| Enantiopure alcohols are the key chiral building blocks for many important chiral pharmaceuticals. Asymmetric reduction of the corresponding prochiral ketones catalyzed by microbial cells or oxidoreductase has been thought as one of the most promising synthesis methods to produce enantiopure alcohols. The main contents of this work are as follows:Acetophenone was chosen as the model substrate for screening the appropriate microbe strain to catalyze the asymmetric reduction of prochiral ketones. Gaseous and liquid acetophenone was supplied as the sole carbon in the microbial isolation. First, the microbe in the soil samples was isolated by culture with adding acetophenone as the sole carbon source. Then the appropriate microbial strains, which could catalyze the asymmetric reduction reaction, were screened by the reduction reaction with yield and e.e. (e.e., enantiometric excess) as the indicators. Eighteen strains were obtained,which could catalyze the asymmetric reduction of acetophenone. Fourteen strains could catalyze asymmetric reduction of acetophenone to R-1-phenyl ethanol. On the contrary, S-1-phenyl ethanol could be obtained with the other 4 strains. #1-2 strain is the best strain in term of yield and e.e.. Furthermore, the reaction characteristics of the asymmetric reduction of acetophenone catalyzed by #1-2 strain were studied. Effects of reaction temperature, reaction buffer pH, substrate concentration, cosubstrate, reaction time on the reaction were investigated. The optimal reduction conditions were obtained: 30℃, pH 6.0, 15 mmol/L acetophenone, reaction for 30 h and using isopropanol as the cosubstrate.Microbial identification was conducted with morphological features, biochemical and physiological test, and 16S rDNA molecular biology identification. The colonies on the plate appeared circular and radial spreading to the surrounding. The mycelium had partitions and was easy to be broken, the aerial mycelium was straight and ramose without spiral structure. The spores were column-shaped. The 16S rDNA sequence was queried in NCBI database with BLAST method. The result showed that the #1-2 strain was very similar with Microbacterium sp. The identities is 99% to Microbacterium sp. In the phylogenetic tree, #1-2 strain was closely related to several strains in Microbacterium sp. All these taxonomic studies of #1-2 strain indicated that it belongs to Microbacterium sp. in Bergy's Manual of Determinative Bacteriology(Eighth Edition). However, there were several differences between #1-2 strain and four strains of Microbacterium sp. in Bergy's Manual of Determinative Bacteriology (Eighth Edition) in morphological and physiological characteristics. So #1-2 strain might be a new member in Microbacterium sp.We tried to isolate and purify the carbonyl reductase from the new screened strain, Microbacterium sp., which could catalyze the asymmetric reduction reaction of acetophenone. First, cells were disintegrated by ultrasonic methodology. Then the carbonyl reductase was partially purified through DEAE ion exchange chromatography and Phenyl Sepharose hydrophobic interaction chromatography. The partially purified enzyme was obtained. Purification folds was 50, yield was 13.5%,. Furthermore, enzymatic characteristics of the new isolated carbonyl reductase were discovered. The results showed that the carbonyl reductase was a NADH dependent reductase. The optimal temperature for the enzyme was 40℃. The optimal pH was 7.0. The enzyme was a good thermal stable reductase. Although pretreated at 60 oC, 70% of the activity could be remained. The enzyme was stable under the pH between 6.0 and 8.0. Cu2+ strongly inhibited enzyme activity, while EDTA didn't affect the activity of the enzyme.
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