摘要
将分离到的野生革耳菌(Panusrudis)漆酶的cDNA序列克隆到毕赤酵母表达载体pPIC9K上,构建成受强启动子AOX1控制的重组表达载体.重组质粒DNA经BglII线性化后,电击转化毕赤酵母GS115细胞,通过G418筛选和PCR鉴定的重组酵母在甲醇诱导后能分泌表达活性漆酶.低温培养是该漆酶基因活性表达的必要条件.培养基中添加400μmol/L的CuSO4最有利于漆酶的活性表达.
A cDNA coding for laccase from Panus rudis was cloned into the vector pPIC9K for heterologous expression in Pichia pastoris under the control of the AOX1 promoter.Pichia pastoris GS115 was transformed by the electroporation method with the recombinant plasmid DNA linearised with BglII.After G418 screening and PCR analysis,the recombinant Pichia strains were grown in liquid medium BMGY and BMMY and secreted active laccase after methanol induction.The production of laccase was relative to the biomass and the pH value of the medium was maintained at pH 5.5-6.0 during the culture course.It was necessary for active laccase production to culture the recombinant Pichia strains under decreased temperature (16 or 20℃).A copper concentration of 400 μM in medium produced the highest level of active laccase.
基金
中国科学院"百人计划"(30225015)
国家自然科学基金资助项目(30370045)
安徽省优秀青年基金资助项目(04043048).
