组成型分泌表达嗜热酸性生淀粉α-淀粉酶的重组粪肠球菌的构建

Construction of recombinant Enterococcus faecalis with constitutive secreted expression of thermoacidiphilic raw starch α-amylase

该研究以高效组成型启动子P10替换pSIP401-gtamyhds中诱导型启动子,构建重组载体pSIP401Z-gtamyhds,并以其为基础框架,分别导入粪肠球菌(Enterococcus faecalis)来源的8种信号肽(s1~s8),采用电转化法将信号肽筛选载体转入具有优良益生特性的粪肠球菌EXW27,构建组成型分泌表达嗜热酸性生淀粉α-淀粉酶Gt-amy的重组粪肠球菌。以发酵上清液中α-淀粉酶活性为评价指标,对8种信号肽进行筛选,并对嗜热酸性生淀粉α-淀粉酶Gt-amy进行分离纯化和酶学性质研究。结果表明,信号肽s7对嗜热酸性生淀粉α-淀粉酶Gt-amy的分泌效率最高,发酵上清液中α-淀粉酶活性达到310 U/m L。重组Gt-amy的最适反应pH值为5.0,在pH 4.0~8.0范围内具有较好的稳定性,最适反应温度为80℃,于80℃的半衰期为3 h,在40℃条件下反应3 h,对玉米淀粉的降解率可达55.8%。

The inducible promoter p SIP401-gtamyhds was replaced by high efficiency constitutive promoter P10, the recombinant vector pSIP401 Zgtamyhds was constructed and used as the basic framework. Eight signal peptides(s1-s8) from Enterococcus faecalis were introduced respectively. The signal peptide screening vector was transferred into E. faecalis EXW27 with excellent probiotics by electrotransformation, and the recombinant E. faecalis secreting and expressing thermophilic acidophilic raw starch α-amylase Gt-amy was constructed. The eight signal peptides were screened by using theα-amylase activity in the fermentation supernatant of recombinant E. faecalis as the evaluation index. The results showed that the signal peptide s7 had the highest efficiency in guiding the secretion of recombinant Gt-amy, and the α-amylase activity in the fermentation supernatant of the corresponding recombinant E. faecalis reached 310 U/ml. The optimal reaction p H of the recombinant Gt-amy was 5.0, Gt-amy exhibited good p H stability in the range of p H 4.0-8.0, and the optimal reaction temperature was 80 ℃, the half-life of Gt-amy at 80 ℃ was 3 h, and the degradation rate of corn starch was up to 55.8% after reaction at 40 ℃ for 3 h.