组合策略提高枯草芽孢杆菌漆酶在大肠杆菌中的胞外表达

Improved extracellular expression of Bacillus subtilis laccase in Escherichia coli using a combined strategy

漆酶是一种多铜氧化酶,在食品、纺织、环境修复和造纸等领域有重要应用。为提高漆酶的发酵水平,该研究将枯草芽孢杆菌(Bacillus subtilis)来源的漆酶(CotA)表达于大肠杆菌(Escherichia coli)BL21(DE3)中,并分别考察了核糖体结合位点、诱导表达条件及共表达裂解蛋白对其表达效率的影响。结果显示,最优核糖体结合位点(ribosome binding site,RBS)重组菌CotA-RBS5胞内CotA表达水平达到1236 U/L,较优化前提高1.75倍。基于正交实验设计确定了最佳诱导表达条件为:待菌体浓度OD_(600)达到0.8时,加入终浓度为2 mmol/L Cu及0.1 mmol/L异丙基-β-D-硫代半乳糖苷后,于30℃诱导。在该条件下,重组菌CotA-RBS5的胞内酶活力提升至7926 U/L,较优化前提升了5.4倍;同时,胞外酶活力达到4984 U/L。共表达裂解蛋白E促进了细胞的裂解。在菌体浓度OD_(600)达到3.5时诱导裂解蛋白E表达,胞外CotA酶活力在发酵30 h达到10283 U/L。研究结果将为漆酶的工业化生产提供了重要的基础数据。

Laccase is a multi-copper oxidase,which has important applications in food,textile,environmental remediation,and papermaking.To improve the fermentation level of laccase,Bacillus subtilis laccase(CotA)was expressed in Escherichia coli BL21(DE3),and the effects of ribosome binding site,induction expression condition,and co-expression of lysis protein on its expression efficiency were investigated.The results showed that the recombinant strain with the optimal ribosome binding site(CotA-RBS5)achieved 1236 U/L of intracellular CotA,1.75 times higher than that of before optimization.Based on the orthogonal experimental,the optimal condition of induction expression was as follows:the final concentration of 2 mmol/L Cuand 0.1 mmol/L IPTG were added when the biomass OD_(600)reached 0.8;then,induction condition was performed at 30℃.Under the optimal condition,the intracellular enzyme activity of the recombinant strain increased to 7926 U/L,5.40 times higher than that of before optimization;meanwhile,the extracellular enzyme activity reached 4984 U/L.Co-expression of lysis protein E promoted cell lysis.With the expression of cleavage protein E was induced at 3.5 of OD_(600),the extracellular CotA activity reached 10283 U/L as it fermented to 30 h.The results will provide important basic data for the industrial production of laccase.