硫色曲霉β-甘露聚糖酶在毕赤酵母中的组成型分泌表达

Constitutive and Secreting Expression of Aspergillus sulphureus β-Mannanase in Pichia pastoris

目的:构建硫色曲霉β-甘露聚糖酶的毕赤酵母组成型分泌表达菌株,研究重组菌株的产酶水平。方法:EcoRⅠ/XbaⅠ双酶切质粒pPIC-mann-opt,琼脂糖凝胶电泳、回收目的基因片段后,与组成型毕赤酵母表达载体pGAPzαA连接,转化大肠杆菌,经筛选获得含有pGAP-mann-opt的重组克隆;提取pGAP-mann-opt,用限制性内切酶BspHⅠ线性化后,转化毕赤酵母X-33感受态细胞,进行Zeocin抗性筛选和PCR鉴定。结果:获得了重组菌株,重组菌株在YPD培养基中摇瓶发酵24h,上清液酶活达到343U/mL,产酶蛋白约为1.0mg/mL。结论:构建的硫色曲霉β-甘露聚糖酶组成型表达菌株具有较好的应用前景。

Objective： To construct Pichia pastoris which can constitutively express β-mannanase of Aspergillus sulphureus, and to study the enzyme-producing level. Methods： The target DNA fragment was generated by digestion of plasmid pPIC-mann-opt with two enzymes EcoR I/Xba I , and it was ligated with constitutive expression vector pGAPzαA. The ligation product was transformed into E.coli, subsequently, the recombinant strain containing pGAP-mann-opt was screened. After the resulting plasmid was isolated and linearized, it was transformed into P.postoris X-33 component cells. Results： The recombinant P.pastoris was screened by Zeocin resistantance and identified with PCR. The enzyme activity and protein concentration of the supernatant reached 343 U/mL and 1.0 mg/mL respectively after the the recombinant P. pastoris was cultured in YPD medium for 24 h. Conclusion： The constructed P.pastoris constitutively expressing β-man-nanase has a better prospect.