Characterization Of Carbonyl Reductase AcCR And Its Molecular Modification And Application In Asymmetric Synthesis Of Chiral Alcohols

Chiral alcohols and their derivatives are important intermediates for synthesis of many chemical products,such as,chiral pharmaceuticals,pesticides and liquid crystal materials.Currently,enzyme and microbial cells as biocatalysts used for the product of chiral alcohols have attracted much more attention.Carbonyl reductase as an important oxidoreductase could be used as biocatalyst to catalyze the asymmetric reduction of carbonyl compounds to obtain optically pure chiral alcohols.Our research group,in preliminary work,has successfully isolated a strain Acetobacter sp.CCTCC M209061which harboring a carbonyl reductase,and the strain shows exclusive anti-Prelog stereoselectivity for asymmetric reduction of many of prochiral carbonyl compounds,possessing broad substrate spectrum and excellent stereoselectivity.Despite the strain has potential to be a versatile biocatalyst,its application was restricted by its low expression level of carbonyl reductase and strain biomass,complicated enzyme system in Acetobacter sp.CCTCC M209061,and expensive medium components including large amount of nitrogen source to maintain its normal growth,as well as the laborious purification procedure.To solve those problems and investigate the enzymatic characterization of carbonyl reductase from Acetobacter sp.CCTCC M209061 for further application,in this dissertation,the carbonyl reductase?AcCR?has been cloned and over-expressed in E.coli by gene engineering methods,followed by systematical investigation of the enzymatic characterization of the carbonyl reductase.Then the molecular modification has been well conducted with site-directed mutation according its enzymatic characterization.Moreover,the carbonyl reductase mutant and glucose dehydrogenase?GDH?gene has been co-expressed in E.coli,and the application of the co-expressed strain to catalyze the asymmetric reduction of carbonyl compounds has been investigated to develop a high efficient system of synthesis of chiral alcohol.The carbonyl reductase gene from Acetobacter sp.CCTCC M209061 was consist of a polynucleotide sequence with the length of 762 bp which contained an intact open reading frame?ORF?,encoding a putative polypeptide of 253 amino acid residues with a predicted molecular weight of about 27 kDa.Multiple sequence alignment analysis showed the AcCR belonged to short chain dehydrogenase/reductase?SDRs?.The AcCR has a typical Rossmann fold,N-terminal has coenzyme-binding pattern of typically G-x-x-x-G-x-G,and an active-site pattern of S-x_n-Y-x-x-x-K was located at mid-chain patterns.The accr was constructed to pGEX-2T which carrying a GST tag could augment the solubility of insoluble recombinant protein.The specific activity of carbonyl reductase in cells and biomass of BL21?DE3??pGEX-accr?reached 425.7 U/g-dw and 1.57 g/L,which were much higher than the wild Acetobacter strain,improving 10.78 and 1.43 times,respectively.The recombinant AcCR was found to be a kind of carbonyl reductase which could utilize both NAD?H?and NADP?H?as coenzyme to execute the reduction of carbonyl compounds and the oxidation of the corresponding alcohols,and the recombinant AcCR was confirmed to be more preferred NADH to NADPH.The enzyme exhibited excellent thermostability with half-lives of 25.8 h at 35°C and 13.9 h at 45°C,respectively.The best pH stability was obtained at pH 6.5 with 80%relative activity after incubating 96 h at 4 ^oC.Mn²⁺,Mg²⁺and Zn²⁺didn't have markedly activation and inhibition of EDTA was also slightly to recombinant AcCR,showing that the recombinant AcCR was a metalion-independent enzyme.Iodoacetamide and?-mercaptoethanol had no significant effect on the activity of the recombinant AcCR,indicating there was no crucial disulfide linkages present in the catalytic groups of the enzyme.The recombinant AcCR exhibited excellent enantioselectivity and broad substrate spectrum with aryl ketones,?-ketoesters and aliphatic ketones with highly stereoselective anti-Prelog reduction of those prochiral carbonyl compounds.Moreover,the reduction activities of carbonyl compounds were obviously higher than those of oxidation activity of alcohols,which was beneficial to proceed reductions to obtain chiral alcohols.The structure characteristic of AcCR was obtained and analyzed by homologous modeling and molecular docking,followed by prediction and semi-rational design of the mutation sites.After site-directed mutation of AcCR,the activity of the mutants towards 4'-chloroacetophenone?mut-E144A/G152L?,ethyl 2-oxo-4-phenylbutyrate?mut-G152L/Y189N?and 2-hydroxy-acetophenone?mut-I147V/G152L?were improved 17.9,61.3 and 17.4 times,respectively.The AcCR mutants were available to catalyze asymmetric reduction of several carbonyl compounds with excellent substrates specificity and enantioselectivity.Under 200mmol/L different substrates,the yield could reached 76.8%-99.1%with>99%of e.e.value.The carbonyl reductase mut-I147V/G152L?mut-AcCR?was successfully expressed in E.coli combined with glucose dehydrogenase?GDH?to construct an efficient whole-cell biocatalyst with coenzyme NADH in situ regeneration.The specific activity of the mut-AcCR in the recombinant strain reached 420.9 U/g-dw.The asymmetric reduction of 2-hydroxy-acetophenone catalyzed by the recombinant strain has been investigated.The optimal buffer pH,reaction temperature,substrate concentration,cells dosage,mole ratio of glucose to HAP were 6.5,35 ^oC,250 mmol/L,15 mg-dw/mL and 1.5,respectively,under which,the initial reaction rate,the yield and the product e.e.were 4.22 mmol/L/min,94.7%and above 99%.Further study the reduction in C₄MIMI·PF₆/buffer system,the optimal substrate concentration was improved to 450 mmol/L,and the initial reaction rate,the yield and the product e.e.were6.74 mmol/L/min,92.0%and>99%.The space time yield enhanced 1.46 times compared with that in buffer system.When the reaction was conducted in 100 mL preparative scale in C₄MIMI·PF₆/buffer system,a promising and competitive product yield was achieved?90.5%?with a changeless e.e.value.This study not only provides an in-depth understanding of enzymatic characterization of carbonyl reductase from Acetobacter sp.,but also improves activity of AcCR by molecular modification,opening an opportunity to friendly synthesis of alcohols for industrial production with the high efficient whole-cell catalyst.