Sortase A-mediated oligomers of (S)-carbonyl reductase Ⅱ suitable for biotransformation of (S)-phenyl-1, 2-ethanediol

Abstract:

[Objective] To obtain oligomers of (S)-carbonyl reductase Ⅱ with strong activity and stability, we used sortase A from Staphylococcus aureus as molecular "stapler" to conjugate (S)-carbonyl reductase Ⅱ. The (S)-carbonyl reductase Ⅱ oligomers efficiently catalyzed the biotransformation of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol.[Methods] We cloned sortase A gene from S. aureus genome and expressed it in Escherichia coli. The recombinant enzyme was purified through Ni-affinity and gel filtration chromatography. Meanwhile, we added GGGGSLPETGG to C terminus of (S)-carbonyl reductase Ⅱ using genetic techniques, and purified recombinant SCRⅡ-GGGGSLPETGG to homogeneity. We determined the optimal reaction conditions of sortase A-mediated ligation of (S)-carbonyl reductase Ⅱ to oligomers. The enzyme characteristics of the generated oligomers were studied. And the oligomers-catalyzed biotransformation efficiency of (S)-1-phenyl-1,2-ethanediol was further detected.[Results] Oligomers showed a specific activity of 38.5 U/mg, 6-fold increase compared to (S)-carbonyl reductase Ⅱ. The optimal temperature and pH of ligation reaction by oligomers were 35℃ and 6.0 respectively. The relative activity was maintained over 90% at 50℃ for 1 hour. Denaturation test showed that the denaturation temperature of oligomers was 60.1℃, 10℃ higher than that of (S)-carbonyl reductase Ⅱ. Biotransformation results indicated that oligomers completely transformed 5 g/L 2-hydroxyacetophenone within 3 hours to generate (S)-1-phenyl-1,2-ethanediol with an optical purity of 100%. With oligomers, we reduced transformation duration for 16 folds compared to that of recombinant E. coli-(S)-carbonyl reductase Ⅱ cells.[Conclusion] This work first described sortase A-mediated the ligation of oxidoreductase and significantly improved catalytic efficiency and thermal stability of enzyme, suggesting sortase had great potential application in chiral synthesis.

Key words:Sortase A;molecular ligation;oligomer;oxidoreductase;chiral catalysis