组成型分泌表达蛋白酶的重组粪肠球菌的构建及应用

Construction and application of the recombinant Enterococcus faecalis with constitutive secretion of protease

为构建组成型分泌表达蛋白酶Ker的重组粪肠球菌,采用豆粕固态发酵实验并评估其应用效果。以重组质粒pSIP401-kerhds为基础框架,向其中引入组成型启动子p10和高效分泌信号肽S6,构建重组载体pSIP401Z-s6-kerhds。采用电击转化方法将该重组载体转入具有益生特性的粪肠球菌EXW27来构建重组粪肠球菌。重组粪肠球菌的胞外蛋白酶的比酶活力为125.37 U/mL。重组蛋白酶Ker的最适反应温度为40℃,在pH 5.0~10.0范围内具有较高的相对酶活力和稳定性,并对胆盐溶液具有较好的耐受性,可适用于动物肠道环境。豆粕固态发酵实验表明,与粪肠球菌EXW27相比,重组粪肠球菌可以更有效降解豆粕中蛋白质。该研究构建了既具有益生特性又具有蛋白酶活性的重组粪肠球菌,为开发新型微生态制剂奠定了基础。

The purpose of this study was to construct a recombinant Enterococcus faecalis with constitutive secretory expression of protease Ker and evaluate its application effect by solid state fermentation of soybean meal.In this study,the recombinant plasmid pSIP401-kerhds was constructed and used as the basic framework.The recombinant vector pSIP401Z-s6-kerhds was constructed by the introduction of the constitutive promoter p10 and efficient signal peptide S6.The recombinant E.faecalis was constructed by transferring the recombinant vector pSIP401Z-s6-kerhds into the excellent probiotics E.faecalis EXW27.The extracellular protease activity of the recombinant E.faecalis reached 125.37 U/mL.The optimal reaction temperature of recombinant protease Ker was 40℃and the high relative activity and stability was obtained at pH 5.0~10.0.Meanwhile,protease Ker exhibited good tolerance to bile salt solution.The enzymatic properties of Ker made it applicable to the intestinal environment of animals.Compared with E.faecalis EXW27,the recombinant E.faecalis could degrade the protein of soybean meal more effectively.In this study,the recombinant E.faecalis with the characters of probiotic and high protease activity was constructed.This stain showed its potential as a novel probiotic.