Dissertation > Biological Sciences > Microbiology

The Screening of a Stereoselective Reduction Strain of Fluorenone and the Isolation of Its Key Reductase

Author YuanWeiGuang
Tutor YangQing
School Dalian University of Technology
Course Biochemistry and Molecular Biology
Keywords Biotransformation Asymmetric Reduction Streptomyces Carbonyl reductase Lumefantrine
Type Master's thesis
Year 2010
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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 .

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