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
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摘要
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-28 a(+)-CwlJ was successfully constructed. The results showed that the size of CwlJ gene was440 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.
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-28 a(+)-CwlJ was successfully constructed. The results showed that the size of CwlJ gene was440 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.
引文
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[13]晁雅熙,王淑艳,吴谡琦,等.海洋弧菌(Vibrio sp.QD-5)褐藻胶裂解酶基因的克隆和生物信息学分析[J].海洋科学进展,2018,36(2):290-300.
[14]黄庆,唐乐丽,王晓萌,等.β-葡萄糖苷酶Bgl2238生物信息学分析[J].基因组学与应用生物学,2018,37(4):1457-1464.
[15]张鑫,赵勇,谭海东,等.稻瘟病菌单加氧酶lpmo M1基因的克隆及生物信息学分析[J].中国酿造,2015,34(11):35-40.
[16]韩占品,任健,于玮玮,等.花椰菜ERF114基因的克隆及Gateway技术构建表达载体[J].西南农业学报,2018,31(6):1128-1134.
[17]EMANUELSSON O,BRUNAK S,HEIJNE G V,et al.Locating proteins in the cell using Target P,Signal P and related tools[J].Nat Protoc,2007,2(4):953-971.
[18]王梦雅,赵学亮,胡红莲,等.山羊Claudin-1基因编码蛋白的生物信息学分析[J].畜牧与兽医,2018,50(7):18-23.
[19]WILKINSON D L,HARRISON R G.Predicting the solubility of recombinant proteins in Escherichia coli[J].Nat Biotechnol,1991,9(5):443-448.
[20]张颋,王菊芳,冯延叶,等.可溶性预测模型在包涵体复性中的应用[J].南方医科大学学报,2009,29(11):2156-2160.
[2]BLACK E P,SETLOW P,HOCKING A D,et al.Response of spores to high-pressure processing[J].Compr Rev Food Sci Food Safety,2007,6(4):103-119.
[3]BAGYAN I,SETLOW P.Localization of the cortex lytic enzyme Cwl J in spores of Bacillus subtilis[J].J Bacteriol,2002,184(4):1219-1224.
[4]CHIRAKKAL H,O'ROURKE M,ATRIH A,et al.Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination[J].Microbiology,2002,148(8):2383-2392.
[5]PAIDHUNGAT M,SETLOW B,DANIELS W B,et al.Mechanisms of induction of germination of Bacillus subtilis spores by high pressure[J].Appl Environ Microbiol,2002,68(6):3172-3175.
[6]PANDEY R,TER B A,VISCHER N O,et al.Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution[J].Food Microbiol,2015,45:63-70.
[7]ISHIKAWA S,YAMANE K,SEKIGUCHI J.Regulation and characterization of a newly deduced cell wall hydrolase gene(Cwl J)which affects germination of Bacillus subtilis spores[J].J Bacteriol,1998,180(6):1375.
[8]MAKINO S,MORIYAMA R.Hydrolysis of cortex peptidoglycan during bacterial spore germination[J].Med Sci Monit,2002,8(6):RA119.
[9]章中,胡小松,廖小军,等.温压结合超高压处理对芽孢杀灭作用的研究进展[J].高压物理学报,2013,27(1):147-152.
[10]任欣,娄阁,沈群.高压热处理对复合芝麻酱品质特性的影响[J].中国食品学报,2016,16(8):140-148.
[11]REINEKE K,MATHYS A,HEINZ V,et al.Mechanisms of endospore inactivation under high pressure[J].Trends Microbiol,2013,21(6):296-304.
[12]陈力学,曾照芳,康格非.生物信息学在基因组研究中的应用[J].国际检验医学杂志,2003,24(6):339-340.
[13]晁雅熙,王淑艳,吴谡琦,等.海洋弧菌(Vibrio sp.QD-5)褐藻胶裂解酶基因的克隆和生物信息学分析[J].海洋科学进展,2018,36(2):290-300.
[14]黄庆,唐乐丽,王晓萌,等.β-葡萄糖苷酶Bgl2238生物信息学分析[J].基因组学与应用生物学,2018,37(4):1457-1464.
[15]张鑫,赵勇,谭海东,等.稻瘟病菌单加氧酶lpmo M1基因的克隆及生物信息学分析[J].中国酿造,2015,34(11):35-40.
[16]韩占品,任健,于玮玮,等.花椰菜ERF114基因的克隆及Gateway技术构建表达载体[J].西南农业学报,2018,31(6):1128-1134.
[17]EMANUELSSON O,BRUNAK S,HEIJNE G V,et al.Locating proteins in the cell using Target P,Signal P and related tools[J].Nat Protoc,2007,2(4):953-971.
[18]王梦雅,赵学亮,胡红莲,等.山羊Claudin-1基因编码蛋白的生物信息学分析[J].畜牧与兽医,2018,50(7):18-23.
[19]WILKINSON D L,HARRISON R G.Predicting the solubility of recombinant proteins in Escherichia coli[J].Nat Biotechnol,1991,9(5):443-448.
[20]张颋,王菊芳,冯延叶,等.可溶性预测模型在包涵体复性中的应用[J].南方医科大学学报,2009,29(11):2156-2160.