Coupled Recombinant System Co-expressing Stereospecific Carbonyl Reductase And Glucose Dehydrogenase For Asymmetric Synthesis Of(R)-4-Methoxyl-1-Phenylethanol

Recently, optically pure chiral alcohols and their derivatives can be employed for the synthesis of many chiral pharmaceuticals and functional materials. It is a hotspot for many researchers to prepare them through biological methods in mild reaction conditions these days. The enantiopure(R)-4-methoxyl-1-phenylethanol(MOPE) is a key intermediate of many synthetic drugs. Meanwhile,(R)-MOPE can be used for the synthesis of chiral 3-aryl-3-substituted propanoic acids with antiinflammation activity. Up to now, information on the bio-catalytic resolution of MOPE to get enantiopure(R)-MOPE using bio-catalytic methods is not too much, and the conversion rate of(R)-MOPE is low. Carbonyl reductase which is needed in preparation and biological catalytic asymmetric reduction of chiral aromatic alcohols is facing problems such as coenzyme regeneration difficulties and low reaction efficiency in industrial production.Before,the 4- methoxy phenylethyl-1-ketone(MOAP) carbonyl reductase SCRII which was obtained from Candida parapsilosis CCTCCM 203011 previously, the function for catalyzing(R)-MOPE of carbonyl reductase(SCRII) was modified by site-directed mutagenesis, which overcome the deficiency of the wild type. Concerning theses issues above, also in order to maintain abundant NADPH supplement, the mutated SCRII was co-expressed with a glucose dehydrogenase(gdh) from Bacillus sp. YX-1. Finally, the whole-cell system and cell-free extract system were investigated with MOAP as substrate respectively. The main results were shown as follows:(1) An important amino acid site(A220) was selected for mutagenesis by the study of amino acid sequence and protein structure alignment. The efficient synthesis of(R)-MOPE was achieved with recombinant E. coli SCRII-A220 D. Then the co-expression plasmid harboring both A220 D and gdh was constructed and transformed into E. coli. The results showed that the catalytic activity of co-expression recombinant E. coli BL21/p ET-SCRII-A220D-SD-AS-gdh was increased.(2) By investigating the effects of bio-mediated reaction conditions, the involved conditions such as substrate concentration, cell concentration, reaction temperature and initial p H value were optimized for enhancement of yield of desired product. When using MOAP(10 g×L-1) as substrate, the catalytic reaction will take 30 h in the alkaline buffer solution by 125 g×L-1recombinant cells. Consequently, the optically pure(R)-MOPE was obtained with optical purity of 98%e.e. and increased yield of 82%.(3) In order to further improve the substrate concentration and catalytic efficiency, cell-free system was constructed. This cell-free system was characterized by using glucose and trace NADP+ to conduct asymmetric reduction. The optimum conditions for the cell-free reaction were as follows: the concentration of the recombinant cells was 150 g×L-1. The concentrations of the NADP+ and glucose were 0.05 m M and 20 g×L-1, respectively. Consequently, the product was obtained with optical purity of 98%e.e. and the yield of 80% when using the cell-free system. Above all, the substrate concentration has improved up to 20 g×L-1 with 12 h of reaction.