水产学报2017，Vol.41Issue(10)：1552-1561,10.DOI:10.11964/jfc.20161110614
解淀粉芽孢杆菌HZ-1510壳聚糖酶基因的克隆、重组表达及其活性
Cloning, expression and activity analysis of chitosanase from Bacillus amyloliquefaciens HZ-1510
摘要
Abstract
To study the hydrolysis activity of chitosanase, in this report, the coding sequence of chitosanse from Bacillus amyloliquefaciens HZ-1510 was cloned, and glutathione S-transferase (GST) fused-chitosanase was expressed and purified fromEscherichia coli. Furthermore, we analysed the signal peptide, amino acid sequence and its three-dimensional structure. In the end, its chitosan hydrolysis activity was measured. The chitosanase gene consisted of an open reading frame of 837 bp which encoded a protein of 279 amino acids with predicted molecular weight of 31.45 ku. The protein contained a signal peptide with a cleavage site located between the 36th and 37th amino acids. The deduced amino acid sequence homology analysis of the chitosanase revealed that it belonged to GH46 family. The recombinant chitosanase has been purified to homogeneity by using only GST column chromatography. The optimal pH and temperature for the chitosan hydrolysis activity was 5.5 and 55 °C, respectively. The enzyme activity was increased in the presence of Mn2+, Ca2+ and Mg2+. However, Fe3+, Ag+, Cu2+, Ba2+ and K+ could inhibit its activity. Furthermore, its activity could be increased in the presence of 0.1 mmol/L Zn2+ while it was inhibited by 2.0 mmol/L Zn2+. This study explored the optimal condition for hydrolytic activity of chitosanase and established a theoretical basis for its industrial application.关键词
解淀粉芽孢杆菌/壳聚糖酶/基因克隆/蛋白表达/酶活性Key words
Bacillus amyloliquefaciens/chitosanase/gene cloning/protein expression/activity分类
生物科学引用本文复制引用
段静,林蠡,刘广鑫,董晏君,周洋,李锦铨,刘小玲,周萌,梁日深,赵丽娟..解淀粉芽孢杆菌HZ-1510壳聚糖酶基因的克隆、重组表达及其活性[J].水产学报,2017,41(10):1552-1561,10.基金项目
湖北省科技支撑计划(2015BBA228) （2015BBA228）
武汉市科技局基金(2016020101010089) （2016020101010089）
广东省海洋与渔业局基金(2015-033)Special Fund for Science and technology from Hubei Province (2015BBA228) （2015-033）
Fund from Wuhan Science and Technology Bureau (2016020101010089) （2016020101010089）
Special Funds from the Administration of Ocean and Fisheries of Guangdong Province (A201512C003) （A201512C003）
