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|Title:||Bioreduction with efficient recycling of NADPH by coupled permeabilized microorganisms|
|Authors:||Zhang, W. |
|Citation:||Zhang, W., O'Connor, K., Wang, D.I.C., Li, Z. (2009-02). Bioreduction with efficient recycling of NADPH by coupled permeabilized microorganisms. Applied and Environmental Microbiology 75 (3) : 687-694. ScholarBank@NUS Repository. https://doi.org/10.1128/AEM.01506-08|
|Abstract:||The glucose dehydrogenase (GDH) from Bacillus subtilis BGSC 1A1 was cloned and functionally expressed in Escherichia coli BL21(pGDH1) and XL-1 Blue(pGDH1). Controlled permeabilization of recombinant E. coli BL21 and XL-1 Blue with EDTA-toluene under optimized conditions resulted in permeabilized cells with specific activities of 61 and 14 U/g (dry weight) of cells, respectively, for the conversion of NADP + to NADPH upon oxidation of glucose. The permeabilized recombinant strains were more active than permeabilized B. subtilis BGSC 1A1, did not exhibit NADPH/NADH oxidase activity, and were useful for regeneration of both NADH and NADPH. Coupling of permeabilized cells of Bacillus pumilus Phe-C3 containing an NADPH- dependent ketoreductase and an E. coli recombinant expressing GDH as a novel biocatalytic system allowed enantioselective reduction of ethyl 3-keto-4,4,4-trifluorobutyrate with efficient recycling of NADPH; a total turnover number (TTN) of 4,200 mol/mol was obtained by using E. coli BL21(pGDH1) as the cofactor- regenerating microorganism with initial addition of 0.005 mM NADP +. The high TTN obtained is in the practical range for producing fine chemicals. Long-term stability of the permeabilized cell couple and a higher product concentration were demonstrated by 68 h of bioreduction of ethyl 3-keto-4,4,4- trifluorobutyrate with addition of 0.005 mM NADP + three times; 50.5 mM (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate was obtained with 95% enantiomeric excess, 84% conversion, and an overall TTN of 3,400 mol/mol. Our method results in practical synthesis of (R)-ethyl 3-hydroxy-4,4,4- trifluorobutyrate, and the principle described here is generally applicable to other microbial reductions with cofactor recycling. © 2009, American Society for Microbiology. All Rights Reserved.|
|Source Title:||Applied and Environmental Microbiology|
|Appears in Collections:||Staff Publications|
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