Isolation and Characterization of Microorganisms for Asymmetric Reduction of Prochiral2-Aminoacetophenone
|School||Tianjin University of Science and Technology|
|Course||Microbial and Biochemical Pharmacy|
|Keywords||Biocatalysis asymmetric reduction carbonyl reductase 2-aminoacetophenone 2-amino-1-phenylethanol|
Many derivatives of2-amino-l-phenylethanol are useful as important adrenergic drugs. Three microbes which could asymmetric reduce2-aminophenone were isolated from soil through two sreening methods. The microbial identification was conducted with morphological features and molecular biology identification.The optimum cultivation conditions and reaction conditions were studied. The main contents of this work are as follows:1)2-aminoacetophenone was supplied as the sole carbon and energy source in screening that52strains were gotten. Through TLC and HPLC, two strains named1403and4802were used for next reserch. They can reduce2-aminoacetophenone to (R)-or (5)-2-amino-l-phenylethanol, and the e.e. could reach to99%and70%especially. Microbial identification was conducted with morphological features and ITS-5.8S rDNA molecular biology identification. The results showed that the4802strain was very similar with Galactomyces sp and the1403strain was very similar with Fusarium sp.The conditions for culture and transformation of both strains were studied. The optimal cultivation time of both strains were24h; the optimized concentrations of substrate and the resting cell were5g/L and20g/L(dry wt) for strain1403,3g/L and80g/L(dry wt) for strain4802, respectively.Both of them can convert2-chloroacetophenone,2-bromoacetophenone,2-hydroxyacetophenone,and acetophenone to corresponding alcohols. When α-hydroxyacetophenone was served as substrate, both of the strains can produce (S)-2-hydroxy-1-phenylethanol and the e.e. could reached to99%.2) In this part,2-aminoacetophenone was set as limiting factor in screening for microbes which could catalyze the asymmetric reduction reaction from2-aminoacetophenone to2-amino-1-phenylethanol. Finally,6303was chosen as the best strain in term of yield and e.e.. The strains can reduce2-aminoacetophenone to (R)-2-amino-1-phenylethanol, the e.e. and the yield could reach to99%and31%especially. Microbial identification was also conducted with morphological features and ITS-5.8S rDNA、26SrDNA D1/D2sequences analysis. The result both showed that the6303was similar with Candida sp.and the identities were100%.The experiment of6303was used to optimize parameters of the bioreduction conditions. The optimum carbon source was10g/L glucose.nitrogen source was10g/L yeast exact; and the optimum cultivation conditions were:inoculum size of2%, initial pH6.5,and30℃The optimum reaction conditions of the asymmetric reduction of2-amino acetophenone were as follows:temperature45℃, buffer pH8, cell concentration 200g/L(wet wt).