信号肽优化在枯草芽孢杆菌体系中对脂肪酶LipS分泌表达的影响  

Effects of Signal Peptides' s Optimization on the Secretion of Lipase S in Bacillus subtilis

作　　者：周勇[1] 徐刚[1] 杨立荣[1] 吴坚平[1] 

机构地区：[1]浙江大学化学工程与生物工程学院,杭州310027

出　　处：《中国生物工程杂志》2015年第9期42-49,共8页China Biotechnology

基　　金：国家"973"计划(2011CB710800);国家"863"计划(2011AA02A209);国家自然科学基金(20936002)资助项目

摘　　要：枯草芽孢杆菌具有良好的安全性和优异的外分泌能力,是目前常用的脂肪酶异源分泌表达体系。在枯草体系中如何提高脂肪酶分泌量是目前的研究热点。该体系中信号肽与目的基因不匹配和目标蛋白总表达量不高是导致脂肪酶LipS分泌表达效果较差的重要原因。针对这些问题,依据信号肽结构特征的不同(正电荷与疏水氨基酸个数的差异)对枯草芽孢杆菌体系中Sec和Tat这两个主要分泌途径的信号肽进行了筛选,结果显示Tat途径中的phoD信号肽分泌效果明显优于其他信号肽。在此基础上,通过替换使用诱导型表达载体、突变优化phoD信号肽,并考察了表达条件的影响,显著提高了脂肪酶LipS的分泌表达量,发酵液中转酯酶活达到62.07U/L,占总酶活的62.30%,较原始分泌表达体系的分泌量提高了13.7倍。Due to its safety and excellent exocrine capacity,Bacillus subtilis is used as the heterologous expression system of lipase currently. How to improve the secretion level of lipase in Bacillus subtilis has become a research hotspot. The expression of LipS in Bacillus subtilis has the problems that the signal peptide of LipS can＇t secrete itself well and the expression level of LipS isn＇t very high. To solve these problems,the screening of different signal peptides with different structure in Sec pathway and Tat pathway maybe a good method. The results show the signal peptide phoD can secrete the LipS more well than the other signal peptides. Then the strategies such as changing the expression system with an inducible expression system is took,optimizing the signal peptide phoD and expression conditions,the secretion level of LipS increased significantly. The secreted lipase activity reached 62. 07 U / L,accounting for 62. 30% of the total lipase activity. Compared with the extracellular activity of the original system,the new system increased about 13. 7 times.

关 键 词：脂肪酶 信号肽 筛选 分泌表达 

分 类 号：Q814[生物学—生物工程]