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

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.