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[1]蔡少丽,邹有土,黄建忠,等.定点突变对扩展青霉脂肪酶热稳定性的改善[J].应用与环境生物学报,2013,19(01):43-47.[doi:10.3724/SP.J.1145.2013.00043]
　CAI Shaoli,ZOU Youtu,HUANG Jianzhong,et al.Improvement of Thermostability of Penicillium expansum Lipase by Site-directed Mutagenesis[J].Chinese Journal of Applied & Environmental Biology,2013,19(01):43-47.[doi:10.3724/SP.J.1145.2013.00043]
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定点突变对扩展青霉脂肪酶热稳定性的改善()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
19卷
期数:
2013年01期
页码:
43-47
栏目:
研究论文
出版日期:
2013-02-25

文章信息/Info

Title:
Improvement of Thermostability of Penicillium expansum Lipase by Site-directed Mutagenesis
作者:
蔡少丽; 邹有土; 黄建忠; 林琳
（1福建师范大学工业微生物教育部工程研究中心 福州 350108）
（2福建师范大学生命科学学院 福州 350108）
（3福建省现代发酵技术工程研究中心 福州 350108）
Author(s):
CAI Shaoli; ZOU Youtu; HUANG Jianzhong; LIN Lin
(1Engineering Research Center of Industrial Microbiology, Ministry of Education, Fujian Normal University, Fuzhou 350108, China)
(2College of Life Sciences, Fujian Normal University, Fuzhou 350108, China)
(3Engineering Research Center of Fujian Modern Ferment Technology, Fujian Normal University, Fuzhou 350108, China)
关键词:
扩展青霉; 脂肪酶; 定点突变; 毕赤酵母; 热稳定性
Keywords:
Penicillium expansum;  lipase;  site-directed mutagenesis;  Pichia pastoris;  thermostability
分类号:
Q814 : Q939.9
DOI:
10.3724/SP.J.1145.2013.00043
文献标志码:
A
摘要:
利用重叠延伸PCR方法对扩展青霉脂肪酶基因进行体外定点突变，构建了K115R与随机突变体ep8双突变的重组质粒pAO815-ep8-K115R. 将该质粒电转化到毕赤酵母GS115中进行异源表达，获得双突变体脂肪酶PEL-ep8-K115R-GS. 与野生型脂肪酶PEL-GS和随机突变体脂肪酶PEL-ep8-GS进行比较，发现双突变体脂肪酶PEL-ep8-K115R-GS在40 ℃温浴30 min后残余酶活为89%，比野生型脂肪酶PEL-GS和随机突变体脂肪酶PEL-ep8-GS分别提高54%和27%；双突变体脂肪酶PEL-ep8-K115R-GS的Tm值为42.2 ℃，比野生型脂肪酶PEL-GS提高3.5 ℃，比随机突变脂肪酶PEL-ep8-GS提高2.0 ℃. 研究结果表明对扩展青霉脂肪酶的分子改造能提高其热稳定性.
Abstract:
In order to improve the thermostability of Penicillium expansum lipase (PEL), the lipase gene was mutated by site-directed mutagenesis. A recombinant plasmid pAO815-ep8-K115R which contains double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate another mutation site K115R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8-K115R can secret the double-mutant lipase PEL-ep8-K115R-GS into the medium when it was induced by methanol. Thermostability analysis revealed that the residual activity of the double-mutant lipase PEL-ep8-K115R-GS after incubated at 40 ℃ for 30 min was 54% and 27% higher than that of the wild type lipase PEL-GS and the random-mutant lipase PEL-ep8-GS respectively. Tm of the double-mutant lipase PEL-ep8-K115R-GS was 42.2 ℃, 3.5 ℃ higher than that of the wild type lipase PEL-GS, and 2.0 ℃ higher than that of the random-mutant lipase PEL-ep8-GS.

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备注/Memo

备注/Memo:
国家自然科学基金项目（30270033）、福建省自然科学基金项目（B0120001，C0410009）和福建工业微生物研发平台建设项目（2006H0085）资助 Supported by the National Nature Science Foundation of China (No. 30270033), the Nature Science Foundation of Fujian Province (Nos. B0120001, C0410009) and the Platform Establishing Project for Industrial R & D of Fujian Province (No. 2006H0085)
更新日期/Last Update: 2013-02-26