Genome Mining Of Carbonyl Reductases And Their Applications In The Synthesis Of Chiral 1-Phenyl-1,2-Ethanediol

Chiral vicinal diols are very important compounds,and they have been used in many fields,such as the synthesis of drugs,the synthesis of agricultural chemicals,the preparation of functional materials,and the addition of spices and essential oils.Preparation of chiral vicinal diols by chemical methods need complex reaction steps,expensive toxic chemical reagent and environmental unfriendly.Catalyzing asymmetric reduction of α-hydroxy ketones to preparation of chiral vicinal diols by biological methods because of its high catalytic activity,stereoselectivity and environmental friendly,had attracted more and more attention.In this paper,two carbonyl reductases(BDHA and GoSCR)with high activity and high selectivity toward2-hydroxyacetophenone were discovered from a carbonyl reductase box preserved in our lab,and the two carbonyl reductases were purified and characterized.Furthermore,the enzymes coupling system of carbonyl reductase and glucose dehydrogenase were constructed to improve the catalytic efficiency.This paper provided a new method for the preparation of chiral 1-phenyl-1,2-ethanediol.First,two carbonyl reductases BDHA and GoSCR with high selectivity and activity toward 2-hydroxyacetophenone were discovered from twenty carbonyl reductases which were cloned by genome mining from Bacillus subtilis and Gluconobacter.The obtained carbonyl reductases were recloned in vector pET28 a and expressed as soluble protein with high level in E.coli.Second,the obtained carbonyl reductases were purified and the enzyme properties were characterized by 2-HAP as the substrate.The results showed that the optimum reaction pH of BDHA was 6.0 and the optimal reaction temperature was 40oC;BDHA showed residual activity of above 80% over a period of 18 h at pH7.0;BDHA was sensitive to high temperature,the rapid inactivationat was observed at temperature over 40 oC,but the residual enzyme activity could be retained more than 60% for 18 h at 30oC;BDHA maximum activity was 2.1 U/mg,the KM value was 1.0 mM,the kcat value was 1.3 s-1,the kcat/KM value was 1.3 s-1mM-1;BDHA was sensitive to dimethyl sulfoxide(DMSO)and showed >70% of its initial activity when the DMSO concentration was kept with in 15%(v/v);BDHA had good substrate tolerance,even the substrate concentration increase up to 200 mM,it still kept in >95%of its initial activities.The optimum pH of GoSCR was 6.0 and the optimum temperature was 45oC;GoSCR showed residual activity of above 85% over a period of 18 h at pH7.0 and the stability in alkaline condition was higher than BDHA;the thermal stability of GoSCR was lower than that of BDHA,and the residual activity could be retained more than 60% for 10 h at 20oC;Go SCRmaximum activity was 1.1 U/mg about 50% of BDHA,KM value was 0.8 mM,kcat value was 0.5 s-1,kcat/KM value was 0.6 s-1mM-1;high organic solvents concentration of dimethyl sulfoxide would make it lose most of GoSCR enzyme activity,and Go SCR showed >50% of its initial activity when the DMSO concentration was kept with in 15%(v/v);when the substrate concentration increase up to 200 mM,GoSCR still kept in >95% of its initial activities,so the enemzy revealed strong substrate tolerance.Third,in order to solve the consumption of coenzyme of carbonyl reductase in the reaction process,carbonyl reductase coupled with glucose dehydrogenase from Bacillus subtilis for cofactor regeneration was constructed.The recombinant Escherichia coli expressing BDHA,GoSCR and GDH were broken and centrifuged to obtained crude enzyme.The carbonyl reductase system with cofactor regeneration was constructed in vitro with the mixture of the cell-free extracts.The results showed that in vitro bioreductions of2-hydroxyacetophenone by double enzyme coupling systems BDHA/GDH and GoSCR/GDH,without the addition of coenzyme,the substrate conversion rates were greatly improved,the products(R)-and(S)-1-phenyl-1,2-glycol were obtained with >99% yield,>99% ee.The results showed that the cofactors originated from E.coli cells were efficiently regenerated by glucose dehydrogenase.Because the whole cell reaction have the advantages of rapid preparation of catalyst,regeneration of intracellular cofactor and low cost of downstreamtreatment.Last,the co-expression system of carbonyl reductase and glucose dehydrogenase was constructed,and the reaction conditions of co-expression system were optimized.The results showed that E.coil(BDHA-GDH)catalysed 2-hydroxyacetophenone asymmetric reduction,the optimum reaction temperature range was 20-35 oC,the optimum reaction pH range was 6.0-8.0.E.coil(GoSCR-GDH)catalysed 2-hydroxyacetophenone asymmetric reduction,the optimum reaction temperature range was 25-30 oC,the optimum reaction pH range was 7.0-8.0.Both E.coil(BDHA-GDH)and E.coil(GoSCR-GDH)had high substrate tolerance.And when the substrate concentration was 100 mM,the optimum cell dosage of E.coil(BDHA-GDH)and E.coil(GoSCR-GDH)were 10 g cdw/L.In the process of the whole cell catalytic reaction,E.coil(BDHA-GDH)and E.coil(GoSCR-GDH)catalysed 400 mM2-hydroxyacetophenone(54 g/L)with no additional cofactor and at the optimal reaction conditions,the conversions were above 99% yield with 99% ee,and space-time conversion rate up to 18 g·L-1·h-1,respectively.This study provided a new method for the industrial preparation of chiral 1-phenyl-1,2-ethanediol,which has potential industrial application value.