The Screening of a Stereoselective Reduction Strain of Fluorenone and the Isolation of Its Key Reductase
|School||Dalian University of Technology|
|Course||Biochemistry and Molecular Biology|
|Keywords||Biotransformation Asymmetric Reduction Streptomyces Carbonyl reductase Lumefantrine|
Chiral alcohol synthesis of pharmaceuticals , agricultural chemicals , food , and other fine chemical intermediates and raw materials . Carbonyl asymmetric reduction is an important way for the preparation of chiral alcohols . This study screened able to effectively catalyze the prochiral fluorenone high stereoselective reduction of the Streptomyces strains , the preliminary separation and purification to obtain the carbonyl reductase which play a key role , and to provide a new source of biocatalyst for the asymmetric reduction of fluorenone and technology base. The main contents of the paper are as follows: (1) from natural soil actinomycetes library screening can effectively catalyze 2 - chloro- fluorenone restore Streptomyces strain Streptomyces resistomycificus DUT002 . Strain was transformed to obtain the enantiomerically pure dextrose 2 - chloro- lumefantrine , the absolute conformation of the R type . (2) determining the the Streptomyces DUT002 conversion of prochiral fluorenone characteristics . Strains of domesticated cultured for 4 generations , reducing capacity increased to 2.4 times . The optimal reaction conditions for the strains 30 ℃, pH 7.5. As an organic co-solvent , the carbonyl reduction activity increased by 43% to 10% (V / V) DMSO . Maltose to 5% (W / V) as a cosubstrate , providing system of a hydrogen donor and energy promoting the reduced coenzyme NAD (P) H regeneration , the activity can be increased by 30% . Metal ions Al3, of Zn2 and anion NO3 - , H2PO4 - , HPO42 - can effectively improve the vitality of the reaction . Streptomyces DUT002 substrate spectrum, including the 2 - substituted fluorenone . Substituted group is F , Cl , Br, I , CH3 when yield is 92% , 91%, 87%, 81% and 93% ee values ??were GT ; 99 % , 100 % , GT ; 99 % 92% and 74%. (3) preliminary separation and purification of the Streptomyces DUT002 carbonyl reductase . Tris buffer, lysed cell lysate having fluorenone restore vigor, reducing coenzyme NADP (H) dependent on the active group was divided into proteins . Precipitated by (NH4) 2SO4 , BY SEPHADEX G -25 desalting , SOURCE 30Q anion -exchange chromatography , 2 ', 5' - ADP -Sepharose 4B affinity separation and SDS polyacrylamide gel electrophoresis , to obtain a molecular weight of 31kDa protein subunit , the separation factor of 366 times .