枯草芽孢杆菌角蛋白酶kerC基因在毕赤酵母中的高效表达

doi:10.7506/spkx1002-6630-201309053

食品科学 ›› 2013, Vol. 34 ›› Issue (9): 262-266.doi: 10.7506/spkx1002-6630-201309053

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 625014

收稿日期:2012-08-06 修回日期:2013-04-02 出版日期:2013-05-15 发布日期:2013-05-07
通讯作者: 陈惠 E-mail:sheephanxueyi@sina.com

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 625014, China

Received:2012-08-06 Revised:2013-04-02 Online:2013-05-15 Published:2013-05-07
Contact: CHEN Hui E-mail:sheephanxueyi@sina.com

摘要/Abstract

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。

关键词: 枯草芽孢杆菌, 角蛋白酶, kerC基因, 毕赤酵母, 高效表达

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 was purified as determined by SDS-PAGE.

Key words: Bacillus subtilis, keratinase, kerC gene, Pichia pastoris, overexpression

中图分类号:

Q814

韩学易，唐自钟，王丽华，陈惠*. 枯草芽孢杆菌角蛋白酶kerC基因在毕赤酵母中的高效表达[J]. 食品科学, 2013, 34(9): 262-266.

HAN Xue-yi，TANG Zi-zhong，WANG Li-hua，CHEN Hui*. Overexpression of Bacillus subtilis Keratinase Gene (kerC) in Pichia pastoris[J]. FOOD SCIENCE, 2013, 34(9): 262-266.

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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

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