唾液乳杆菌BSH1及其突变体的底物特异性  

Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius

作　　者：毕洁[1,2,3] 方芳[1] 仇钰莹 杨庆利[3] 陈坚[1,2] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学粮食发酵工艺与技术国家工程实验室,江苏无锡214122 [3]山东省花生研究所,山东青岛266100

出　　处：《生物工程学报》2014年第3期445-454,共10页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31100064);江苏省优势学科建设工程项目资助~~

摘　　要：为了解析胆盐水解酶催化中心中关键氨基酸位点与其底物特异性的关系,以大肠杆菌pET-20b(+)表达系统为分子改造平台,采用理性设计,结合氨基酸定点突变的方法,成功构建了唾液乳杆菌Lactobacillus salivarius胆盐水解酶BSH1的7种突变体。通过对比L.salivarius BSH1及其突变体对6种结合胆盐的底物特异性表明,7种突变体对不同的结合胆盐的水解活性有所改变。结果说明,Cys2和Thr264分别是BSH1催化TCA和GCA的关键残基,且对酶的催化活性的保持具有关键作用。其中,高保守性的氨基酸位点Cys2不是BSH1唯一的活性位点,而其他突变的氨基酸位点可能作为BSH1的结合位点参与了底物的结合,也可能影响了底物进入BSH1活性中心的通道或底物结合口袋的体积与形状,进而影响了BSH1对不同结合胆盐的水解活性。In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase （BSH1） were constructed by using the Escherichia coli pET-20b（＋） gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH 1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.

关 键 词：唾液乳杆菌 胆盐水解酶 定点突变 同源建模 底物特异性 

分 类 号：Q936[生物学—微生物学]