枯草芽孢杆菌角蛋白酶kerC基因在毕赤酵母中的高效表达  

Overexpression of Bacillus subtilis Keratinase Gene (kerC) in Pichia pastoris

作　　者：韩学易[1] 唐自钟[1] 王丽华[1] 陈惠[1] 

机构地区：[1]四川农业大学生命科学与理学院,四川雅安625014

出　　处：《食品科学》2013年第9期262-266,共5页Food Science

摘　　要：以角蛋白酶基因为研究对象,构建毕赤酵母(Pichia pastoris)稳定的高效表达系统。采用PCR方法从枯草芽孢杆菌(Bacillus subtilis)B-3中克隆得到角蛋白酶基因kerC,将其构建在毕赤酵母表达载体pPICZαA上,得到重组质粒pPICZαA-kerC,转入毕赤酵母X-33中,成功实现了角蛋白酶基因kerC在真核表达系统中的高效表达。重组菌株以体积分数1%的甲醇诱导培养108h后,酶活力可达32.31U/mL,提高了11.15倍。重组酶最适温度为60℃,最适pH值为7.5,反应体系中3mmol/L的Co2+、Fe2+和Ca2+能明显增强其酶活力。SDS-PAGE检测表明,重组酶分子质量约为31kD。A stable and highly effective expression system was constructed for the expression of keratinase gene（kerC） in Pichia pastoris.kerC gene was cloned from Bacillus subtilis B-3 by PCR,inserted into the expression vector pPICZαA and transformed into Pichia pastoris X-33.The results showed that the kerC gene was over expressed successfully in the eukaryotic expression system.The maximum keratinase yield of the recombinant strains reached 32.31 U/mL in the culture supernatant grown for 108 h by 1% methanol induction,a 11.15-fold increase in comparison to that obtained with the prokaryotic expression strain BL21-pET-kerC.The optimal temperature and pH of the recombinant keratinase were 60 ℃ and 7.5,respectively.It was strongly stimulated by Co2＋,Fe2＋ and Ca2＋ despite being completely inhibited by Fe3＋.The recombinant keratinase with a molecular mass of 31 kD was purified as determined by SDS-PAGE.

关 键 词：枯草芽孢杆菌 角蛋白酶 kerC基因 毕赤酵母 高效表达 

分 类 号：Q814[生物学—生物工程]