枯草芽孢杆菌分泌载体构建及其对脂肪酶A的分泌表达

Construction of Secretory Expression Vectors for Bacillus subtilis and Secretion of Lipase A

[目的]构建含不同信号肽编码序列的枯草芽孢杆菌分泌表达载体,并研究这些载体对脂肪酶A的分泌表达效率。[方法]克隆得到枯草芽孢杆菌168株的脂肪酶A编码基因,同时扩增得到蛋白质NprE、Bpr、YweA的信号肽编码序列,构建了脂肪酶A分泌表达载体pMA5-estA、pMA5-nprE-estA、pMA5-bpr-estA和pMA5-yweA-estA,并转化到枯草芽孢杆菌168株中得到相应的重组分泌表达菌株。[结果]对4株重组表达菌株进行摇瓶培养,所有菌株都实现了脂肪酶A的分泌,20 h后,168(pMA5-bpr-estA)的细胞外脂肪酶A的酶活力达到1.68 U/ml,约占该菌株脂肪酶A总表达量的86%。[结论]枯草芽孢杆菌Bpr信号肽对脂肪酶A具有较高的分泌效率。

[Objective]The aim of this study was to develop different secretory expression vectors for B.subtilis using different signal peptide coding sequences,and then to study the efficiency of these vectors in secretion of lipase A.[Method] The lipase A coding gene was cloned from B.subtilis 168.The signal peptide coding sequences of B.subtilis NprE,Bpr and YweA were amplified,and the secretory expression vectors pMA5-estA,pMA5-nprE-estA,pMA5-bpr-estA and pMA5-yweA-estA were constructed.These vectors were transferred B.subtilis 168 for construction of recombinant strains.[Result] These 4 recombinant strains were cultivated by flask shaking and all them achieved the secretion of lipase A.After 20 h of cultivation,the extracellular enzymatic activity of lipase A expressed by 168（pMA5-bpr-estA） was 1.68 U/ml,which accounted for about 86% of the total enzymatic activity of lipase A synthesized.[Conclusion]The signal peptide of B.subtilis Bpr had better efficiency for secretion of lipase A.