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时间:2019-03-07
《乙醛还原酶工程菌的构建以及与葡萄糖脱氢酶工程菌偶联还原制备r-chbe》由会员上传分享,免费在线阅读,更多相关内容在教育资源-天天文库。
1、化工进展·1120·CHEMICALINDUSTRYANDENGINEERINGPROGRESS2010年第29卷第6期乙醛还原酶工程菌的构建以及与葡萄糖脱氢酶工程菌偶联还原制备R-CHBE金永琴,徐娴,毛政,何冰芳(南京工业大学生物与制药工程学院,江苏南京210009;浙江省食品药品监督管理局培训中心,浙江杭州310012)摘要:R4一氯一3一羟基丁酸乙酯(R—CHBE)是多种药物的手性中间体,可由4一氯乙酰乙酸乙酯(COBE)立体选择性还原制备,而辅酶高效再生是绝大多数氧化还原酶实际应用的瓶颈
2、。本研究从赭色掷抱酵母细胞ZJU010中克隆出NADPH依赖型乙醛还原酶ARI基因(口fr),构建重组大肠杆菌E.coliBL21~pET-air,并与葡萄糖脱氢酶的基因工程茵(BL21IpET-gdh)进行偶联转化COBE。合理调整双菌双酶比例,当BL21~pET-air和BL21/pET-gdh的湿菌重比为6:1时,CHBE的产量迟到最高,无需添加辅酶,转化22h摩尔转化率达到95.88%,产物R—CHBE的对映体过量值ee为96%,较好地实现了双酶偶联制备R—CHBE。关键词:NADPH再生
3、;葡萄糖脱氢酶;乙醛还原酶;R4一氯一3一羟基丁酸乙酯中图分类号:Q55;Q78;TQ203文献标识码:A文章编号:1000—6613(2010)06一l120—05ProductionofR-CHBEusingtworecombinantscarryingaldehydereductasecouplingGDHgeneJINYongqin,XUXian,MAOZheng,HEBingfang(CollegeofBiotechnologyandPharmaceuticalEngineering,N
4、anjingUniversityofTechnology,Nanjing210009,Jiangsu,China;ZhejiangFoodandDrugAdministrationTrainingCenter,Hangzhou310012,Zhejiang,China)Abstract:R—Ethyl一4.chloro一3一hydroxybutanoate(R—CHBE)iSusedasachiralbuildingblockforthesynthesisofpharmaceuticaltarge
5、tcompounds.RCHBEcanbeproducedbytheasymmetricreductionofethyl4一chloro一3一oxobutanoate(COBE).However,effectivecofactorregenerationisakeystepfortheapplicationofmostoxidoreductases.Inthisresearch,theNADPH—dependentaldehydereductasegene(air)wasclonedfromthe
6、genomeofsporobolomycessalmonicolorZJU010,andtherecombinantE.colfBL21/pET_以f,strainwasconstructed.AsymmetricreductionofCOBEtoCHBEwasperformedusingtherecombinantE.coliBL21/pET_nf,couplingwithE.coliBL21/pET-gdh.Thehighestreductionactivitywasobtainedbyrec
7、ombinantrestingcellsat6:1weightratioofE.coliBL21/pET_口f,withEcolfBL21/pET-gdh.Yieldandtheenantiomericexcess(ee)ofR—CHBEreachedto95.88%and96%,respectively,after22hbioconversionwithoutaddingcoenzymeNADPHorNADP.R.CHBEcouldbeefectlyproducedusingtherecombi
8、nantE.coliBL21~pET-aircouplingwithE.coliBL21IpET-gdhforNADPHregenerantion.Keywords:NADPHregeneration;GDH:ARI;R—CHBE氧化还原酶广泛用于催化制备手性醇、羟基收稿日期:2009—12—28:修改稿日期:2010.叭.O7。基金项目:国家973计划资助项目(2009CB724700)。酸、氨基酸等重要医药中间体,大多数的氧化还原第一作者简介:金永琴(1982一),女,硕士研究
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