Study On Recombinant Expression Of Anti-Prelog Carbonyl Reductase From Rhodococcus Sp. And Its Biosynthesis Of Chiral Alcohols

Chiral alcohol play vital roles in pharmaceuticals,functional environmental materials,pesticides,flavors due to their unique optical properties.In recent years,with the development of biotechnology,the synthesis of chiral alcohol catalyzed by biological enzyme has become one of the most promising methods.Carbonyl reductase belongs to the family of oxidoreductase,catalyzing the reduction of carbonyl compounds to produce corresponding chiral alcohols.However,there are some problems of low catalytic substrate concentration and poor stereoselectivity in many biocatalysts,limiting its industrial production.Therefore,besides to the modification of the existing catalytic enzymes,it is essential to explore new carbonyl reductase.In this paper,we screened bacterial strains that could convert to?R?-1-phenyl-1,2-ethanediol??R?-PED?.A recombinant Escherichia coli efficiently expressing the gene of carbonyl reductase from the screened strain was constructed.The carbonyl reductase was purified and characterised.Then,the regeneration of coenzyme NADH was achieved by coupling coenzyme regeneration system.Finally,the biphasic catalytic conversion system was optimized and an efficient bioreduction process for producing?R?-PED was established,which laid the foundation for the industrial application of?R?-PED.The main results were summarized as follows:?1?A bacterial strain HBU-SI7 isolated from the soil samples was screened by using?-hydroxyacetophenone?2-HAP?as the substrate,which was capable of enantioselective reduction of 2-HAP to?R?-PED.HBU-SI7 was identified as Rhodococcus sp.by bacterial morphological observation and 16S rDNA analysis.The bioconversion of 2-HAP reached 90.1%with the substrate concentration of 0.5 g/L while 99.3%of e.e.for?R?-PED was achieved.?2?A carbonyl reductase gene rhadh was isolated from the genome of HBU-SI7.The carbonyl reductase RhADH follow the anti-prelog rule.The DNA fragments of rhadh and plasmid pGEX-4T-1were ligated and transformed into E.coli.A recombinant strain E.coli/pGEX-4T-1-rhadh was obtained.Recombinant protein RhADH was purified by GSTrap HP affinity chromatography column.SDS-PAGE analysis showed that the molecular weight of RhADH was 36 kDa.RhADH exhibited specific activities of 110 U/mg with purification factor of 900-fold and recovery of 29.8%.Enzymatic assays revealed that Rh ADH had moderate heat resistance and acid tolerance.The optimal reaction temperature and pH were 45?and 7.5,respectively.Metal ions had great influence on enzyme activity.Cu²⁺?2 mmol/L?completely inhibited RhADH enzyme activity,while Zu²⁺?2 mmol/L?significantly promoted the enzyme activity.RhADH displayed excellent biocatalytic activity and high enantioselectivity toward several different carbonyl compounds.?3?The formate dehydrogenase gene fragment cpfdh was amplified from the C.parapsilosis and the recombinant strain E.coli/pET28a-cpfdh was constructed.Recombinant protein CpFDH was achieved by affinity column chromatography.RhADH-CpFDH coupling system was established to realizing the regeneration of coenzyme NADH in situ.When the enzyme activity ratio of RhADH:CpFDH was 1:10,the bioconversion of 2-HAP reached the highest of 98.3%.?4?The result of the optimization of the enzyme-coupled catalytic system was showed that dibutyl phthalate was the best organic phase in phosphate buffer aqueous solvent.This system reduced the inhibition of substrate and product and promoted the catalytic efficiency.In the two-phase system,the optimal ratio of water/organic phase was 3:1 and the optimal substrate concentration was 40 g/L.The highest bioconversion efficiency was 99.0%with the e.e.value of 99%.It concluded that the two-phase system significantly improved the asymmetric reduction efficiency.