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Improve thermostability of Bacillus sp. TS chitosanase through structure-based alignment.

ABSTRACT: Chitosanases can catalyze the release of chitooligosaccharides which have a number of medical applications. Therefore, Chitosanases are good candidates for large-scale enzymatic synthesis due to their favorable thermostability properties and high catalytic efficiency. To further improve the thermostability of a chitosanase from Bacillus sp. TS, which has a half-life of 5.32 min, we mutated specific serine residues that we identified as potentially relevant through structure comparison with thermophilic CelA from Clostridium thermocellum. Out of a total of 15 mutants, three, namely S265G, S276A, and S347G, show higher thermostability. Their half-lives at 60 °C were calculated as 34.57 min, 36.79 min and 7.2 min. The K_m values of S265G, S276A and S347G mutants show substrate binding ability comparable to that of the wild-type enzyme, while the S265G mutant displays a significant decrease of enzymatic activities. Additionally, we studied the synergistic effects of combined mutations, observing that all double mutants and the triple mutant are more stable than the wild-type enzyme and single mutants. Finally, we investigated the mechanisms which might give a reasonable explanation for the improved thermostability via comparative analysis of the resulting 3D structures.

SUBMITTER: Zhou Z

PROVIDER: S-EPMC8339078 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.

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Improve thermostability of Bacillus sp. TS chitosanase through structure-based alignment.

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