|本期目录/Table of Contents|

[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]
点击复制

定点突变对扩展青霉脂肪酶热稳定性的改善()
分享到：

《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.

参考文献/References:

1 Hasan F, Shah AA, Hameed A. Industrial applications of microbial lipases [J]. Enzyme Microbial Technol, 2006, 39 (2): 235-251
2 Treichel H, de Oliveira D, Mazutti MA, Di Luccio M, Oliveira JV. A Review on microbial lipases production [J]. Food Bioprocess Technol, 2010, 3 (2): 182-196
3 郑毅, 黄建忠, 施巧琴, 翁丽星, 吴松刚. 中温碱性脂肪酶的研究——Ⅲ. 扩展青霉PF868变株碱性脂肪酶的纯化及其酶学性质[J]. 工业微生物, 1996, 26 (3): 15-19 [Zheng Y, Huang JZ, Shi QQ, Weng LX, Wu SG. Studies on alkaline-mesophile lipase Ⅲ. Purification and some properties of alkaline lipase from Penicillum expansum PF898 [J]. Ind Microbiol, 1996, 26 (3): 15-19]
4 林琳, 谢必峰, 杨冠珍, 施巧琴, 林奇英, 谢联辉, 吴祥甫, 吴松刚. 扩展青霉PF898碱性脂肪酶cDNA的克隆及序列分析[J]. 中国生物化学与分子生物学学报, 2002, 18 (1): 32-37 [Lin L, Xie BF, Yang GZ, Shi QQ, Lin QY, Xie LH, Wu XF, Wu SG. Cloning and sequence analysis of cDNA encoding alkaline lipase from Penicillum expansum PF898 [J]. Chin J Biochem Mol Biol, 2002, 18 (1): 32-37]
5 林琳, 谢必峰, 杨冠珍, 施巧琴, 林奇英, 谢联辉, 吴松刚, 吴祥甫. 扩展青霉PF898碱性脂肪酶基因组DNA的克隆及序列分析[J]. 中国生物化学与分子生物学学报, 2003, 19 (1): 12-16 [Lin L, Xie BF, Yang GZ, Shi QQ, Lin QY, Xie LH, Wu SG, Wu XF. Cloning and sequence analysis of DNA encoding alkaline lipase from Penicillum expansum PF898 [J]. Chin J Biochem Mol Biol, 2003, 19 (1): 12-16]
6 李生强, 袁彩, 林琳. 扩展青霉碱性脂肪酶基因的表达载体的构建和表达[J]. 福建农林大学学报(自然科学版), 2003, 32: 259-262 [Li SQ, Yuan C, Lin L. Expression vector construction and expression of alkaline lipase gene from Penicillum expansum [J]. J Fujian Agric For Univ (Nat Sci Ed), 2003, 32: 259-262]
7 袁彩, 林琳, 施巧琴, 吴松刚. 扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达[J]. 生物工程学报, 2003, 19 (2): 231-235 [Yuan C, Lin L, Shi QQ, Wu SG. Overexpression of Penicillum expansum lipase in Pichia pastoris [J]. Chin J Biotechnol, 2003, 19 (2): 231-235]
8 Bian CB, Yuan C, Lin L, Lin JH, Shi XL, Ye XM, Huang ZX, Huang MD. Purification and preliminary crystallographic analysis of a Penicillium expansum lipase [J]. Biochim Biophys Acta, 2005, 1752: 99-102
9 Bian CB, Yuan C, Chen LQ, Meehan EJ, Jiang LG, Huang ZX, Lin L, Huang MD. Crystal structure of a triacylglycerol lipase from Penicillium expansum at 1.3 Å determined by sulfur SAD [J]. Proteins: Struct Funct Bioinformatics, 2010, 78 (6): 1601-1605
10 Beisson F, Tiss A, Rivière C, Verger R. Methods for lipase detection and assay: a critical review [J]. Eur J Lipid Sci Technol, 2000, 102 (2): 133-153
11 Zhao HM, Arnold FH. Directed evolution converts subtilisin E into a functional equivalent of thermitase [J]. Protein Eng, 1999, 12 (1): 47-53
12 Nagao T, Shimada Y, Sugihara A, Tominaga Y. Increase in stability of Fusarium heterosporum lipase [J]. J Mol Catal B: Enzym, 2002, 17: 125-132
13 Kamal MZ, Ahmad S, Molugu TR, Vijayalakshmi A, Deshmukh MV, Sankaranaryanan R, Rao NM. In vitro evolved non-aggregating and thermostable lipase: structural and thermodynamic investigation [J]. J Mol Biol, 2011, 413: 726-741
14 Gatti-Lafranconi P, Caldarazzo SM, Villa A, Alberghina L, Lotti M. Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase [J]. FEBS Lett, 2008, 582 (15): 2313 -2318
15 Khurana J, Sirgh R, Kaur J. Engineering of Bacillus lipase by directed evolution for enhanced thermal stability: effect of isoleucine to threonine mutation at protein surface [J]. Mol Biol Rep, 2011, 38: 2919-2926
16 Ruslan R, Rahman RNZRA, Leow TC, Ali MSM, Basri M, Salleh AB. Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 [J]. Intern J Mol Sci, 2012, 13: 943-960
17 Le QAT, Joo JC, Yoo YJ, Kim YH. Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge [J]. Biotechnol Bioeng, 2012, 109 (4): 867-876
18 Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, D’Amico S, Dumont J, Garsoux G, Georlette D, Hoyoux A, Lonhienne T, Meuwis MA, Feller G. Cold-adapted enzymes: from fundamentals to biotechnology [J]. Trends Biotechnol, 2000, 18 (3): 103-107
19 Wells JA. Additivity of mutational effects in proteins [J]. Biochemistry, 1990, 29 (6): 8509-8517
20 Minagawa H, Kaneko H. Effect of double mutation on thermostability of lactate oxidase [J]. Biotechnol Lett, 2000, 22: 1131-1133

相似文献/References:

[1]张晓梅,窦文芳,许泓瑜,等.一株产脂肪酶嗜盐菌的鉴定及耐盐机理[J].应用与环境生物学报,2010,16(01):100.[doi:10.3724/SP.J.1145.2010.00100]
　ZHANG Xiaomei,DOU Wenfang,XU Hongyu,et al.Identification of a Lipase Producing Halophilic Strain and Its Halophilic Mechanism[J].Chinese Journal of Applied & Environmental Biology,2010,16(01):100.[doi:10.3724/SP.J.1145.2010.00100]
[2]张银波,江木兰,万霞,等.环境基因组DNA脂肪酶基因克隆与分析[J].应用与环境生物学报,2010,16(02):269.[doi:10.3724/SP.J.1145.2010.00269]
　ZHANG Yinbo,JIANG Mulan,WAN Xia,et al.Cloning and Analysis of a Lipase Gene from Environmental Genome DNA[J].Chinese Journal of Applied & Environmental Biology,2010,16(01):269.[doi:10.3724/SP.J.1145.2010.00269]
[3]吴尧,万霞,张银波,等.白地霉（lip42）脂肪酶的纯化及酶学性质[J].应用与环境生物学报,2010,16(05):710.[doi:10.3724/SP.J.1145.2010.00710]
　WU Yao,WAN Xia,ZHANG Yingbo,et al.Purification and Properties of a Lipase from Geotrichum candidum lip42[J].Chinese Journal of Applied & Environmental Biology,2010,16(01):710.[doi:10.3724/SP.J.1145.2010.00710]
[4]付玮,李友国,李一星.基于黄单胞菌冰核蛋白N端的表面展示体系建立[J].应用与环境生物学报,2014,20(03):351.[doi:10.3724/SP.J.1145.2014.10001]
　FU Wei,LI Youguo,LI Yixing.Establishment of cell surface display system based on N-domain of ice nucleation protein of Xanthomonas[J].Chinese Journal of Applied & Environmental Biology,2014,20(01):351.[doi:10.3724/SP.J.1145.2014.10001]
[5]马抒晗,张玮佳,吴茜,等.高产脂肪酶菌株的筛选鉴定及酶学、转酯特性[J].应用与环境生物学报,2014,20(04):602.[doi:10.3724/SP.J.1145.2014.01028]
　MA Shuhan,ZHANG Weijia,WU Qian,et al.Lipase-producing strain: isolation, identification, enzymology and transesterification[J].Chinese Journal of Applied & Environmental Biology,2014,20(01):602.[doi:10.3724/SP.J.1145.2014.01028]

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