单加氧酶基因筛选表达及其在苯甲亚砜中的合成应用

Cloning and expression of monooxygenase genes and their application in the synthesis of phenyl methyl sulfoxide

目的克隆和表达假单胞菌菌株Pseudomonas monteilii CCTCC M2013683的单加氧酶基因并研究其在手性亚砜合成中的应用。方法根据假单胞菌菌株Pseudomonas monteilii CCTCC M2013683基因组序列,选取3个单加氧酶基因设计特异性的PCR引物,利用PCR技术,以菌株基因组DNA为模板扩增基因片段,构建重组质粒p ET-32a-pmmo,转化至大肠杆菌BL21(DE3)中,诱导表达目的蛋白。以构建的重组菌为生物催化剂,苯甲硫醚为底物进行催化反应。结果扩增获得了3个目的基因并构建了相应的重组质粒,诱导表达的3个重组蛋白中有2个(pmmo-3546、pmmo-6814)具有活性,可催化苯甲硫醚转化为R构型的苯甲亚砜。结论成功获得了具有R-选择性催化苯甲硫醚活性的单加氧酶重组蛋白。

Objective Cloning and expression of monooxygenase genes of Pseudomonas monteilii CCTCC M2013683 were performed to investigate their application in the synthesis of chiral sulfoxides. Methods Based on the genome sequence of Pseudomonas monteilii CCTCC M2013683, three monooxygenase genes were selected and specific PCR primers were designed. Afterwards, PCR technology was applied to amplify these genes using genom- ic DNA as template. Recombinant plasmids pET- 32a -pmmo were then constructed and transformed into E. coli BI21 （DE3） for recombinant proteins expression. Finally, the genetically engineered bacteria expressing recombi- nant monooxygenase proteins were used as biocatalysts to catalyze thioanisole substratc. Results Three target genes were amplified and corresponding plasmids were constructed. Recombinant proteins pmmo -3546 and pmmo -6814 exhibit high activity towards thioanisole to form chiral sulfoxide with R - selectivity. Conclusion Recombinant mo- nooxygenase proteins with R -selectivity catalytic activity for thioanisole were successfully obtained.