植物乳杆菌BBE7的胆盐水解酶基因在毕赤酵母中的分泌表达

Secretory expression of bile salt hydrolase gene from Lactobacillus plantarum BBE7 in Pichia pastoris

由于用大肠杆菌对植物乳杆菌来源的bsh基因进行胞内和胞外表达时,它们很容易形成包涵体,而且大肠杆菌的内毒素会显限制BSH的应用,所以利用毕赤酵母这一高效的表达系统对其进行了分泌表达.首先用融合PCR的方法将信号肽α-factor与BSH连接,然后再克隆到质粒pPIC9K上,得到重组质粒pPIC9K-α-factor-BSH.重组质粒经过SalⅠ线性化后转化至毕赤酵母GS115.表达产物经SDS-PAGE分析和酶活测定发现,重组BSH的相对分子质量(Mr)和胞外酶活分别为37.0×103和1.08 U mL-1.通过正交实验[L9(34)]对发酵条件优化后,胞外总酶活达到2.69 U mL-1,比原来提高了1.49倍.研究为胆盐水解酶的分离纯化、酶学性质研究以及大规模生产奠定了一定的基础.

When bile salt hydrolase （bsh） genes from Lactobacillus plantarum are expressed intracellularly and extracellularly by Escherichia coli, they usually form inclusion bodies. Therefore, Piehia pastoris, an efficient expression system, was used to achieve the secretory expression of bsh. Firstly, signal peptide a-factor was ligated with bsh by fusion PCR, and the fragment obtained was then cloned into vector pPIC9K, resulting in a recombinant plasmid pPIC9K-a-factor-BSH. Finally, this plasmid was linearized by Sal I and transformed into Pichia pastoris GS115. As analyzed by SDS-PAGE and BSH assay, the molecular weight and extracellular activity of recombinant BSH was determined to be 37.0 × 103 and 1.08 U mL-1, respectively. After optimization of fermentation conditions using orthogonal experimental design （L9 （34））, the extracellutar BSH activity was 2.69 U mL-1, 1.49 times higher than before. This lays a solid foundation for purification, characterization and large-scale production of BSH.