S-2-氯丙酸脱卤酶在毕赤酵母中的重组表达  

Cloning and Expression of S-2-chloropropionic Acid Dehalogenase in Pichia pastoris

作　　者：秦迎春[1] 杨立荣[1] 徐刚[1] 吴坚平[1] 

机构地区：[1]浙江大学化学工程与生物工程学系,杭州310027

出　　处：《中国生物工程杂志》2013年第8期24-30,共7页China Biotechnology

基　　金：国家"973"计划(2011CB710800);国家"863"计划(2011AA02A209);国家自然科学基金面上项目(21076187)资助项目

摘　　要：根据S-2-氯丙酸脱卤酶的基因序列设计引物,克隆重组大肠杆菌中的S-2-氯丙酸脱卤酶基因。将目的基因片段构建到pPIC9K载体,经Sac I单酶切后转化到毕赤酵母GS115中,通过G418抗性YPD平板筛选高拷贝重组子,甲醇诱导成功实现胞外活性表达。重点考察了基因拷贝数和甲醇浓度的影响,结果表明重组子含有12个拷贝,甲醇浓度为1%时较佳,重组脱卤酶的酶活可达236U/L。重组毕赤酵母具有很好的遗传稳定性。对该酶最适反应温度及pH进行研究,表明其最适反应温度为50℃,最适pH为9.5。The primers of S-2-chloropropionic acid dehalogenase （DehlI-B2） gene was designed by its gene sequence and was cloned into pPIC9K shuttle plasmid. The recombinant plasmid was linearized by enzyme Sac I and transformed into GS115 yeast strain by electroporation. Screening of multiple copies of the transformants was performed on YPD medium containing different concentrations of geneticin G418. After screening, recombinants with different copies were obtained. The research of copy number and methanol concentrations effects on recombinant DehlI-B2 indicated that one obtained highest activity containing 12 copy numbers at the concentration of methanol was 1%. The expressed recombinant DehlI-B2 activity was up to 236U/L. Additionally, recombinant DehlI-B2 showed the quite good genetic stability. The optimum temperature and pH of the recombinant DehlI-B2 was 50℃ and 9.5, respectively.

关 键 词：S-2-氯丙酸脱卤酶 毕赤酵母 胞外活性表达 高拷贝 

分 类 号：Q784[生物学—分子生物学]