不同启动子调控羰基还原酶基因在枯草芽胞杆菌中的表达水平
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摘要
为了实现羰基还原酶基因mldh在枯草芽胞杆菌中的高效表达,以摩氏摩根菌Morganella morganii CMCC(B)49208染色体DNA为模板,PCR扩增得到目的基因mldh,分别与启动子PQ和启动子p43进行连接,构建不同启动子组合的表达载体PHY-p43-mldh、PHY-PQ-mldh、PHY-p43-p43-mldh和PHY-p43-PQ-mldh,化学法转化B.subtilis Wb600后对重组菌细胞破碎液进行SDS-PAGE分析及全细胞生物转化反应实验发现,4种重组菌的转化能力差异显著,其中重组菌B.subtilis Wb600(PHY-p43-p43-mldh)进行全细胞转化反应,转化液中d-伪麻黄碱的浓度最高,达到142.1 mg/L,底物转化率为78.25%,成功实现了羰基还原酶基因mldh在枯草芽胞杆菌中的高效表达。
In order to achieve its high expression in Bacillus subtilis,carbonyl reductase gene mldh was amplified by PCR with the template of chromosome DNA from Morganella morganii CMCC(B) 49208 and connected with promoter PQ and promoter p43 to obtain different expression plasmids PHY-p43-mldh,PHY-PQ-mldh,PHY-p43-p43-mldh,and PHY-p43-PQ-mldh.Subsequently,these recombined vectors were transformed into B.subtilis Wb600.SDS-PAGE analysis of recombinants.And whole cellular biotransformation showed that there was distinctive difference among the four recombinants,in which the B.subtilis Wb600(PHY-p43-p43-mldh) could obtain the highest concentration(142.1 mg/L) of d-pseudoephedrine in conversion solution and the mole ratio of substrate was 78.25%.
In order to achieve its high expression in Bacillus subtilis,carbonyl reductase gene mldh was amplified by PCR with the template of chromosome DNA from Morganella morganii CMCC(B) 49208 and connected with promoter PQ and promoter p43 to obtain different expression plasmids PHY-p43-mldh,PHY-PQ-mldh,PHY-p43-p43-mldh,and PHY-p43-PQ-mldh.Subsequently,these recombined vectors were transformed into B.subtilis Wb600.SDS-PAGE analysis of recombinants.And whole cellular biotransformation showed that there was distinctive difference among the four recombinants,in which the B.subtilis Wb600(PHY-p43-p43-mldh) could obtain the highest concentration(142.1 mg/L) of d-pseudoephedrine in conversion solution and the mole ratio of substrate was 78.25%.
引文
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[13]张鹏华,张梁,卢燕,等.Morganella morganii J-8羰基不对称还原酶的分离纯化及性质研究[J].生物工程学报,2007,23(2):268-272.
[2]刘刚,刑苗,余邵文.耐高温α-淀粉酶在溶原性枯草芽孢杆菌表达系统中的高效表达[J].应用与环境生物学报,2005,11(3):368-372.
[3]董晨,曹娟,张迹,等.耐高温α-淀粉酶基因在枯草芽孢杆菌中的高效表达[J].应用与环境生物学报,2008,14(4):534-538.
[4]佟小雪,牛丹丹,陈献忠,等.地衣芽孢桿菌高温α-淀粉酶异源表达研究[J].生物技术,2009,19(5):11-12.
[5]宇文伟刚,张梁,王正祥,等.羰基还原酶基因与葡萄糖脱氢酶基因在大肠杆菌中的共表达及其在不对称还原产麻黄碱中的初步应用[J].工业微生物,2010,40(3):19-24.
[6]Yun H,Choi H L,Fadnavis N W,et al.Stereospecific synthesis of(R)-2-hydroxy carboxylic acids using recombinant E.coli BL21overexpressing YiaE from Escherichia coli K12 and glucosedehydrogenase from Bacillus subtilis[J].Biotechnol Progr,2005,21(2):366-371.
[7]Xu Z N,Jing K J,Liu Y,et al.High level expression ofrecombinant glucose dehydrogenase and its application in NADPHregeneration[J].J Ind Microbiol Biotechnol,2007,34(1):83-90.
[8]彭艳红,张梁,丁重阳,等.重组枯草芽孢杆菌不对称还原产d-伪麻黄碱[J].生物工程学报,2011,27(7):1082-1091.
[9]Sambrook J,Fritsch EF,Maniatis T.Molecular cloning:a laboratorymanual[M].2nd ed.New York:Cold Spring Harbor LaboratoryPress,1989:20-25.
[10]Ausubel F M,Brent R,Kingston R E,et al.Short protocols inmolecular biology:a compendium of methods from current protocolsin molecular biology[M].New York:John Wiley and Sons Inc,2002:56-80.
[11]Dubnau D.Genetic competence in Bacillus subtilis[J].MicrobiolRev,1991,55(3):395-424.
[12]董世建,石贵阳,卢燕,等.微生物转化法生产d-伪麻黄碱[J].无锡轻工大学学报,2004,23(2):5-7.
[13]张鹏华,张梁,卢燕,等.Morganella morganii J-8羰基不对称还原酶的分离纯化及性质研究[J].生物工程学报,2007,23(2):268-272.