分子模拟指导化学修饰对有机相中脂肪酶的性能强化

doi:10.16085/j.issn.1000-6613.2015.10.032

化工进展 ›› 2015, Vol. 34 ›› Issue (10): 3725-3730.DOI: 10.16085/j.issn.1000-6613.2015.10.032

分子模拟指导化学修饰对有机相中脂肪酶的性能强化

郭丽, 徐刚, 吴坚平, 杨立荣

浙江大学化学工程与生物工程学系生物工程研究所, 浙江杭州 310027

收稿日期:2015-03-24 修回日期:2015-04-07 出版日期:2015-10-05 发布日期:2015-10-05
通讯作者: 徐刚,副教授,研究方向为生物催化与转化。E-mailxugang_1030@zju.edu.cn。
作者简介:郭丽(1990—),女,硕士研究生。
基金资助:
国家973计划(2011CB710800)、国家自然科学基金(20936002)及国家863计划(2011AA02A209)项目。

Performance enhancement of chemically modified lipase in the organic phase guided by molecular simulation

GUO Li, XU Gang, WU Jianping, YANG Lirong

Bioengineering Institute, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, Zhejiang, China

Received:2015-03-24 Revised:2015-04-07 Online:2015-10-05 Published:2015-10-05

摘要/Abstract

摘要： 脂肪酶是一种应用广泛的重要生物催化剂,它对手性底物具有良好的立体选择性,而且能适应包括疏水有机溶剂在内的多种非天然反应介质。提高脂肪酶在非天然环境中的立体选择性已经成为关于脂肪酶的研究热点。然而已有研究在提高选择性的同时,往往伴随催化活力的降低。本文提出一种基于计算机模拟的理性改造方法,能够在不降低脂肪酶活力的情况下大幅提高其在有机介质中的立体选择性。以洋葱假单胞菌脂肪酶(Pseudomonas cepacia lipase,PcL)在正己烷中催化手性仲醇的转酯化反应为模型,借助分子动力学模拟,预测 N-乙酰咪唑(NAI)修饰 PcL的效果。蛋白质谱证实产生主要影响的是位于 PcL活性口袋内的 Tyr²⁹ 残基。进一步通过实验验证,发现经 NAI 修饰酪氨酸残基(Tyr)后 PcL催化拆分手性仲醇的对映体选择性最高从 E = 12.6 提高到 48.1,同时催化活力也有所提高。

关键词: 化学修饰, 模拟, 手性仲醇, 对映体选择性, 脂肪酶

Abstract: Lipase is one of the most important biocatalysts because of its stereoselectivity towards chiral substrates and reasonable adaptiveness of non-conventional reaction media, including hydrophobic organic solvents. Increasing the selectivity of lipase in such non-conventional environments has been a continuous hotspot. However, current researches on improving lipase selectivity usually cause critical decrease of catalytic activity. In this work, a novel computation- directed approach to enhance lipase enantioselectivity in organic media without losing activity was introduced. Stereoselective acylation of chiral secondary alcohols catalyzed by PcL(Pseudomonas cepacia lipase) in n-hexane was taken as the model reaction. According to the results from molecular dynamics simulation, when the Tyr²⁹ inside the PcL catalytic cavity was modified by N-acetylimidazole (NAI), the enantioselectivity of the lipase was significantly increased. The simulation result was agreed with the experimental data. MALDI-TOF-MS confirmed the Tyr²⁹ modified position, and kinetic resolution of chiral secondary alcohols by NAI-modified PcL displayed an increase of enantioselectivity ratio from 12.6 to 48.1 and higher activity.

Key words: chemical modification, simulation, chiral secondary alcohol, enantioselectivity, lipase

中图分类号:

O643

郭丽, 徐刚, 吴坚平, 杨立荣. 分子模拟指导化学修饰对有机相中脂肪酶的性能强化[J]. 化工进展, 2015, 34(10): 3725-3730.

GUO Li, XU Gang, WU Jianping, YANG Lirong. Performance enhancement of chemically modified lipase in the organic phase guided by molecular simulation[J]. Chemical Industry and Engineering Progree, 2015, 34(10): 3725-3730.

参考文献

[1] 崔丽娟,徐刚,孟枭,等. 多元电解质对脂肪酶有机相拆分炔戊醇的激活[J]. 化工进展,2014,33(8):2150-2154.

[2] 戴晓庭,孟枭,徐刚,等. 酰基供体对动态动力学拆分1-四氢萘胺的影响[J]. 化工进展,2014,33(9):2421-2424.

[3] Bajaj A,Lohan P,Jha P N,et al. Biodiesel production through lipase catalyzed transesterification:An overview[J]. J. Mol. Catal. B:Enzym.,2010,62(1):9-14.

[4] Fan Y X,Qian J Q. Lipase catalysis in ionic liquids/supercritical carbon dioxide and its applications[J]. J. Mol. Catal. B:Enzym.,2010,66(1):1-7.

[5] Laane C,Boeren S,Vos K,et al. Rules for optimization of biocatalysis in organic solvents[J]. Biotechnol. Bioeng.,1987,30(1):81-87.

[6] Yadav G D,Lathi P S. Kinetics and mechanism of synthesis of butyl isobutyrate over immobilized lipases[J]. Biochem. Eng. J.,2003,16(3):245-252.

[7] Lemke K,Lemke M,Theil F. A three-dimension predictive active site model for lipase from Pseudomonas cepacia[J]. J. Org. Chem.,1997,62(18):6268-6273.

[8] Fernandez L R. Lipase from Thermomyces lanuginosus:Uses and prospects as an industrial biocatalyst[J]. J. Mol. Catal. B:Enzym.,2010,62(3):197-212.

[9] Contesini F J,Lopes D B,Macedo G A,et al. Aspergillus sp. lipase:Potential biocatalyst for industrial use[J]. J. Mol. Catal. B:Enzym.,2010,67(3):163-171.

[10] Li N,Zong M H. Lipases from the genus Penicillium:Production,purification,characterization and applications[J]. J. Mol. Catal. B:Enzym.,2010,66(1):43-54.

[11] Rodrigues R C,Fernandez L R. Lipase from Rhizomucor miehei as a biocatalyst in fats and oils modification[J]. J. Mol. Catal. B:Enzym.,2010,66(1):15-32.

[12] Rodrigues R C,Fernandez L R. Lipase from Rhizomucor miehei as an industrial biocatalyst in chemical process[J]. J. Mol. Catal. B:Enzym.,2010,64(1):1-22.

[13] Cabrera Z,Fernandez L G,Fernandez L R,et al. Enhancement of Novozym-435 catalytic properties by physical or chemical modification[J]. Proc. Biochem.,2009,44:226-231.

[14] Palomo J M,Fernandez L G,Guisan J M,et al. Modulation of immobilized lipase enantioselectivity via chemical amination[J]. Adv. Synth. Catal.,2007,349(7):1119-1127.

[15] Rodrigues R C,Berenguer M A,Fernandez L R. Coupling chemical modification and immobilization to improve the catalytic performance of enzymes[J]. Adv. Synth. Catal.,2011,353(13):2216-2238.

[16] Mateo C,Palomo J M,Fernandez L G,et al. Improvement of enzyme activity,stability and selectivity via immobilization techniques[J]. Enzym Microb. Technol.,2007,40(6):1451-1463.

[17] Chen H,Wu J P,Yang L R,et al. A combination of site-directed mutagenesis and chemical modification to improve diastereopreference of Pseudomonas alcaligenes lipase[J]. Biochim. Biophys Acta,2013,1834(12):2494-2501.

[18] Tuomi W V,Kazlauskas R J. Molecular basis for enantioselectivity of lipase from Pseudomonas cepacia toward primary alcohols. Modeling,kinetics,and chemical modification of Tyr29 to increase or decrease enantioselectivity[J]. J. Org. Chem.,1999,64(8):2638-2647.

[19] Kim K K,Song H K,Shin D H,et al. The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor[J]. Sturcture,1997,5(2):173-185.

[20] Peretz A,Pell L,Gofman Y,et al. Targeting the voltage sensor of Kv7.2 voltage-gated K⁺ channels with a new gating-modifier[J]. PANS,2010,107(35): 15637-15642.

[21] Case D A,Darden T A,Cheatham T E,et al. AMBER 11[C]. San Francisco:University of California,2011,

[22] Jorgensen W L,Chandrasekhar J,Madura J D,et al. Comparison of simple potential functions for simulating liquid water[J]. J. Chem. Phys.,1983,79(2):926-935.

[23] Chen C S,Fujimoto Y,Girdaukas G,et al. Quantitative analyses of biochemical kinetic resolutions of enantiomers[J]. J. Am. Chem. Soc.,1982,104(25):7294-7299.

[24] Furth A J,Hope D B. Studies on the chemical modification of the tyrosine residue in bovine neurophysin-II[J]. Biochem. J.,1970,116:545-553.

[25] Norin M,Olsen O,Svendsen A,et al. Theoretical studies of Rhizomucor miehei lipase activation[J]. Protein Eng.,1993,6(8):855-863.

[26] Ru M T,Dordick J S,Reimer J A,et al. Optimizing the salt-induced activation of enzymes in organic solvents:Effects of lyophilization time and water content[J]. Biotechnol. Bioeng.,1999,63(2):233-241.

[27] Gupta N,Rathi P,Gupta R. Simplified para-nitrophenyl palmitate assay for lipases and esterases[J]. J. Med. Chem.,2003,46(4):499-511.

[28] Zehl M,Lescic L,Abramic M,et al. Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry:Localization of the active site serine[J]. J. Mass Spectrom.,2004,39(12):1474-1483.

分子模拟指导化学修饰对有机相中脂肪酶的性能强化

Performance enhancement of chemically modified lipase in the organic phase guided by molecular simulation

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