Co-expressed System Construction And Site-directed Mutagenesis Of Imine Reductase

Imine Reductase(IRED)is a type of oxidoreductase that relies on the coenzyme NADPH.It can catalyze the asymmetric synthesis of chiral amines by imines or ketones and amines,with mild reaction conditions and high stereoselectivity.This unique advantage is crucial for the preparation of chiral amines.In this thesis,by coexpressing an IRED and glucose dehydrogenase(GDH),the regeneration of coenzyme NADPH is achieved,with reduced cost and efficient synthesis.Based on protein structure analysis and site-directed mutagenesis,the enzyme with better properties can be obtained.In this research,ired and gdh gene from Bacillus were cloned into plasmid p ET-28a(+)respectively,to construct recombinant plasmids.Then the co-expression plasmid p ET28a-ired-gdh was constructed using them as templates,and expressed in E.coli BL21(DE3).The enzyme activities were determined to be 68 U/g and 58 U/g respectively.The copexression strain was used to calalyze 2MPN.It was found at 37 °C and p H 8,with 10% methanol,the conversation rate and optical purity were both greater than 95%.Substrate specificity studies revealed that the enzyme could not catalyze the synthesis of solifenacin and rivastigmine intermediates,suggesting that the substrate binding domain of the enzyme is narrow.Site-specific mutations were predicted by Hot Spot Wizard 2.0.Four mutants,M192 L,W196L,G256 A and S259 G were constructed.Mutant M192 L with increased activity and decreased transformation rate indicated that the stability of the mutant was decreased.Mutant W196 L with decreased activity and conversion rate indicated that these sites were indeed key sites for enzyme activity.Mutant G256 A and S259 G with reduced activity and nondecreasing conversion rate indicated the stability has been enhanced.At present,there are few researches on the reductase coenzyme regeneration system and catalytic mechanism of imine reductase.In this study,an imine reductase coenzyme regeneration system was constructed,the key enzyme sites were verified and stains with better enzymatic properties were obtained,which laid the foundation for the scale-preparation of chiral amines and further research of the properties of the enzyme.