigned any GH13 subfamily as yet. It possesses a non-specified immunoglobulin-like domain at its N-terminus mimicking a starch-binding domain (SBD) and the segment MPDLN in its fifth conserved sequence region (CSR) typical, however, for the subfamily GH13_36. The searches through sequence databases resulted in collecting a group of 108 homologs forming a convincing cluster in the evolutionary tree, well separated from all remaining GH13 subfamilies. The members of the newly proposed subfamily share a few exclusive sequence features, such as the “aromatic” end of the CSR-II consisting of two well-conserved tyrosines with either glycine, serine, or proline in the middle or a glutamic acid succeeding the catalytic proton donor in the CSR-III. Concerning the domain N of the representative cyclomaltodextrinase, docking trials with α-, β- and γ-cyclodextrins have indicated it may represent a new type of SBD. This new GH13 subfamily has been assigned the number GH13_46. Full article

(This article belongs to the Special Issue Advances in Amylases)

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Review

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24 pages, 3438 KiB

Open AccessReview

Interfacial Catalysis during Amylolytic Degradation of Starch Granules: Current Understanding and Kinetic Approaches

by Yu Tian, Yu Wang, Yuyue Zhong, Marie Sofie Møller, Peter Westh, Birte Svensson and Andreas Blennow

Molecules 2023, 28(9), 3799; https://doi.org/10.3390/molecules28093799 - 28 Apr 2023

Cited by 7 | Viewed by 2537

Abstract

Enzymatic hydrolysis of starch granules forms the fundamental basis of how nature degrades starch in plant cells, how starch is utilized as an energy resource in foods, and develops efficient, low-cost saccharification of starch, such as bioethanol and sweeteners. However, most investigations on starch hydrolysis have focused on its rates of degradation, either in its gelatinized or soluble state. These systems are inherently more well-defined, and kinetic parameters can be readily derived for different hydrolytic enzymes and starch molecular structures. Conversely, hydrolysis is notably slower for solid substrates, such as starch granules, and the kinetics are more complex. The main problems include that the surface of the substrate is multifaceted, its chemical and physical properties are ill-defined, and it also continuously changes as the hydrolysis proceeds. Hence, methods need to be developed for analyzing such heterogeneous catalytic systems. Most data on starch granule degradation are obtained on a long-term enzyme-action basis from which initial rates cannot be derived. In this review, we discuss these various aspects and future possibilities for developing experimental procedures to describe and understand interfacial enzyme hydrolysis of native starch granules more accurately. Full article

(This article belongs to the Special Issue Advances in Amylases)

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