Development of an active site titration reagent for α-amylases.
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ABSTRACT:
SUBMITTER: Sweeney RP
PROVIDER: S-EPMC8178983 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Complete (Ba-L) and truncated (Ba-S) forms of alpha-amylases from Bacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the alpha-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same alpha-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as alpha-amylase.
| S-EPMC91620 | biostudies-literature
Project description:1. The cell-bound alpha-amylase of Streptococcus bovis has been isolated from other carbohydrases in the cell extract by chromatography on DEAE-cellulose. The enzyme has been compared with the extracellular alpha-amylase produced by this organism. 2. The two amylases had similar action patterns on amylose, the main product being maltotriose with smaller amounts of maltose and a little glucose. 3. The cell-bound amylase hydrolysed maltopentaose and maltohexaose at a similar rate to the hydrolysis of amylose. Maltotetraose was hydrolysed six times more slowly, and maltotriose 280 times more slowly, than amylose. 4. Studies with end-labelled maltodextrins revealed that the cell-bound alpha-amylase preferentially hydrolysed the third linkage from the non-reducing end, liberating maltotriose. The linkage at the reducing end of maltotriose was more easily hydrolysed than the other. 5. Egg-white lysozyme and the extracellular enzymes of Streptomyces albus lysed the cell walls of Streptococcus bovis, releasing amylase into the medium. In the presence of 0.6 m-sucrose 10% of the maximal amylase activity was released by lysozyme. Suspension of the spheroplasts in dilute buffer caused the rupture of the cytoplasmic membrane and the liberation of amylase. 6. A sensitive method for determining the ability of amylases to degrade starch granules is described.
| S-EPMC1206509 | biostudies-other
Project description:Enzymes produced by microorganisms and plants are used as food additives to aid the processing of foods. Identification of the origin of these enzyme products is important for their proper use. Proteinase digestion of α-amylase products, followed by high performance liquid chromatography (HPLC) analysis, was applied to α-amylase from the mold Aspergillus species, the bacteria Bacillus species, and the actinomycetes Saccharomonospora species. Eighteen commercial products of α-amylase were digested with trypsin and endoproteinase Lys-C and HPLC analyzed. For some proteinase/sample combinations, the area of the intact α-amylase peak decreased and new peaks were detected after digestion. The presence and retention times of the novel peaks were used to group the products. The results from this method, called the proteinase digestion-HPLC method, allowed the classification of the α-amylase products into 10 groups, whereas the results from sodium dodecyl sulfate polyacrylamide gel electrophoresis allowed their classification into seven groups.
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Project description:D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.
| S-EPMC1138640 | biostudies-other
Project description:The aim of this study was to isolate novel enzyme-producing bacteria from vegetation samples from East Antarctica and also to characterize them genetically and biochemically in order to establish their phylogeny. The ability to grow at low temperature and to produce amylases and proteases cold-active was also tested. The results of the 16S rRNA gene sequence analysis showed that the 4 Alga rRNA was 100% identical to the sequences of Streptomyces sp. rRNA from Norway and from the Solomon Islands. The Streptomyces grew well in submerged system at 20°C, cells multiplication up to stationary phase being drastically increased after 120 h of submerged cultivation. The beta-amylase production reached a maximum peak after seven days, while alpha-amylase and proteases were performing biosynthesis after nine days of submerged cultivation at 20°C. Newly Streptomyces were able to produce amylase and proteases in a cold environment. The ability to adapt to low temperature of these enzymes could make them valuable ingredients for detergents, the food industry and bioremediation processes which require low temperatures.
| S-EPMC3768780 | biostudies-literature
Project description:The subfamily GH13_1 of alpha-amylases is typical of Fungi, but it is also found in some unicellular eukaryotes (e.g., Amoebozoa, choanoflagellates) and non-bilaterian Metazoa. Since a previous study in 2007, GH13_1 amylases were considered ancestral to the Unikonts, including animals, except Bilateria, such that it was thought to have been lost in the ancestor of this clade. The only alpha-amylases known to be present in Bilateria so far belong to the GH13_15 and 24 subfamilies (commonly called bilaterian alpha-amylases) and were likely acquired by horizontal transfer from a proteobacterium. The taxonomic scope of Eukaryota genomes in databases has been greatly increased ever since 2007. We have surveyed GH13_1 sequences in recent data from ca. 1600 bilaterian species, 60 non-bilaterian animals and also in unicellular eukaryotes. As expected, we found a number of those sequences in non-bilaterians: Anthozoa (Cnidaria) and in sponges, confirming the previous observations, but none in jellyfishes and in Ctenophora. Our main and unexpected finding is that such fungal (also called Dictyo-type) amylases were also consistently retrieved in several bilaterian phyla: hemichordates (deuterostomes), brachiopods and related phyla, some molluscs and some annelids (protostomes). We discuss evolutionary hypotheses possibly explaining the scattered distribution of GH13_1 across bilaterians, namely, the retention of the ancestral gene in those phyla only and/or horizontal transfers from non-bilaterian donors.
| S-EPMC7003070 | biostudies-literature
Project description:We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation.
| S-EPMC8596790 | biostudies-literature
Project description:Starch degradation in chloroplasts requires ?-amylase (BAM) activity, but in Arabidopsis, there are nine BAM proteins, five of which are thought to be catalytic. Although single-gene knockouts revealed the necessity of BAM3 for starch degradation, contributions of other BAMs are poorly understood. Moreover, it is not possible to detect the contribution of individual BAMs in plants containing multiple active BAMs. Therefore, we constructed a set of five quadruple mutants each expressing only one catalytically active BAM, and a quintuple mutant missing all of these BAMs (B-Null). Using these mutants, we assessed the influence of each individual BAM on plant growth and on leaf starch degradation. Both BAM1 and BAM3 alone support wild-type (WT) levels of growth. BAM3 alone is sufficient to degrade leaf starch completely whereas BAM1 alone can only partially degrade leaf starch. In contrast, BAM2, BAM5, and BAM6 have no detectable effect on starch degradation or plant growth, being comparable with the B-Null plants. B-Null plant extracts contained no measurable amylase activity, whereas BAM3 and BAM1 contributed about 70% and 14% of the WT activity, respectively. BAM2 activity was low but detectable and BAM6 contributed no measurable activity. Interestingly, activity of BAM1 and BAM3 in the mutants varied little developmentally or diurnally, and did not increase appreciably in response to osmotic or cold stress. With these genetic lines, we now have new opportunities to investigate members of this diverse gene family.
| S-EPMC7011640 | biostudies-literature
Project description:BACKGROUND:α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included. RESULTS:In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases. CONCLUSIONS:The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.
| S-EPMC6815381 | biostudies-literature
Project description:Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase ?-(1, 4) or ?-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.
| S-EPMC4135293 | biostudies-literature