Journal
BIOTECHNOLOGY JOURNAL
Volume -, Issue -, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.202300010
Keywords
Bacillus subtilis; catalytic activity; chitooligosaccharides; chitosanase; GH46; saturation mutagenesis; thermostability
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Catalysis activity and thermostability are important characteristics of enzymes. By mutating the C-terminal K242 of Bacillus subtilis chitosanase BsCsn46A, the K242P mutant showed significantly increased catalytic activity and thermostability, making it a potential candidate for industrial production of chitooligosaccharide (COS).
Catalysis activity and thermostability are some of the fundamental characteristic of enzymes, which are of great significance to their industrial applications. Bacillus subtilis chitosanase BsCsn46A is a kind of enzyme with good catalytic activity and stability, which can hydrolyze chitosan to produce chitobiose and chitotriose. In order to further improve the catalytic activity and stability of BsCsn46A, saturation mutagenesis of the C-terminal K242 of BsCsn46A was performed. The results showed that the six mutants (K242A, K242D, K242E, K242F, K242P, and K242T) showed increased catalytic activ-ity on chitosan. The catalytic activity of K242P increased from 12971 +/- 597 U mg(-1) of wild type to 17820 +/- 344 U mg(-1), and the thermostability of K242P increased by 2.27%. In order to elucidate the reason for the change of enzymatic properties, hydro-gen network, molecular docking, and molecular dynamics simulation were carried out. The hydrogen network results showed that all the mutants lose their interaction with Asp6 at 242 site, thereby increasing the flexibility of Glu19 at the junction sites of alpha 1 and loop1. Molecular dynamics results showed that the RMSD of K242P was lower at both 313 and 323 K than that of other mutants, which supported that K242P had better thermostability. The catalytic activity of mutant K242P reached 17820.27 U mg(-1), the highest level reported so far, which could be a robust candidate for the industrial application of chitooligosaccharide (COS) production.
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