定点突变提高细胞色素P450 BM-3吲哚羟基化能力

doi:10.3969/j.issn.0438-1157.2013.09.035

化工学报 ›› 2013, Vol. 64 ›› Issue (9): 3331-3337.DOI: 10.3969/j.issn.0438-1157.2013.09.035

定点突变提高细胞色素P450 BM-3吲哚羟基化能力

张彭湃¹, 胡升², 黄俊³, 梅乐和^1,2, 雷引林², 金志华², 姚善泾¹

1. 浙江大学化学工程与生物工程学系, 浙江杭州 310027;
2. 浙江大学宁波理工学院生物与化学工程学院, 浙江宁波 315100;
3. 浙江科技学院生物与化学工程学院, 浙江杭州 310023

收稿日期:2013-01-31 修回日期:2013-03-19 出版日期:2013-09-05 发布日期:2013-09-05
通讯作者: 梅乐和
作者简介:张彭湃(1978- ),男,博士研究生。
基金资助:
国家自然科学基金项目(21176220,31240054);浙江省自然科学基金重点项目(Z12B060008);宁波市重大科技项目(2011C11023)。

Improving indole-hydroxylation ability of cytochrome P450 BM-3 by site-directed mutagenesis

ZHANG Pengpai¹, HU Sheng², HUANG Jun³, MEI Lehe^1,2, LEI Yinlin², JIN Zhihua², YAO Shanjing¹

1. Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, Zhejiang, China;
2. School of Biological and Chemical Engineering, Ningbo Institute of Technology of Zhejiang University, Ningbo 315100, Zhejiang, China;
3. School of Biological & Chemical Engineering, Zhejiang University of Science & Technology, Hangzhou 310023, Zhejiang, China

Received:2013-01-31 Revised:2013-03-19 Online:2013-09-05 Published:2013-09-05
Supported by:
supported by the National Natural Science Foundation of China(21176220,31240054),Zhejiang Provincial Natural Science Foundation(Z13B060008) and Key Technology Research and Development Project of Ningbo(2011C11023).

摘要/Abstract

摘要： 为进一步提高细胞色素P450 BM-3(A74G/F87V/L188Q)对吲哚的羟基化能力,根据酶结构与功能的关系,以突变酶E435T为基础,在168位点引入D168L突变,获得了吲哚羟基化能力得到显著提高的突变酶D168L/E435T。突变酶对吲哚的K_m为1.72 mmol·L^-1(父本2.09 mmol·L^-1),转化数(k_cat)为28.15 min^-1(父本4.04 min^-1),表明D168L定点突变可以略微提高酶对底物的亲和力,但主要的效应是促进了底物的转化速率,这两个效应的综合表现是使酶的催化效率(k_cat K_m^-1)比父本酶提高了8.48倍。此外,产物中副产物靛玉红的比例也降低为1.2%(父本7.3%),这说明该突变酶催化吲哚的区域选择性上也更有利于靛蓝的生成。

关键词: 细胞色素P450 BM-3, 定点突变, 羟基化, 吲哚, 靛蓝

Abstract: According to the relationship between the structure and function of enzyme,it was speculated that the combination of the mutant enzyme D168L and E435T might improve the catalytic ability of indole to generate indigo.The mutant enzyme D168L/E435T was produced by site-directed mutagenesis conducted on the 168th site of the P450 BM-3(A74G/F87V/L188Q/E435T)and it was identified with higher hydroxylation activity toward indole in comparison to its parent enzyme.Concomitantly,its K_m for indole was 1.72 mmol·L^-1 and this value was lower compared to its parent enzyme(2.09 mmol·L^-1).Also with the k_cat=28.15 min^-1,the turnover rate of the mutant enzyme was increasing up to 6.97-fold that of its parent enzyme(4.04 min^-1).The catalytic efficiency(k_catK_m^-1)was correspondingly raised to 8.48 times as much as that of its parent enzyme.Moreover,the proportion of the byproducts indirubin reduced to 1.2%,much lower than that of its parent mutant(7.3%),which indicated that this regioselectivity of mutant enzyme to catalyze indole was more conducive to generating indigo.

Key words: P450 BM-3, site-directed mutagenesis, hydroxylation, indole, indigo

中图分类号:

TQ962.2

张彭湃, 胡升, 黄俊, 梅乐和, 雷引林, 金志华, 姚善泾. 定点突变提高细胞色素P450 BM-3吲哚羟基化能力[J]. 化工学报, 2013, 64(9): 3331-3337.

ZHANG Pengpai, HU Sheng, HUANG Jun, MEI Lehe, LEI Yinlin, JIN Zhihua, YAO Shanjing. Improving indole-hydroxylation ability of cytochrome P450 BM-3 by site-directed mutagenesis[J]. CIESC Journal, 2013, 64(9): 3331-3337.

参考文献 18

[1]	Wu Qi(吴祺).Baeyer and the production of indigo[J].Chemistry(化学通报),2001, 64(8):527-529
[2]	Han Xiaohong(韩晓红),Wang Wei(王伟),Xiao Xingguo(肖兴国).Microbial biosynthesis and biotransformation of indigo and indigo-like pigments[J].Chin.J.Biotech.(生物工程学报),2008,24(6):921-926
[3]	Jia Xiuling(贾秀玲),Cui Yunhua(崔运花),Han Yalan(韩雅岚),Ran Wei(冉唯),Wu Qiumeng(吴秋蒙),He Xu(何旭),Lu Jiahuan(陆佳欢).Development and application of plant indigo and its prospect[J].Prog.Text.Sci.Technol.(纺织科技进展),2011,32(2):24-26
[4]	Ma Qiao(马桥),Qu Yuanyuan(曲媛媛),Zhang Xuwang(张旭旺),Xu Bingwen(许炳雯),Zhou Jiti(周集体).Recent advances in microbial synthesis of indigo[J].Chin.J.Appl.Environ.Biol.(应用与环境生物学报),2012,18(2):344-350
[5]	Rosic N N.Versatile capacity of shuffled cytochrome P450s for dye production[J].Appl.Microbiol.Biotechnol.,2009,82(2):203-210
[6]	Zhang Z G,Liu Y,Guengerich F P.Identification of amino acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using screening of random libraries[J].J.Biotechnol.,2009,139(1):12-18
[7]	Rosic N N,Huang W L,Johnston W A,DeVoss J J,Gillam E M J.Extending the diversity of cytochrome P450 enzymes by DNA family shuffling[J].Gene,2007,395(1/2):40-48
[8]	Gillam E M J,Aguinaldo A M A,Notley L M,Kim D H,Mundkowski R G,Volkov A A,Arnold F H,Soucek P,DeVoss J J,Guengerich F P.Formation of indigo by recombinant mammalian cytochrome P450[J].Biochem. Biophys.Res.Commun.,1999,265(2):469-472
[9]	Li Q S,Schwaneberg U,Fischer P,Schmid R D.Directed evolution of the fatty-acid hydroxylase P450 BM3 into an indole-hydroxylating catalyst[J].Chem.Eur.J.,2000,6(9):1531-1536
[10]	van Vugt-Lussenburg B M A,Stjernschantz E,Lastdrager J,Oostenbrink C,Vermeulen N P E,Commandeur J N M.Identification of critical residues in novel drug metabolizing mutants of cytochrome P450 BM3 using random mutagenesis[J].J.Med.Chem.,2007,50(3):455-461
[11]	Li H M,Mei L H,Urlacher V B,Schmid R D.Cytochrome P450 BM3 evolved by random and saturation mutagenesis as an effective indole-hydroxylating catalyst[J].Appl.Biochem.Biotechnol.,2008,144(1):27-36
[12]	Li Hongmei(李红梅),Mei Lehe(梅乐和).Amino acid mutation effect on indole-hydroxylating ability of cytochrome P450 BM-3[J].J.Chem.Eng. Chinese Univs.(高校化学工程学报),2008,22(2):282-287
[13]	Omura T,Sato R.The carbon monoxide-binding pigment of liver microsomers[J].J.Biol.Chem.,1964,239(7):2370-2378
[14]	Choi S J,Kim M,Kim S I,Jeon J K.Microplate assay measurement of cytochrome P450-carbon monoxide complexes[J].J.Biochem.Mol.Biol.,2003,36(3):332-335
[15]	Narhi L O,Fulco A J.Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium[J].J.Biol.Chem.,1996,26(1):7160-7169
[16]	Gorsky L D,Koop D R,Coon M J.On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450[J].J.Biol.Chem.,1984,259(11):6812-6817
[17]	Ou Zhimin(欧志敏),Jin Zhihua(金志华),Wu Jianping(吴坚平),Yang Lirong(杨立荣),Cen Peilin(岑沛霖).Kinetic model of asymmetric reduction of 2'-chloroacetophenone by Saccharomyces cerevisiae B5 with coenzyme regeneration process[J].J.Chem.Eng. Chinese Univs.(高校化学工程学报),2005,19(4):511-517
[18]	Hu Sheng(胡升),Yu Qing(虞青),Mei Lehe(梅乐和),Yao Shanjing(姚善泾),Jin Zhihua(金志华).Semi-rational directed evolution in improving indole-hydroxylation ability of cytochrome P450 BM-3[J].CIESC Journal(化工学报),2009,60(11):2869-2875

定点突变提高细胞色素P450 BM-3吲哚羟基化能力

Improving indole-hydroxylation ability of cytochrome P450 BM-3 by site-directed mutagenesis

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献 18

相关文章 15

编辑推荐

Metrics

本文评价

[1]	宗迅成, 胡蝶, 阚婷婷, 王婷婷, 邬敏辰. 定点突变提高菜豆环氧水解酶的对映选择性[J]. 化工学报, 2018, 69(10): 4335-4341.
[2]	曾邦定, 黄钦耿, 梁玲, 郭小雷, 王明兹, 施巧琴, 吴松刚. 黄色短杆菌ilvN基因定点突变和ilvBN、ilvC串联表达对L-缬氨酸产量的影响[J]. 化工学报, 2016, 67(7): 2956-2962.
[3]	程德红, 王佳齐, 林杰, 卢声, 郝旭, 路艳华. 离子液体金属配合物吸附废水中活性蓝19染料[J]. CIESC Journal, 2015, 66(S1): 242-246.
[4]	许世民, 郎中敏, 王亚雄, 赫文秀, 梁倩卿. 羟基化多壁碳纳米管/R141b纳米流体核沸腾[J]. 化工学报, 2015, 66(11): 4424-4430.
[5]	陈杰, 贾旭, 于慧敏, 罗晖, 沈忠耀. 耦合末端盐桥与定点突变的重组腈水合酶催化动力学[J]. 化工学报, 2014, 65(7): 2821-2828.
[6]	张彭湃, 胡升, 梅乐和, 雷引林, 金志华, 姚善泾. 一个有利于细胞色素P450 BM-3催化吲哚合成靛蓝的三位点突变酶：D168L/E435T/V445A[J]. 化工学报, 2014, 65(4): 1374-1380.
[7]	冯筱晴1.2，唐龙1，王瑞瑞1，王羚竹1，宋国强1. 1-苯基吲哚的合成[J]. 化工进展, 2013, 32(10): 2486-2490.
[8]	叶招莲1，张仁熙2，陈娴1，席志楠1. 微波无极准分子光/H₂O₂氧化降解含N-杂环化合物[J]. 化工进展, 2012, 31(02 ): 440-447.
[9]	冯勇，吴德礼，马鲁铭. 结构态亚铁羟基化合物还原预处理印染废水的效果和机制 [J]. CIESC Journal, 2011, 62(7): 2033-2041.
[10]	马光和;何建波. 靛蓝磺化物还原隐色和氧化脱色的薄层光谱电化学研究 [J]. CIESC Journal, 2010, 61(4): 923-928.
[11]	王宇红, 袁联群, 俞磊, 项婷婷. 镧、钒取代MCM-41分子筛的结构表征及其在苯酚羟基化反应中的催化性能 [J]. 化工学报, 2010, 61(10): 2565-2572.
[12]	陈素琴，黄向红. 2-丁基-6-硝基吲哚的合成 [J]. CIESC Journal, 2009, 28(9): 1639-.
[13]	徐贞贞;陈忠林;贲岳;齐飞;沈吉敏. 羟基化锌催化臭氧化水中痕量p-CNB的动力学和机理 [J]. CIESC Journal, 2009, 60(7): 1687-1692.
[14]	胡升, 虞青, 梅乐和, 姚善泾, 金志华. 细胞色素P450 BM-3羟基化吲哚能力的半理性改造 [J]. 化工学报, 2009, 60(11): 2869-2875.
[15]	刘民;高健;郭新闻;王祥生;李灿;冯兆池. 气固相法制备的高效Ti-ZSM-5沸石的表征和催化性能 [J]. CIESC Journal, 2006, 57(4): 791-798.