产几丁质酶侧孢短芽孢杆菌的筛选及其酶学性质研究

Isolation of a Chitinase-producing Strain Brevibacillus laterosporus and Its Enzymatic Properties

筛选并鉴定高产几丁质酶的芽孢杆菌菌株,旨在研究其酶学特性为高效利用几丁质酶资源奠定基础。分离纯化产几丁质酶芽孢杆菌,将目的菌株的几丁质酶基因异源表达并纯化后研究其酶学参数。考虑到菌株的生物安全性,选择侧孢短芽孢杆菌CDY64为进一步研究对象,将其几丁质酶基因表达于大肠杆菌BL21中。酶学研究表明,纯化后的几丁质酶最适反应温度为60℃,在p H6.0-8.0范围内均表现出良好的活性;使用胶体几丁质作为底物时Km与kcat值分别为5.85μmol/L和29.27 S-1。结果表明,侧孢短芽孢杆菌CDY64所产几丁质酶在高温下具有良好的催化能力,在体外对多种植物病原真菌表现出了良好的拮抗作用。

This study aims to screen and identify Bacillus strain producing high yield of chitinase and to study its enzymatic properties for laying groundwork in the efficient utilization of chitinase resources. Firstly,the Bacillus producing chitinase was isolated and purified,and then chitinase gene in the target strain was heterologously expressed,and enzymatic parameters of the purified chitinase were assayed at optimal conditions. Considering the bio-safety of the strain,Brevibacillus laterosporus CDY64 was selected as study object,and its chitinase（Chi72A） was expressed in Escherichia coli BL21（DE3）. Enzymatic studies revealed that the purified Chi72A by Ni-NTA affinity presented solid activity at pH range 6.0-8.0,and the optimal temperature of its activity was 60℃;and the Km and kcat of Chi72A were 5.85 μmol/L and 29.27 S-1,respectively while using colloidal chitin as substrate. In conclusion,Chi72A from CDY64 had high catalytic ability at high temperature and exhibited high antifungal activity against many phytopathogenic fungi in vitro.