Isolation and classification of Bacillus sp. strains to mine genes encoding perhydrolysis activity

Abstract:

[Background] Subtilisin carlsberg, acetyl xylan esterase and cephalosporin acetyl hydrolase from Bacillus sp. display high perhydrolysis activity in previous reports, and are worth developing for commercial application. [Objective] To produce perhydrolase for enzymatic synthesis of peroxyacetic acid in the future, series of hydrolase with promising perhydrolysis activity-encoding genes were cloned from Bacillus sp. strains isolated from rhizosphere soil samples and natto soybean products. [Methods] Protease-producing thermotolerant Bacillus sp. strains were screened from rhizosphere soil samples and natto soybean products using directed-screening medium, and then identified using 16S rRNA gene sequencing. Three full-length hydrolase genes (including subtilisin carlsberg, acetyl xylan esterase and cephalosporin acetyl hydrolase) were cloned from Bacillus sp. strains and then analyzed using sequence alignment. [Results] Total 85 protease-producing pseudo-Bacillus sp. strains was screened, and then identified and classified into four groups: Bacillus subtilis, Bacillus cereus, Bacillus pumilus and Bacillus megaterium. Three full-length hydrolase genes, subtilisin carlsberg gene (encoding 381 aa) from B. subtilis NSYT-3, acetyl xylan esterase gene (encoding 320 aa) from B. pumilus OSLJ-13, and cephalosporin acetyl hydrolase gene (encoding 318 aa) from B. subtilis NSYT-3, were cloned. All of the simulated 3D structure of subtilisin carlsberg, acetyl xylan esterase, and cephalosporin acetyl hydrolase were coincident with the typical structural characterization of α/β hydrolase fold enzymes. [Conclusion] Cloning and expression of hydrolase with perhydrolysis activity-encoding genes from Bacillus sp. strains will lay the foundation for the enzymatic synthesis technology of peroxyacetic acid.