假单胞菌甲苯双加氧酶在手性亚砜合成中的活性分析
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摘要
[目的]从蒙氏假单胞菌CCTCC M2013683中克隆出甲苯双加氧酶基因,通过基因克隆与重组表达,获得重组酶,初步探讨其在手性亚砜合成中的活性。[方法]利用基因克隆技术获得甲苯双加氧酶基因的重组质粒,通过热激法转化到大肠杆菌BL21(DE3)中,用IPTG进行可溶性蛋白的诱导表达,用SDS-PAGE凝胶电泳检测可溶性蛋白的表达情况。将表达有重组蛋白的整细胞作为催化剂,催化氧化苯甲硫醚,用气相色谱检测苯甲亚砜的产率。[结果]基因tod-c1和基因tod-c2成功地连接在了p ETDUET-1载体上,重组表达菌株表达出了大量可溶性蛋白(Tod-C1/C2)。甲苯双加氧酶催化氧化2 mmol/L苯甲硫醚底物,最终得到了产率为0.6%的苯甲亚砜。[结论]获得了甲苯双加氧酶重组蛋白,并应用该蛋白催化获得了产率为0.6%的苯甲亚砜。
[Objective] Cloning and recombinant expressing toluene dioxygenase genes from Pseudomonas monteilii CCTCC M2013683 for the biocatalytic synthesis research of chiral sulfoxide. [Methods] Gene cloning technology was used to obtain the recombinant plasmid of toluene dioxygenase genes. The recombinant plasmid was transformed into Escherichia coli BL21( DE3) expression strain by heat shock,and the soluble protein was induced by IPTG. The expression of soluble protein was detected by SDS-PAGE gel electrophoresis. The whole cell expressed the recombinant protein was used as a catalyst to catalyze the oxidation of methyl phenyl sulfide. The yield of methyl phenyl sulfoxide was detected by gas chromatography. [Results]Two genes( tod-c1 and tod-c2) were cloned into the plasmid p ETDUET-1. The soluble recombinant proteins( Tod-C1/C2) were successfully expressed. The recombinant toluene dioxygenase was able to catalyze the oxidation of 2 mmol/L methyl phenyl sulfide,yielding 0. 6% of methyl phenyl sulfoxide. [Conclusion] Recombinant protein of toluene dioxygenase was expressed and 0. 6% yield of methyl phenyl sulfoxide was obtained catalyzed by this enzyme.
[Objective] Cloning and recombinant expressing toluene dioxygenase genes from Pseudomonas monteilii CCTCC M2013683 for the biocatalytic synthesis research of chiral sulfoxide. [Methods] Gene cloning technology was used to obtain the recombinant plasmid of toluene dioxygenase genes. The recombinant plasmid was transformed into Escherichia coli BL21( DE3) expression strain by heat shock,and the soluble protein was induced by IPTG. The expression of soluble protein was detected by SDS-PAGE gel electrophoresis. The whole cell expressed the recombinant protein was used as a catalyst to catalyze the oxidation of methyl phenyl sulfide. The yield of methyl phenyl sulfoxide was detected by gas chromatography. [Results]Two genes( tod-c1 and tod-c2) were cloned into the plasmid p ETDUET-1. The soluble recombinant proteins( Tod-C1/C2) were successfully expressed. The recombinant toluene dioxygenase was able to catalyze the oxidation of 2 mmol/L methyl phenyl sulfide,yielding 0. 6% of methyl phenyl sulfoxide. [Conclusion] Recombinant protein of toluene dioxygenase was expressed and 0. 6% yield of methyl phenyl sulfoxide was obtained catalyzed by this enzyme.
引文
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[15]Zhigang Zhang,Richard Lonsdale,Joaquin Sanchis,et al.Extreme synergistic mutational effects in the directed evolution of a Baeyer-Villiger monooxygenase as catalyst for asymmetric sulfoxidation[J].Journal of the American Chemical Society,2014,136(49):17262-17272.
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[17]Tisana Nitisakulkan,Shota Oku,Daizo Kudo,et al.Degradation of chloroanilines by toluene dioxygenase from Pseudomonas putida T57[J].Journal of Bioscience&Bioengineering,2014,117(3):292-297.
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[19]María Agustina Vila,Mariana Pazos,César Iglesias,et al.Toluene dioxygenase‐catalysed oxidation of benzyl azide to benzonitrile:mechanistic insights for an unprecedented enzymatic transformation[J].Chembiochem A European Journal of Chemical Biology,2015,17(4):291-295.
[2]Imene Boussouar,Qian Jin Chen,Xue Chen,et al.Single nanochannel platform for detecting chiral drugs[J].Analytical Chemistry,2017,89(2):1110-1116.
[3]Fernanda S.Correia-Melo,Samantha S.Silva,Lucas Araújo-de-Freitas,et al.S-(+)-ketamine-induced dissociative symptoms as a traumatic experience in patients with treatment-resistant depression[J].Revista Brasileira De Psiquiatria,2017,39(2):188-189.
[4]Sahid Hussain,Dhrubajyoti Talukdar,Saitanya K.Bharadwaj,et al.VO2F(dmpz)2:a new catalyst for selective oxidation of organic sulfides to sulfoxides with H2O2[J].Cheminform,2013,44(10):6512-6515.
[5]Stephanie G.Burton.Oxidizing enzymes as biocatalysts[J].Trends in Biotechnology,2003,21(12):543-549.
[6]Jung-Min Choi,Sang-Soo Han,Hak-Sung Kim.Industrial applications of enzyme biocatalysis:Current status and future aspects[J].Biotechnology Advances,2015,33(7):1443-1454.
[7]Xiao Hong Zhai,Yuan Hui Ma,Dun Yue Lai,et al.Development of a whole-cell biocatalyst with NADPH regeneration system for biosulfoxidation[J].Journal of Industrial Microbiology&Biotechnology,2013,40(8):797-803.
[8]Peng Fei Gao,Ai Tao Li,Heng Hiang Lee,et al.Enhancing enantioselectivity and productivity of P450-catalyzed asymmetric sulfoxidation with an aqueous/ionic liquid biphasic system[J].Acs Catalysis,2014,4(10):3763-3771.
[9]Maria Laura Mascotti,Martin Alejandro Palazzolo,Elizabeth Lewkowicz,et al.Expanding the toolbox for enantioselective sulfide oxidations:Streptomyces strains as biocatalysts[J].Biocatalysis&Agricultural Biotechnology,2013,2(4):399-402.
[10]María-Isabel Ramos-González,Arie Ben-Bassat,MaríaJesús Campos,et al.Genetic engineering of a highly solvent-tolerant Pseudomonas putida strain for biotransformation of toluene to p-hydroxybenzoate[J].Applied&Environmental Microbiology,2003,69(9):5120-5127.
[11]Kyoung Lee,Brand J M,David Gibson.Stereospecific sulfoxidation by toluene and naphthalene dioxygenases[J].Biochemical&Biophysical Research Communications,1995,212(1):9-15.
[12]Yong Zheng Chen,Jun Rui Zhuo,Dai Jun Zheng,et al.Stereoselective oxidation of sulfides to optically active sulfoxides with resting cells of Pseudomonas monteilii CCTCC M2013683[J].Journal of Molecular Catalysis B Enzymatic,2014,106:100-104.
[13]Jia Wei Yang,Dai Jun Zheng,Bao Dong Cui,et al.RNA‐seq transcriptome analysis of a Pseudomonas strain with diversified catalytic properties growth under different culture medium[J].Microbiologyopen,2016,5(4):626-636.
[14]Daijun Zheng,Min Yang,Junrui Zhuo,et al.Regio-and stereoselective benzylic hydroxylation to synthesize chiral tetrahydroquinolin-4-ol and tetrahydro-1H-benzo[b]azepin-5-ol with Pseudomonas plecoglossicidas[J].Journal of Molecular Catalysis B Enzymatic,2014,110:87-91.
[15]Zhigang Zhang,Richard Lonsdale,Joaquin Sanchis,et al.Extreme synergistic mutational effects in the directed evolution of a Baeyer-Villiger monooxygenase as catalyst for asymmetric sulfoxidation[J].Journal of the American Chemical Society,2014,136(49):17262-17272.
[16]Jian Bo Wang,Adriana Ilie,Manfred T.Reetz.Chemo-and stereoselective cytochrome P450-BM3 catalyzed sulfoxidation of 1‐thiochroman-4-ones enabled by directed evolution[J].Advanced Synthesis&Catalysis,2017,359(12):2056-2060.
[17]Tisana Nitisakulkan,Shota Oku,Daizo Kudo,et al.Degradation of chloroanilines by toluene dioxygenase from Pseudomonas putida T57[J].Journal of Bioscience&Bioengineering,2014,117(3):292-297.
[18]Derek R.Boyd,Narain D.Sharma,John F.Malone,et al.Toluene dioxygenase-catalysed synthesis and reactions of cis-diol metabolites derived from 2-and 3-methoxyphenols[J].Journal of Organic Chemistry,2015,80(7):3429-3439.
[19]María Agustina Vila,Mariana Pazos,César Iglesias,et al.Toluene dioxygenase‐catalysed oxidation of benzyl azide to benzonitrile:mechanistic insights for an unprecedented enzymatic transformation[J].Chembiochem A European Journal of Chemical Biology,2015,17(4):291-295.