枯草芽孢杆菌芽孢皮层裂解酶CwlJ基因的克隆及酶生物信息学分析

Cloning and bioinformatics analysis of cortex lytic enzyme CwlJ gene in spores of Bacillus subtilis

高压热杀菌(HPTS)技术是一种重要的食品杀菌技术,能有效杀灭细菌芽孢。分离纯化出芽孢皮层裂解酶CwlJ可用于研究HPTS杀灭芽孢的机理。通过基因克隆得到了皮层裂解酶基因CwlJ,并构建了表达载体pET-28a(+)-CwlJ。结果表明,CwlJ基因大小为440 bp,编码142个氨基酸;生物信息学分析表明,皮层裂解酶CwlJ为亲水性的碱性不稳定蛋白;皮层裂解酶CwlJ中α-螺旋占26.06%,β-延伸链占21.83%,β-转角占4.93%,无规则卷曲占47.18%;不是分泌性蛋白,形成包涵体的可能性为68.11%;与苏云金芽孢杆菌(Bacillus thuringiensis)、苏云金芽孢杆菌(Bacillus thuringiensis)Bt18247的同类酶CwlJ亲缘关系最近。该研究为后续皮层裂解酶CwlJ基因的表达、分离纯化以及阐明HPTS下皮层肽聚糖水解机制奠定了理论基础。

High pressure thermal sterilization (HPTS) is an important food sterilization technology, which can effectively kill bacterial spores. Isolation and purification of cortex lytic enzyme CwlJ in spores can be used to study the mechanism of killing spores by HPTS. In this paper, the cortical lyase gene CwlJ was cloned and the expression vector pET-28a(+)-CwlJ was successfully constructed. The results showed that the size of CwlJ gene was 440 bp, encoding 142 amino acids. Bioinformatics analysis showed that the cortex lytic enzyme CwlJ was a hydrophilic alkaline unstable protein;alpha-helix, beta-elongation chain, beta-angle and irregular curl accounted for 26.06%, 21.83%, 4.93%, 47.18% in the cortex lytic enzyme CwlJ, respectively. The cortex lytic enzyme CwlJ was non-secretory protein, and the possibility of forming inclusion body was 68.11%. It had the closest relationship with a similar enzyme CwlJ in Bacillus thuringiensis and B. thuringiensis Bt18247. The study laid a theoretical foundation for the subsequent expression, isolation and purification of the cortex lytic enzyme CwlJ gene and elucidation of the hydrolysis mechanism of subcortical peptidoglycan by HPTS.