| Optically active alcohols have been recognized as key chiral intermediates for synthesizing fine chemicals and pharmaceuticals.Carbonyl reductase-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols due to the high chemo-,enantio-,and regioselectivity of the enzymes.However,the majority of prochiral ketones used in industrial applications are structurally complex.These include aryl ketones,ketoesters and heterocyclic ketones bearing bulky substituents.Substrate size-induced deficiency of enzyme activity is still a challenge for the enzymatic reduction of ketones.Therefore,it is necessary to develop suitable enzymes for substrates with bulky groups,for the production of corresponding chiral alcohols with high optical purity.In this study,a series of stereospecific carbonyl reductases towards sterically hindered substrates were identified and constructed.The catalytic property and stereoselectivity of the enzymes were further evaluated towards various aryl ketones,ketoesters and heterocyclic ketones,achieving the highly stereoselective synthesis of various pharmaceutical intermediates.Besides,the effects of reaction conditions on asymmetric reduction of both ethyl 2-oxo-4-phenylbutyrate(OPBE)and l-benzyl-3-pyrrolidone were explored.The main results are as follows:(1)Aldo-keto reductases from Candida albicans(CaCR),Saccharomyces cerevisiae(ScCR),Kluyvero.yces marxianus(KmCR)were newly identified through gene mining.And another two aldo-keto reductases from C.parapsilosis(CPR-C1,CPR-C2)were also identified.Recombinant Escherichia coli BL21(DE3)/pET-28a(+)-CaCR,E.coli BL21(DE3)/pET－28a(+)-ScCR,E.coli BL21(DE3)/pET-28a(+)-KmCR were constructed and actively expressed by IPTG induction.Thus,we constructed a ketoreductase toolbox towards bulky substrates.In addition,two mutants of carbonyl reductase RCR,F285A/W286A and W116A,also served as members of the toolbox.By Ni-NTA affinity chromatography,purified enzymes that showed a single band on SDS-PAGE were obtained.(2)The capability of the toolbox in catalyzing reduction of bulky carbonyl compounds was investigated using various substrates,including aryl ketones,ketoesters and heterocyclic ketones.The results showed that each carbonyl reductase exhibited catalytic activity to one or more of the tested substrates.Among the enzymes,ScCR showed a broader substrate spectrum compared to the others.Most of the enzymes were potential to reduce ethyl benzoylformate.Regarding Km values related to substrate association,we also provided insights into the specificity and preference of certain enzymes.(3)The enantioselectivities of the recombinant carbonyl reductases catalyzing asymmetric reduction were evaluated.The results showed that substrates were converted to the corresponding chiral alcohols,in line with the demand of stereo-configuration for application.Enantiopure(R)-methyl mandelate,(R)-ethyl mandelate,(R)-ethyl-2-hydroxy-4－phenylbutyrate((R)-HPBE),ethyl(S)-3-hydroxy-3-phenylpropanoate’ and(S)-1-benzyl-3－pyrrolidinol(>99%e.e.)were obtained.(4)The effects of reaction system components and conditions on the asymmetric reduction of OPBE and l-bcnzyl-3*pyrrolidone were investigated.The results showed that when OPBE was reduced by F285A/W286A,the yield of 80.3%and the optical purity of more than 99%e.e were obtained under the conditions including 1 g·L-1 substrate,isopropanol(3.5%,v+v-1),pH 7.0,30℃,and 12 h.And in the case of l-benzyl-3-pyrrolidone by W116A,the yield of 75.2%and the optical purity of more than 99%e.e was obtained under the conditions including 0.5 g·L-1 of substrate and isopropanol(60%,v·v-1). |
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