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Alpha-amylase, the major form of amylase with secondary carbohydrate binding sites, is a crucial enzyme throughout the growth period and life cycle of angiosperm. In rice, alpha-amylase isozymes are critical for the formation of the storage starch granule during seed maturation and motivate the stored starch to nourish the developing seedling during seed germination which will directly affect the plant growth and field yield. Alpha-amylase has not yet been studied intensely to understand its classification, structure, expression trait, and expression regulation in rice and other crops. Among the 10-rice alpha-amylases, most were exclusively expressed in the developing seed embryo and induced in the seed germination process. During rice seed germination, the expression of alpha-amylase genes is known to be regulated negatively by sugar in embryos, however positively by gibberellin (GA) in endosperm through competitively binding to the specific promoter domain; besides, it is also controlled by a series of other abiotic or biotic factors, such as salinity. In this review, we overviewed the research progress of alpha-amylase with focus on seed germination and reflected on how in-depth work might elucidate its regulation and facilitate crop breeding as an efficient biomarker.

Currently, the main α-amylase family GH13 has been divided into 47 subfamilies in CAZy, with new subfamilies regularly emerging. The present in silico study was performed to highlight the groups, represented by the maltogenic amylase from Thermotoga neapolitana and the α-amylase from Haloarcula japonica , which are worth of creating their own new GH13 subfamilies. This enlarges functional annotation and thus allows more precise prediction of the function of putative proteins. Interestingly, those two share certain sequence features, e.g. the highly conserved cysteine in the second conserved sequence region (CSR-II) directly preceding the catalytic nucleophile, or the well-preserved GQ character of the end of CSR-VII. On the other hand, the two groups bear also specific and highly conserved positions that distinguish them not only from each other but also from representatives of remaining GH13 subfamilies established so far. For the T. neapolitana maltogenic amylase group, it is the stretch of residues at the end of CSR-V highly conserved as L-[DN]. The H. japonica α-amylase group can be characterized by a highly conserved [WY]-[GA] sequence at the end of CSR-II. Other specific sequence features include an almost fully conserved aspartic acid located directly preceding the general acid/base in CSR-III or well-preserved glutamic acid in CSR-IV. The assumption that these two groups represent two mutually related, but simultaneously independent GH13 subfamilies has been supported by phylogenetic analysis as well as by comparison of tertiary structures. The main α-amylase family GH13 has thus been expanded by two novel subfamilies GH13_48 and GH13_49. Key points • In silico analysis of two groups of family GH13 members with characterized representatives • Identification of certain common, but also some specific sequence features in seven CSRs • Creation of two novel subfamilies—GH13_48 and GH13_49 within the CAZy database

Amylase is amongst the most indispensable enzymes that have a large number of applications in laboratories and industries. Mostly, α-amylase is synthesized from microbes such as bacteria, fungi and yeast. Due to the high demand for α-amylase, its synthesis can be enhanced using recombinant DNA technology, different fermentation methods, less expensive and good carbon and nitrogen sources, and optimizing the various parameters during fermentation, e.g., temperature, pH and fermentation duration. Various methods are used to measure the production and activity of synthesized α-amylase like iodine, DNS, NS and dextrinizing methods. The activity of crude α-amylase can be elevated to the maximum level by optimizing the temperature and pH. Some metals also interact with α-amylase and increase its activity like K+, Na+, Mg2+ and Ca2+. Some industries such as starch conversion, food, detergent, paper, textile industries and fuel alcohol production extensively utilize α-amylase for their various purposes.

Mario Falchi, Philippe Froguel and colleagues report association of a multi-allelic copy number variant encompassing the salivary amylase gene AMY1 with body mass index and risk of obesity. Common multi-allelic copy number variants (CNVs) appear enriched for phenotypic associations compared to their biallelic counterparts 1 , 2 , 3 , 4 . Here we investigated the influence of gene dosage effects on adiposity through a CNV association study of gene expression levels in adipose tissue. We identified significant association of a multi-allelic CNV encompassing the salivary amylase gene ( AMY1 ) with body mass index (BMI) and obesity, and we replicated this finding in 6,200 subjects. Increased AMY1 copy number was positively associated with both amylase gene expression ( P = 2.31 × 10 −14 ) and serum enzyme levels ( P < 2.20 × 10 −16 ), whereas reduced AMY1 copy number was associated with increased BMI (change in BMI per estimated copy = −0.15 (0.02) kg/m 2 ; P = 6.93 × 10 −10 ) and obesity risk (odds ratio (OR) per estimated copy = 1.19, 95% confidence interval (CI) = 1.13–1.26; P = 1.46 × 10 −10 ). The OR value of 1.19 per copy of AMY1 translates into about an eightfold difference in risk of obesity between subjects in the top (copy number > 9) and bottom (copy number < 4) 10% of the copy number distribution. Our study provides a first genetic link between carbohydrate metabolism and BMI and demonstrates the power of integrated genomic approaches beyond genome-wide association studies.

α-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4–7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α) 8 -barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α) 7 -barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.

Cold-adapted enzymes from psychrophilic species show the general characteristics of being more heat labile, and having a different balance between enthalpic and entropic contributions to free energy barrier of the catalyzed reaction compared to mesophilic orthologs. Among cold-adapted enzymes, there are also examples that show an enigmatic inactivation at higher temperatures before unfolding of the protein occurs. Here, we analyze these phenomena by extensive computer simulations of the catalytic reactions of psychrophilic and mesophilic α-amylases. The calculations yield temperature dependent reaction rates in good agreement with experiment, and also elicit the anomalous rate optimum for the cold-adapted enzyme, which occurs about 15 °C below the melting point. This result allows us to examine the structural basis of thermal inactivation, which turns out to be caused by breaking of a specific enzyme-substrate interaction. This type of behaviour is also likely to be relevant for other enzymes displaying such anomalous temperature optima. Enzymes from organisms inhabiting cold environments (psychrophiles) have adapted to catalyzing chemical reactions at near freezing temperatures. Here – using molecular dynamics simulations – the authors analyze cold adaptation of psychrophilic α-amylase and provide the structural basis for its low anomalous temperature optimum: the increased mobility of a surface loop involved in substrate interaction.

This study focuses on synthesizing a new series of isoxazolinyl-1,2,3-triazolyl-[1,4]-benzoxazin-3-one derivatives 5a–5o. The synthesis method involves a double 1,3-dipolar cycloaddition reaction following a “click chemistry” approach, starting from the respective [1,4]-benzoxazin-3-ones. Additionally, the study aims to evaluate the antidiabetic potential of these newly synthesized compounds through in silico methods. This synthesis approach allows for the combination of three heterocyclic components: [1,4]-benzoxazin-3-one, 1,2,3-triazole, and isoxazoline, known for their diverse biological activities. The synthesis procedure involved a two-step process. Firstly, a 1,3-dipolar cycloaddition reaction was performed involving the propargylic moiety linked to the [1,4]-benzoxazin-3-one and the allylic azide. Secondly, a second cycloaddition reaction was conducted using the product from the first step, containing the allylic part and an oxime. The synthesized compounds were thoroughly characterized using spectroscopic methods, including 1H NMR, 13C NMR, DEPT-135, and IR. This molecular docking method revealed a promising antidiabetic potential of the synthesized compounds, particularly against two key diabetes-related enzymes: pancreatic α-amylase, with the two synthetic molecules 5a and 5o showing the highest affinity values of 9.2 and 9.1 kcal/mol, respectively, and intestinal α-glucosidase, with the two synthetic molecules 5n and 5e showing the highest affinity values of −9.9 and −9.6 kcal/mol, respectively. Indeed, the synthesized compounds have shown significant potential as antidiabetic agents, as indicated by molecular docking studies against the enzymes α-amylase and α-glucosidase. Additionally, ADME analyses have revealed that all the synthetic compounds examined in our study demonstrate high intestinal absorption, meet Lipinski’s criteria, and fall within the required range for oral bioavailability, indicating their potential suitability for oral drug development.

Background Amylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box–Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied. Results The substrate optimization for α-amylase production by the Box – Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae , as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L. Conclusions The process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect ( p <  0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

Alpha-amylase (AMY)- and beta-amylase (BAM)-mediated starch degradation plays central roles in carbohydrate metabolism and participates extensively in the regulation of a wide range of biological processes, including growth, development and stress response. However, the AMY and BAM genes in tea plants (Camellia sinensis) are poorly understood, and the biological functions of these genes remain to be elucidated. In this study, three CsAMY and nine CsBAM genes from tea plants were identified based on genomic and transcriptomic database analyses, and the genes were subjected to comprehensive bioinformatic characterization. Phylogenetic analysis showed that the CsAMY proteins could be clustered into three different subfamilies, and nine CsBAM proteins could be classified into four groups. Putative catalytically active proteins were identified based on multiple sequence alignments, and the tertiary structures of these proteins were analyzed. Ciselement analysis indicated that CsAMY and CsBAM were extensively involved in tea plant growth, development and stress response. In addition, the CsAMY and CsBAM genes were differentially expressed in various tissues and were regulated by stress treatments (e.g., ABA, cold, drought and salt stress), and the expression patterns of these genes were associated with the postharvest withering and rotation processes. Taken together, our results will enhance the understanding of the roles of the CsAMY and CsBAM gene families in the growth, development and stress response of tea plants and of the potential functions of these genes in determining tea quality during the postharvest processing of tea leaves.

In this study, we investigated MalS, a periplasmic α-enzyme from Escherichia coli K12, known for its unique biochemical properties related to polysaccharide utilization. Evolutionarily, MalS has inherited the glycosyl hydrolase catalytic domain from the glycoside hydrolase family 13, with the protein sequences highly conserved across Enterobacteria , including Salmonella and Shigella . MalS exhibited optimal activity at 65 °C, significantly higher than other E. coli enzymes. Although its reaction pattern resembled that of typical α-amylases, its catalytic efficiency on polysaccharides was notably lower. Intriguingly, MalS demonstrated a strong binding affinity for various glucose polymers, including β-cyclodextrin and glycogen, which significantly enhanced its thermostability. Despite full-length MalS binding strongly to glycogen, neither its N-terminal domain, predicted by AlphaFold2 to belong to the Carbohydrate-Binding Module family 69, nor the remaining parts of the enzyme showed binding affinity toward polysaccharides. Kinetic studies revealed that MalS had a 2.5-fold lower K m and 1.4-fold higher catalytic efficiency toward glycogen compared to amylopectin, which contrasts starkly with pancreatic α-amylases. However, over prolonged reactions, glycogen hydrolysis by MalS was slower than that of amylopectin. In the early initial stage, MalS predominantly degraded glycogen to maltopentaose (G5) rather than maltohexaose (G6) as usual. Taken together, these findings suggest MalS may play a role in recognizing glycogen-type polysaccharides in the bacterial periplasm during adaptation to new environments. Given the crucial role of glycogen in the survival and infection processes of pathogenic bacteria, understanding MalS’s interaction with glycogen-type polysaccharides could offer valuable insights into bacterial survival mechanisms and their ability to infect hosts. Key points • MalS has unique structure and properties but conserved among many enterobacteria • Binding of MalS with polysaccharides significantly enhanced its thermostability • Unlike other amylases, MalS showed 2.5-fold lower K m on glycogen than amylopectin Graphical Abstract