The Screening Of A Stereoselective Reduction Strain Of Fluorenone And The Isolation Of Its Key Reductase

Chiral alcohols are important intermediates and raw materials of pharmaceuticals, agrochemicals, food products and other fine chemicals. Asymmetric reduction of carbonyl compounds is the efficient way to produce chiral alcohols. In this study, a Streptomyces strain with highly prochiral fluorenone reducing activity was obtained and a preliminary trial on isolation of the relevant reductase was carried out. This work may provide a new path for fluorenone asymmetric reduction.The main findings of this dissertation include:(1) Strain Streptomyces resistomycificus DUT002, which was screened from soil, showed efficient carbonyl reducing activity and could be used as a biocatalyst for the asymmetric reduction of 2-chloro-fluorenone. The product 2-chloro-fluorenol was enantiomerically pure. The optical rotation exhibited dextrorotation, and the absolute configuration was deduced to be R.(2) The characteristics of prochiral compounds reduction by the strain DUT002 were determined. The fourth generation of strain DUT002 by domestication increased the activity of reduction by 2.4 folds. The optimal temperature and pH was 30℃and pH 7.5, respectively. 10%(V/V) of organic co-solvent DMSO increased the carbonyl reductive capacity by 43%. 5%(W/V) of co-substrate maltose could be used as energy and hydrogen sources to up the reductive capacity by 30%. Metallic ions Al3+, Zn2+ and anions NO3-, H2PO4-, HPO42- increased the reaction yield rate. The substrate spectrum of strain DUT002 included 2-substituted-fluorenone. The strain could reduce 2-fluoro-, chloro-, bromo-, iodo-and methyl-fluorenone with yields of 92%,91%,87%,81%,93%and e.e. values of>99%,100%, >99%,92%,74%.(3) The carbonyl reductase from the strain DUT002 were isolated. The fluorenone reductive activity was in cell lysates dissolved with Tris buffer. The biocatalyst showed NADP(H)-dependent activity, and was isolated through ammonium sulfate recipitation, Sephadex G-25 desalting chromatography, SOURCE 30Q anion exchange chromatography,2', 5'-ADP-Sepharose 4B isolation and SDS polyacrylamide gel electrophoresis. The isolated subunit was 31kDa accordind to SDS-PAGE analysis and purified by 336 folds based on activity measurement.