褐藻胶裂解酶在枯草芽胞杆菌中的重组表达及催化特性研究  

Recombinant Expression of Alginate Lyase in Bacillus subtilis and Catalytic Properties Characterization

作　　者：李星霖 吴雯 徐翊文 李恒 龚劲松 徐国强[1,4] 史劲松 许正宏 LI Xing-lin;WU Wen;XU Yi-wen;LI Heng;GONG Jin-song;XU Guo-qiang;SHI Jin-song;XU Zheng-hong(Schl.of Biotech.,Jiangnan Uni.,Wuxi 214122;Schl.of Life Sci.&Health Engin.Jiangnan Uni.,Wuxi 214122;Key Lab.of Carbohydrate Chem.&Biotech.,Minist.of Educ.,Wuxi 214122;Res.Ctr.for Cereal Fermen.&Food BioMenufact.,Jiangnan Uni.,Wuxi 214122)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学生命科学与健康工程学院,江苏无锡214122 [3]江南大学糖化学与生物技术教育部重点实验室,江苏无锡214122 [4]江南大学粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122

出　　处：《微生物学杂志》2021年第6期18-26,共9页Journal of Microbiology

基　　金：江苏省重点研发项目(BE2018622);江苏省现代农业面上项目(BE2019349);扬州市现代农业项目(YZ2019029)。

摘　　要：为拓展褐藻胶裂解酶在高效、安全的枯草芽胞杆菌体系中的表达,成功构建一株海洋来源的贝特氏菌(Cobetia sp.)WG-007褐藻胶裂解酶枯草芽胞杆菌工程菌Bacillus subtilis WB600/pMA5-aly-cob,对主要发酵条件进行优化,并对重组酶Aly-Cob的酶学性质进行表征。结果表明,优化后的工程菌培养温度和初始pH分别为30℃和7.0,发酵培养基为添加15 g/L甘油和30 g/L酵母浸膏的TB培养基。在此条件下,摇瓶发酵48 h褐藻胶裂解酶Aly-Cob酶活为58.62 U/mL,是优化前的2.7倍。重组酶Aly-Cob最适反应温度和pH分别为40℃和7.0,Mg^(2+)、Ca^(2+)、Na^(+)和K^(+)对酶活有促进作用,该酶可特异性地降解褐藻胶及其片段。研究拓展了褐藻胶裂解酶基因在枯草芽胞杆菌中的表达,为褐藻胶裂解酶的应用提供补充和参考。In order to expand the recombinant expression of alginate lyase(AL)genes in the effective and secure system of Bacillus subtilis,an AL engineered Bacillus subtilis strain WB600/pMA5-aly-cob with the AL gene originated from marine Cobetia sp.WG-007 was constructed.The main fermentation conditions were optimized and the enzymatic properties were characterized.The results showed that the optimized cultivating temperature and initial pH for recombinant bacteria were 30℃and 7.0,respectively.The optimized lower-cost medium formula was TB medium with addition of 15 g/L glycerol and 30 g/L yeast extract.Under these conditions,the enzymatic activity of AL reached 58.62 U/mL 48 h after been cultivated,about 2.7 times higher than that before optimized.The optimum temperature and pH of Aly-Cob were 40℃and 7.0,respectively.Mg^(2+),Ca^(2+),Na^(+)and K^(+) had improvement on the enzymatic activity.Aly-Cob can degrade alginate and its fragments specifically.This study expands the expression of AL gene in B.subtilis,which could provide a supplementary basis and reference for the application of AL.

关 键 词：褐藻胶裂解酶 重组表达 枯草芽胞杆菌 发酵优化 酶学性质 海洋微生物 

分 类 号：Q939.96[生物学—微生物学]