Biocatalysis has become a useful tool for organic synthesis, especially enantioselective synthesis. Biocatalytic oxidoreductions are important reactions for asymmetric syntheses of enantiopure compounds that are useful and valuable synthons and pharmaceutical intermediates. However, their practical applications are often limited due to the requirement of stoichiometric amount of the expensive nicotinamide cofactor NAD(P)H. Methods for enzymatic cofactor recycling have been developed based on two concepts: the “coupled substrates” one utilizes a single enzyme such as an alcohol dehydrogenase for the reduction of a carbonyl-containing substrate and for the simultaneous oxidation of an alcohol co-substrate to regenerate NAD(P)H; the “coupled enzymes” concept requires an enzyme for an oxidoreduction and a second enzyme for another reaction to regenerate the cofactor. These concepts have been applied with isolated enzymes as well as whole cells containing the necessary enzyme(s). While approaches based on isolated enzymes are expensive, approaches based on whole cells depend on the amount of available intracellular cofactor which may be limiting and cannot be altered by the addition of extracellular cofactor.

We recently succeeded in efficient enantioselective bioreduction with the recycling of NADPH by the use of permeabilized microbial cells. The cytoplasmic membrane of a microorganism was made permeable for NAD(P)H and NAD(P)+ without the release of the desired enzyme by controlled treatment with a detergent and/or an organic solvent. Such permeabilized cells enable the use of the externally added cofactor for efficient catalysis, cofactor recycling, easy substrate access, and easy product release; moreover, they are cheap, easily available in large amounts, active for longer period, and can be used repeatedly. Reduction of ethyl 3-keto-4,4,4-trifluorobutyrate to (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate with the recycling of NADPH for 4200 times was achieved by the use of permeabilized cells of B. pumilus Phe-C3 containing both (R)-selective NADPH-dependent ketoreductase for the reduction and glucose-6-phosphate-dehydrogenase for the NADPH regeneration.[1] Cofactor recycling for 1620 times in the same bioreduction was achieved by a new approach: the use of “coupled permeabilized microorganisms” – one (B. pumilus Phe-C3) contains the ketoreductase for the reduction and the other (Bacillus subtilis BGSC 1A1) contains glucose dehydrogenase for the regeneration of NADPH.[2]

REFERENCES:

[1] Zhang, J.; Witholt, B.; Li, Z. Adv. Syn. Catal. 2006, 348, 429-433.

[2] Zhang, J.; Witholt, B.; Li, Z. Chem. Commun. 2006, 398-400.