枯草芽孢杆菌角蛋白酶kerc基因在毕赤酵母中的高效表达.pdfVIP

262 2013, Vol.34, No.09 食品科学 ※生物工程 枯草芽孢杆菌角蛋白酶kerC基因 在毕赤酵母中的高效表达 韩学易，唐自钟，王丽华，陈 惠* ( 四川农业大学生命科学与理学院，四川 雅安 6250 14) 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检测表明，重组酶分子质 量约为31kD 。 关键词：枯草芽孢杆菌；角蛋白酶；kerC基因；毕赤酵母；高效表达 Overexpression of Bacillus subtilis Keratinase Gene (kerC) in Pichia pastoris * HAN Xue-yi ，TANG Zi-zhong，WANG Li-hua，CHEN Hui (College of Life and Basic Sciences, Sichuan Agricultural University, Ya ’an 6250 14, China) Abstract ：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