S-2-氯丙酸脱卤酶的定向进化及其应用

中国生物工程杂志

2012, Vol. 32

Issue (05): 66-72

技术与方法

S-2-氯丙酸脱卤酶的定向进化及其应用

刘鹏, 林春娇, 杨立荣, 徐刚, 吴坚平

浙江大学化学工程与生物工程学系杭州 310027

Directed Evolution and Application of S-2-CPA Dehalogenase

LIU Peng, YANG Chun-jiao, YANG Li-rong, XU Gang, WU Jian-ping

Zhejiang University, Hangzhou 310027, China

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摘要：

为提高S-2-氯丙酸脱卤酶的活力,通过易错PCR的方法将源于假单胞菌(Pseudomonas sp. CGMCC 3267)的脱卤酶(DehII)进行定向进化,进化酶DehII-B2的比活提高3.9倍。同源模建两者的三维结构,并与底物进行分子对接。结果表明,突变位点为A7I,进化酶DehII-B2的结合能比原始酶降低了1.4kcal/mol,活性中心Asp10与底物α碳原子的距离缩短了0.321 6nm,因而加快了酶反应速率、提高了酶比活。目前,该酶的比活高于实验室已得酶。与原始酶相比,其最适温度和热稳定性略有增加,最适pH和pH稳定性没有明显变化。对该酶的应用做了初步的探索,结果表明在40℃,60mmol/L底物浓度下反应10h,转化率达到49.6%,ees>99.9%,因此该酶有一定的应用价值。

关键词： 2-氯丙酸; 脱卤酶; 定向进化; 分子对接

Abstract:

To enhance the functionality of dehalogenase from Pseudomonas sp. CGMCC 3267, Used error-prone PCR to create mutants with improved specific activity. Through error-prone PCR and high-throughput screening method, a desirable mutant DehII-B2 was obtained. Compare to initial strain, the specific activity of DehII-B2 was increased 3.9-fold. Three-dimensional structures of DehII-S and DehII-B2 were constructed by homology modeling, and molecular docking of dehalogenase and S-2-CPA. The results suggested that the mutant site is Ala7, the binding energy of DehII-B2 is reduced 1.4kcal/mol than DehII-S, and the distance of active site Asp10 and the α carbon atom of substrate is shorten 0.321 6nm, thus accelerate the enzyme reaction rate and increased enzyme specific activity. At present, the specific activity of DehII-B2 was higher than all enzyme in our lab. According to the results, the optimum pH and pH-stability were not changed, but the optimum temperature and thermo-stability of DehII-B2 were slightly increased than those of initial strain. The preliminary application of DehII-B2 was studied, and the best result was obtained at 40℃, 60mmol/L 2-CPA reacting for 10h.

Key words: 2-CPA Dehalogenase Directed evolution Molecular docking

收稿日期: 2012-02-09 出版日期: 2012-05-25

ZTFLH:

Q556

基金资助:

国家"973"计划(2011CB710805)、国家自然科学基金面上项目(21076187)资助项目

通讯作者: 吴坚平 E-mail: wjp@zju.edu.cn

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引用本文:

刘鹏, 林春娇, 杨立荣, 徐刚, 吴坚平. S-2-氯丙酸脱卤酶的定向进化及其应用[J]. 中国生物工程杂志, 2012, 32(05): 66-72.

LIU Peng, YANG Chun-jiao, YANG Li-rong, XU Gang, WU Jian-ping. Directed Evolution and Application of S-2-CPA Dehalogenase. China Biotechnology, 2012, 32(05): 66-72.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I05/66

[1] Li Y F, Kurihara T. Mass spectrometric analysis of the reactions catalyzed by L-2-haloacid dehalogenase mutants and implications for the roles of the catalytic amino acid residues. Journal of Molecular Catalysis B-Enzymatic, 2003, 23:337-345.

[2] Arai R, Kukimoto-Niino M. Crystal structure of the probable haloacid dehalogenase PH0459 from Pyrococcus horikoshii OT3. Protein Science, 2006, 15:373-377.

[3] Nakamura T, Yamaguchi A. Roles of K151 and D180 in L-2-haloacid dehalogenase from Pseudomonas sp. YL: analysis by molecular dynamics and Ab initio fragment molecular orbital calculations. Journal of Computational Chemistry, 2009, 30:2625-2634.

[4] Smith J M, Harrison K. Purification and Characterization of D-2-Haloacid Dehalogenase from Pseudomonas-Putida Strain Aj1/23. Journal of General Microbiology, 1990, 136:881-886.

[5] Ridder I S, Rozeboom H J. Three-dimensional structure of L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10 complexed with the substrate-analogue formate. Journal of Biological Chemistry, 1997, 272:33015-33022.

[6] Littlechild J A, Rye C A. Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii. Extremophiles, 2009, 13:179-190.

[7] McFadden J M, Frehywot G L, Townsend C A. A flexible route to (5R)-thiolactomycin, a naturally occurring inhibitor of fatty acid synthesis. Org. Lett., 2002, 4(22): 3859-3862.

[8] 林春娇. C2/C3卤代酸脱卤酶的筛选及克隆表达. 杭州:浙江大学,化工系,2011. Lin C J. Screening, Cloning and Expression for C2/C3 haloacid dehalogenases. Hangzhou: ZheJiang University, Department of Chemical Engineering, 2011.

[9] 项炯华, 吴坚平, 王能强, 等. 2-氯丙酸脱卤酶产酶菌种的筛选及酶学性质研究. 化学反应工程与工艺, 2005, 21(6): 537-541. Xiang J H, Wu J P, Wang N Q, Yang L R. Screening of 2-chloropropionic acid dehalogenase-production microorganisms and its enzymatic characteristics. Chem Reaction Eng And Tech, 2005, 21(6): 537-541.

[10] 黄培堂. 分子克隆实验指南. 第三版, 北京: 科学出版社.2005. Huang P T. Molecular Cloning (A laboratory Manual). 3rd ed, Beijing:Science Press. 2005.

[11] 郭尧君. 蛋白质电泳实验技术. 北京: 科学出版社.1999 Guo Y J. Experimental Technology of protein Electrophoresis. Beijing: Science Press. 1999.

[12] 杨安钢, 毛积芳, 药立波. 生物化学与分子生物学实验技术. 北京: 高教出版社. 2001. Yang A G, Mao J F, Yao L B. Biochemisty and Molecular Biology Protocol. Beijing: High Education Press. 2001.

[13] 张春桃, 卢茂芳, 刘恒言. DCC/DMAP催化制备乙酰阿魏酸苯丙醇酯. 中国实用医学, 2010, 5(23): 37-38. Zhang C T, Lu M F, Liu H Y. DCC/DMAP catalyze to synthesize Phenylpropanol ester of acetyl ferulic acid. China Prac. Med., 2010, 5(23): 37-38.

[14] Li Y F, Yasuo H, Tomomi F, et al. Crystal structures of reaction intermediates of L-2-haloacid dehalogenase and implications for the reaction mechanism. The Journal of Biological Chemistry. 1998, 273(24): 15035-15044.

[15] 胡凤娟, 王旭曼, 刘大岭,等. 具蛋白酶抗性的Armillariella tabescens β-甘露聚糖酶MAN47的分子定向改造. 中国生物工程杂志, 2011, 31(10): 75-82. Hu F J, Wang X M, Liu D L, et al. Directional molecular rebuilding of β-mannanase MAN47 with trypsin-resistance from armillariella tabescens. China Biotechnology, 2011, 31(10): 75-82.

[16] Kim M S, Lei X G. Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR. Appl Microbiol Biotechnol, 2008, 79: 69-75.

[17] Kobayashi R, Hirano N, Kanaya S, et al. Enhancement of the enzymatic activity of Escherichia coli acetyl esterase by random mutagenesis. Journal of Molecular Catalysis B: Enzymatic, 2010, 67: 155-161.

[18] Ridder I S, Rozeboom H J, Kalk K H, et al. Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase. Journal of Biological Chemistry, 1999,274: 30672~30678.

[19] 施碧红, 陈明, 陈文静,等. 基于易错PCR定向进化扩展青霉Penicillium expansum FS1884碱性脂肪酶. 中国生物工程杂志, 2011, 31(11): 64-68. Shi B H, Chen M, Chen W J, et al. Directed evolution of alkaline lipase from Penicillium expansum FS1884 by error-prone PCR. China Biotechnology, 2011, 31(11): 64-68.

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