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[1]王康,吴志革,赵伟睿,等.3-甾酮-9α-羟基化酶基因的表达及其催化合成9α-羟基雄甾-4-烯-3,17-二酮[J].生物加工过程,2018,16(06):13-18.[doi:10.3969/j.issn.1672-3678.2018.06.003]
 WANG Kang,WU Zhige,ZHAO Weirui,et al.Construction,expression of 3-ketosteroid 9α-hydroxylase for 9α-OH-AD synthesis[J].Chinese Journal of Bioprocess Engineering,2018,16(06):13-18.[doi:10.3969/j.issn.1672-3678.2018.06.003]
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3-甾酮-9α-羟基化酶基因的表达及其催化合成9α-羟基雄甾-4-烯-3,17-二酮()
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《生物加工过程》[ISSN:1672-3678/CN:32-1706/Q]

卷:
16
期数:
2018年06期
页码:
13-18
栏目:
出版日期:
2018-11-30

文章信息/Info

Title:
Construction,expression of 3-ketosteroid 9α-hydroxylase for 9α-OH-AD synthesis
文章编号:
1672-3678(2018)06-0013-06
作者:
王康12吴志革1赵伟睿13胡升1麻菊美12王进波1姚善泾2梅乐和12
1.浙江大学 宁波理工学院 生物与化学工程学院,浙江 宁波 315100; 2.浙江大学 化学工程与生物工程学院,浙江 杭州 310027; 3.浙江大学 生物系统工程与食品科学学院,浙江 杭州 310058
Author(s):
WANG Kang12WU Zhige1ZHAO Weirui13HU Sheng1MA Jumei12WANG Jinbo1YAO Shanjing2MEI Lehe12
1.School of Biotechnology and Chemical Engineering,Ningbo Institute of Technology,Zhejiang University,Ningbo 315100,China; 2.Department of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,China; 3.College of Biosystems Engineering and Food Science,Zhejiang University,Hangzhou 310058,China
关键词:
雄甾-4-烯-317-二酮 9α-羟基雄甾-4-烯-317-二酮 3-甾酮-9α-羟基化酶 羟基化 甾体药物
分类号:
Q78;Q819
DOI:
10.3969/j.issn.1672-3678.2018.06.003
文献标志码:
A
摘要:
研究应用于甾体9α-羟基化的高效催化剂和催化体系是实现甾体药物9α位羟基高效合成的关键。本文中,笔者对来源于分支杆菌(Mycobacterium tuberculosis)H37Rv 和红平红球菌(Rhodococcus erythropolis)SQ1的3-甾酮-9α-羟基化酶基因(rekshA,mtkshA)进行优化,并对2个基因的催化活性进行检测。通过构建工程表达菌株,以雄甾-4-烯-3,17-二酮(AD)为底物,对制备9α-羟基雄甾-4-烯-3,17-二酮(9α-OH-AD)的催化反应进行了探索。结果表明:基因序列优化显著提升了ReKshA和MtKshA蛋白的可溶性表达,其中ReKshA具有较好的雄甾-4-烯-3,17-二酮9α位羟基化活性。用TB培养基培养ReKshA的工程表达菌株BL21(DE3)-pET28a-rekshA-yh,在30 ℃、500 μmol/L底物浓度、0.8%(体积分数)乙醇为助溶剂、0.1 mmol/L IPTG诱导浓度、底物与诱导剂同时加入到工程菌液中的条件下,9α-OH-AD得率达到97.09%。

参考文献/References:

[1] 产启银,常继发,曲卫国,等.一种制备9α-羟基雄烯二酮的方法:201310259697.X[P].2013-10-09.
[2] ANDOR A,JEKKEL A,HOPWOOD D A,et al.Generation of useful insertionally blocked sterol degradation pathway mutants of fast-growing Mycobacteria and cloning,characterization,and expression of the terminal oxygenase of the 3-ketosteroid 9α-hydroxylase in Mycobacterium smegmatis mc2155[J].Appl Environ Microbiol,2006,72(10):6554-6559.
[3] 魏长龙,赵树欣,王珊,等.Nocardia canicruria BF313催化甾体9α-羟基化发酵工艺优化[J].食品工业科技,2014,35(18):320-323.
[4] HOLLAND H L.Recent advances in applied and mechanistic aspects of the enzymatic hydroxylation of steroids by whole-cell biocatalysts[J].Steroids,1999,64:178-186.
[5] MAHATO S B,GARAI S.Advances in microbial steroid biotransformation[J].Steroids,1997,62:332-345.
[6] VANDER GEIZE R,HESSELS G I,VAN GERWEN R,et al.Molecular and functional characterization of kshA and kshB,encoding two components of 3-ketosteroid 9α-hydroxylase,a class IA monooxygenase,in Rhodococcus erythropolis strain SQ1[J].Mol Microbiol,2002,45(4):1007-1018.
[7] ARNELL R,JOHANNISSON R,LINDHOLM J,et al.Biotechnological approach to the synthesis of 9α-hydroxylated steroids[J].Prep Biochem Biotechnol,2007,37(4):309-321.
[8] HU Y M,VAN DER GEIZE R,BESRA G S,et al.3-Ketosteroid 9α-hydroxylase is anessential factor in the pathogenesis of Mycobacterium tuberculosis[J].Mol Microbiol,2010,75(1):107-121.
[9] STRIJEWSKI A.The steroid-9α-hydroxylation system from Nocardia species[J].Eur J Biochem,1982,128:125-135.
[10] DONOVA M V,GULEVSKAYA S A,DOVBNYA D V,et al.Mycobacterium sp.mutant strain producing 9α-hydroxy and rostenedione from sitosterol[J].Appl Genet Microbiol Biotechnol,2005,67(5):671-678.
[11] 姚抗.分枝杆菌甾醇转化机制的解析及其代谢工程改造应用于制备重要甾药中间体的研究[D].上海:华东理工大学,2014.
[12] PETRUSMA M,DIJKHUIZEN L,VAN DER GEIZE R.Rhodococcus rhodochrous DSM 43269 3-ketosteroid 9α-hydroxylase,a two-component iron-sulfur-containing monooxygenase with subtle steroids substrate specificity[J].Appl Environ Microbiol,2009,75(16):5300-5307.
[13] 魏巍.Mycobacterium neoaurum NwIB-01降解甾醇母核关键酶3-甾酮-Δ1-脱氢酶和3-甾酮-9α-羟化酶基因的鉴定及其基因工程改造[D].上海:华东理工大学,2010.
[14] GROTE A,HILLER K,SCHEER M,et al.JCat:a novel tool to adapt codon usage of a target gene to its potential expression host[J].Nucleic Acids Res,2005,33:W526-W531.

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备注/Memo

备注/Memo:
收稿日期:2017-11-15修回日期:2018-06-11
基金项目:国家自然科学基金(81502421、31670804); 中国博士后科学基金(2016M592003); 国家星火计划(2015GA701022); 浙江省自然科学基金重点项目(LZ13B060002); 浙江省自然科学基金(Q14C070004、LY16B060008); 宁波市科技惠民计划(2015C50042); 宁波市自然科学基金(2014A610214)
作者简介:王康(1993—),男,山东济宁人,硕士研究生,研究方向:酶工程; 梅乐和(联系人),教授,E-mail:meilh@zju.edu.cn
引文格式:王康,吴志革,赵伟睿,等.3-甾酮-9α-羟基化酶基因的表达及其催化合成9α-羟基雄甾-4-烯-3,17-二酮[J].生物加工过程,2018,16(6):13-18.
WANG Kang,WU Zhige,ZHAO Weirui,et al.Construction,expression of 3-ketosteroid 9α-hydroxylase for 9α-OH-AD synthesis[J].Chin J Bioprocess Eng,2018,16(6):13-18..
更新日期/Last Update: 2018-11-30