Abstract The CAZy database is a web-server for sequence-based classification of carbohydrate-active enzymes that has become the worldwide and indispensable tool for scientists engaged in this research field. It was originally created in 1991 as a classification of glycoside hydrolases (GH) and currently, this section of CAZy represents its largest part counting 172 GH families. The present Opinion paper is devoted to the specificity of α-amylase (EC 3.2.1.1) and its occurrence in the CAZy database. Among the 172 defined GH families, four, i.e. GH13, GH57, GH119 and GH126, may be considered as the α-amylase GH families. This view reflects a historical background and traditions widely accepted during the previous decades with respect to the chronology of creating the individual GH families. It obeys the phenomenon that some amylolytic enzymes, which were used to create the individual GH families and were originally known as α-amylases, according to current knowledge from later, more detailed characterization, need not necessarily represent genuine α-amylases. Our Opinion paper was therefore written in an effort to invite the scientific community to think about that with a mind open to changes and to consider the seemingly unambiguous question in the title as one that may not have a simple answer.

Abstract Sucrose-active enzymes belonging to the glycoside hydrolase (GH) family 70 are attractive tools for the synthesis of oligosaccharides, polysaccharides or glycoconjugates. However, their thermostability is an important issue for the development of robust and cost-effective enzyme-based processes. Indeed, GH70 enzymes are mesophilic and no thermophilic representatives have been described so far. Furthermore, structurally guided engineering is a challenge given the size of these proteins (120 to 250 kDa) and their organization in five domains. Herein, we have investigated the possible role of the domain C in the stability of GH70 enzymes. The alternansucrase (ASR) is the most stable enzyme of the GH70 family. Structural comparison of ASR to other GH70 enzymes highlighted the compactness of its domain C. We assumed that this atypical structure might be involved in the stability of this enzyme and decided to introduce this domain in another much less stable GH70 enzyme of known three-dimensional structure, the branching sucrase GBD-CD2. The chimeric GBD-CD2 exhibited a lower specific activity on sucrose substrate but its specificity was unchanged with the enzyme remaining specific for the branching of dextran via α-1,2 linkage formation. Interestingly, the chimera showed a higher melting temperature and residual activity than the wild-type enzyme after 10 min incubation at 30 °C showing that the domain C can affect GH70 enzyme stability and could be a potential target of both random or rational mutagenesis to further improve their stability.

Abstract Pozol is a beverage made with maize dough that is prepared after boiling the kernels in limewater, causing a decrease in soluble sugars, with starch being the main fermentable carbohydrate in the dough. Previously, Streptococcus infantarius ssp. infantarius 25124 (Sii-25124) was identified as the most amylolytic bacteria isolated in this product. Analysis of Sii-25124 amylolytic enzymes revealed two amylases, a cytoplasmic α-amylase of 55.7 kDa and an extracellular amylopullulanase of 246.3 kDa, with two catalytic domains, one typical of an α-amylase and another typical of a pullulanase/glycogen debranching enzyme. Characterization of the joint activity of both enzymes using Sii-25124 cell lysate supernatant demonstrated stability between 30 °C and 45°C, and pH stability in a range between 6.8 and 8.0. The joint activity of Sii-25124 amylases showed a fast production of reducing sugars when starch was used as the substrate. In contrast, reducing sugar production from amylopectin was lower, but it steadily increased throughout the reaction time. The amylopullulanase produced by Sii-25124 hydrolyzes the starch in the dough to produce low molecular weight oligosaccharides, which may be transported into Sii-25124 cells, so that intracellular α-amylase hydrolyzes them to mono- and disaccharides. Amylopullulanase production by Sii-25124 could be an example of a specialized enzyme that successfully dominates starchy food fermentation.

Abstract Cyclodextrin-hydrolyzing enzymes are widespread in bacteria and archaea where they play their roles in carbohydrates metabolism. They were previously characterized as cyclodextrinases, neopullulanases and maltogenic amylases. In the Carbohydrate-Active enZyme (CAZy) database, most of these enzymes are grouped into the GH13_20 subfamily of the α-amylase family GH13. Here, we have summarized the information available on the substrate specificity, structural features, physiological roles and applications of cyclodextrin-preferring glycoside hydrolases. These enzymes form a distinct group in the α-amylase family. Members of this distinct group possess an extra extension at the N-terminus, which causes a modification of the active site geometry thus making these enzymes more specific for smaller molecules like cyclodextrins than for macromolecules such as starches or pullulan. Multi-substrate specificity, hydrolytic as well as transglycosylation activities make these enzymes attractive for applications in the food and pharmaceutical industries. We have tried here to collect information available on their biochemical properties, three-dimensional structures, physiological roles and potential applications.

Abstract The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

Abstract The 4,6-α-glucanotransferases of the glycoside hydrolase family 70 can convert starch into isomaltooligosaccharides (IMOs). However, no thermostable 4,6-α-glucanotransferases have been reported to date, limiting their applicability in the starch conversion industry. Here we report the identification and characterization of a thermostable 4,6-α-glucanotransferase from Bacillus coagulans DSM 1. The gene was cloned and the recombinant protein, called BcGtfC, was produced in Escherichia coli. BcGtfC is stable up to 66 °C in the presence of substrate. It converts debranched starch into an IMO product with a high percentage of α-1,6-glycosidic linkages and a relatively high molecular weight compared to commercially available IMOs. Importantly, the product is only partly and very slowly digested by rat intestine powder, suggesting that the IMO will provide a low glycaemic response in vivo when applied as food ingredient. Thus, BcGtfC is a thermostable 4,6-α-glucanotransferase suitable for the industrial production of slowly digestible IMOs from starch.

Abstract Glycogen is a natural polysaccharide with a dendrimer structure, in which glucose is frequently branched and polymerized. Functionalizing the numerous non-reducing ends on the molecular surface of glycogen could be expected to enable its use in various fields. We developed a method for enzymatically synthesizing a suitable form of glycogen from sucrose by using sucrose phosphorylase and branching enzyme, both of which belong to the α-amylase family, as well as glucan phosphorylase. We refer to this enzymatically synthesized glycogen as the glucan dendrimer (GD). We then selectively modified the non-reducing ends on the surface of GD particles by using the reaction of glucan phosphorylase with various hexose 1-phosphates. Modifying the non-reducing ends of GD with glucuronic acid or glucosamine added negative and positive charges to the GD particles. In addition, we found that glucuronic acid and/or glucosamine residues at the non-reducing ends can be used to covalently conjugate functional substances, such as sugar chains, proteins, and peptides to the surface of GD particles. GD and modification of its non-reducing ends represent versatile platforms for pharmaceutical applications of polysaccharides.

Abstract α-Amylases from a huge number of sources have been isolated and characterised but very few of them meet the demands of the industries. The industrial processes take place under conditions hostile to biocatalysts thus increasing the industrial demand for a highly stable enzyme in good titre level. Improved understanding of biomolecular aspects of α-amylases has led to the advanced understanding of their catalytic nature. Enzymes with high stability are obtained from extremophiles. Extensive studies have demonstrated the importance of regulating expression and catalytic efficiency of nonextremophiles through genetic engineering, directed evolution and chemical modifications. The inability to culture most microorganisms in the environment by standard methods has also led to the focus on the development of metagenomics for getting improved biocatalytic functions. The present review aims to compile the studies reported by researchers in manipulating nonextremophiles and improving stability through directed evolution, metagenomics and protein engineering.

Abstract Bacteria in the human gut including Ruminococcus bromii and Eubacterium rectale encode starch-active enzymes that dictate how these bacteria interact with starch to initiate a metabolic cascade that leads to increased butyrate. Here, we determined the structures of two predicted secreted glycoside hydrolase 13 subfamily 36 (GH13_36) enzymes: ErAmy13B complexed with maltotetraose from E. rectale and RbAmy5 from R. bromii. The structures show a limited binding pocket extending from –2 through +2 subsites with limited possibilities for substrate interaction beyond this, which contributes to the propensity for members of this family to produce maltose as their main product. The enzyme structures reveal subtle differences in the +1/+2 subsites that may restrict the recognition of larger starch polymers by ErAmy13B. Our bioinformatic analysis of the biochemically characterized members of the GH13_36 subfamily, which includes the cell-surface GH13 SusG from Bacteroides thetaiotaomicron, suggests that these maltogenic amylases (EC 3.2.1.133) are usually localized to the outside of the cell, display a range of substrate preferences, and most likely contribute to maltose liberation at the cell surface during growth on starch. A broader comparison between GH13_36 and other maltogenic amylase subfamilies explain how the activity profiles of these enzymes are influenced by their structures.

Abstract A psychrophilic and halophilic bacterial isolate, Shewanella sp. ISTPL2, procured from the pristine Pangong Lake, Ladakh, Jammu and Kashmir, India, was used for the production and characterization of the psychrophilic and alkalophilic α-amylase enzyme. The α-amylase is a critical enzyme that catalyses the hydrolysis of α-1,4-glycosidic bonds of starch molecules and is predominately utilized in biotechnological applications. The highest enzyme activity of partially purified extracellular α-amylase was 10,064.20 U/mL after 12 h of incubation in a shake flask at pH 6.9 and 10 °C. Moreover, the maximum intracellular α-amylase enzyme activity (259.62 U/mL) was also observed at 6 h of incubation. The extracellular α-amylase was refined to the homogeneity with the specific enzyme activity of 36,690.47 U/mg protein corresponding to 6.87-fold purification. The optimized pH and temperature for the α-amylase were found to be pH 8 and 4 °C, respectively, suggesting its stability at alkaline conditions and low or higher temperatures. The amylase activity was highly activated by Cu2+, Fe2+ and Ca2+, while inhibited by Cd2+, Co2+ and Na2+. As per our knowledge, the current study reports the highest activity of a psychrophilic α-amylase enzyme providing prominent biotechnological potential.