Sortase A-mediated Oligomers Of Carbonyl Reductases With Enhanced Themostability Catalyze The Biotransformation Of Chiral Alcohols Efficiently

An NADPH-dependent(S)-carbonyl reductase II(SCRII)from Candida parapsilosis CCTCC M203011 catalyzed asymmetric reduction of 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol with low efficiency and poor thermostability.In this study,we incorporated recognition sequence of sortase A(SrtA)at C-terminus of SCRII,and constructed SrtA-mediated SCRII oligomers with strengthened functionality and thermostability.The purified oligomers were applied to efficient preparation of(S)-1-phenyl-1,2-ethanediol,where substrate and cofactor were 2-hydroxyacetophenone and NADPH,respectively.Then SrtA-mediated technique was subjected to the other 8 oxidoreductases to construct a genenic platform for improving thermal stability and catalytic efficiency of oxidoreductases.The main resuts were as follows:(1)Core sequence of SrtA gene were amplified from genome of Staphylococcus aureus ST451.Recombinant plasmid pET21a-srtA was constructed and overexpression of SrtA in Escherichia coli BL21(DE3)was achieved.Primers were designed to incorporate GGGGSLPETGG to C-terminus of SCRII.The recombinant pET28a-scrII-mtf was transformed into Escherichia Coli BL21(DE3).Induction and expression results showed that GGGGSLPETGG tag had no impact on the expression of SCRII.SrtA-mediated ligation of SCRII-mtf was optimized.When SrtA concentrastion was 1 mg·m L-1,Ca2+ concentration was 10 mmol·L-1,optimal SrtA-medicated ligation temperature was 25 ?,the ligation reaction was incubated for 36 h,almost all SCRII-mtf was consumed.The ligation products were yielded with the highest production.The ligation products were mainly dimers and trimers.The SrtA-mediated oligomers were applied for further research after they were purified using Superdex 200.(2)Enzymatic properties of SCRII oligomers were determined.When at 35 ?,pH 6.0,SCRII-mtf showed a slight improvement in catalytic activity than SCRII.SCRII oligomers had a specific activity of 38.5 U·mg-1 towards 2-hydroxyacetophenone,with 6-fold improvement compared to SCRII.SCRII oligomers displayed the best catalytic performance at 50 ?.After incubation for 1 h at 50 ?,SCRII oligomers remained over 90% and about 50% the whole activity,indicating that oligomerisation of SCRII significantly improved thermal stability.SCRII,SCRII-mtf and SCRII oligomers showed similar pH dependence.But SCRII oligomers exhibited much better pH tolerance than SCRII and SCRII-mtf.Compared to SCRII,Km value of SCRII oligomers was reduced for 3.3 folds,reflecting that SCRII oligomers had an improved affinity towards 2-hydroxyacetophenone.(3)The optimal conditions of SCRII oligomers-catalyzed biotransformation were determined.As a whole,SCRII oligomers biotransformed(S)-1-phenyl-1,2-ethanediol with the highest yield of over 90% at 35 ? and pH 6.0 within 2 h.The substrate 2-hydroxyacetophenone was transformed to(S)-1-phenyl-1,2-ethanediol by oligomers with a yield of 100% and an optical purity of 100% within 3 h.(4)The other 8 oxidoreductases were selected for the construction of SrtA-mediated oligomers.The 8 oxidoreductases had oligomerization at different levels.Circular dichroism spectrum revealed Tm values of oxidoreductase oligomers were improved by 5 ?-12 ?.The specific activity of oxidoreductase oligomers was enhanced by 5-11 folds than their corresponding monomers.The yields of chiral alcohols catalyzed by SrtA-mediated oligomers were improved by 25%-285% compared to their corresponding monomers within 6 h.