植物根际芽胞杆菌菌种资源采集及其具有过水解催化活性水解酶基因的克隆

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

摘要:

【背景】芽胞杆菌源枯草杆菌蛋白酶(subtilisin carlsberg)、乙酰基木聚糖酯酶(acetyl xylan esterase)和头孢菌素乙酰水解酶(cephalosporin acetyl hydrolase)具有较高的过水解催化活性，有商业开发价值。【目的】挖掘芽胞杆菌菌株中具有过水解酶催化活性的水解酶蛋白基因，为后续制备过水解酶及酶法合成过氧乙酸奠定基础。【方法】利用定向筛选培养基，从植物根际及纳豆产品中筛选产蛋白酶芽胞杆菌候选菌株，并利用RFLP及16S rRNA基因对其进行鉴定。从蛋白酶高产芽胞杆菌菌株中克隆枯草杆菌蛋白酶、乙酰木聚糖酯酶和头孢菌素乙酰水解酶的全长基因。【结果】从植物根际土壤及纳豆产品中共分离到85个候选菌株，RFLP及16S rRNA基因鉴定结果表明候选菌株均为芽胞杆菌，分别属于Bacillus subtilis、Bacillus cereus、Bacillus pumilus和Bacillus megaterium四个类群。从B. subtilis NSYT-3克隆的枯草杆菌蛋白酶基因编码的多肽链全长381个氨基酸，从B. pumilus OSLJ-3克隆得到的乙酰基木聚糖酯酶基因编码的多肽链全长320个氨基酸，从B. subtilis NSYT-3克隆的头孢菌素乙酰水解酶基因编码的多肽链全长318个氨基酸，3D结构模拟表明这3个酶蛋白均具有α/β水解酶折叠家族蛋白结构特点。【结论】芽胞杆菌源具过水解催化活性水解酶基因的克隆，为后续开发酶法合成过氧乙酸工艺奠定了基础。

关键词:根际细菌，芽胞杆菌，过水解催化活性，枯草杆菌蛋白酶，乙酰木聚糖酯酶，头孢菌素乙酰水解酶

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.

Key words:Rhizosphere bacteria, Bacillus sp., perhydrolysis activity, subtilisin carlsberg, Acetyl xylan esterase, Cephalosporin acetyl hydrolase