Efficient Regeneration of NADPH in a 3-Enzyme Cascade Reaction by in situ Generation of Glucose 6-Phosphate from Glucose and Pyrophosphate

We report here a promising method to regenerate NADPH (nicotinamide adenine dinucleotide phosphate) using the intermediate formation of glucose 6-phosphate (G6P) from glucose and pyrophosphate (PPi) catalyzed by the acid phosphatase from Shigella flexneri (PhoN-Sf). The G6P formed is used in turn by glucose 6-phosphate dehydrogenase (G6PDH) to mediate the reduction of NADP(+) to NADPH. The method was tested in a one-pot system with three enzymes in which the NADPH generated was subsequently used by an alcohol dehydrogenase (ADH) from Lactobacillus kefir or Thermoanaerobium brockii to catalyze the enantioselective reduction of acetophenone to R-(+)-1-phenylethyl alcohol or S-(-)-1-phenylethyl alcohol, respectively with NADP(+) as starting cofactor. We were able to synthesize 50 mL of 50 mM R-(+)-1-phenylethyl alcohol in the presence of 5 mM PPi and 0.4 mM NADP(+). The substoichiometric amount of PPi needed demonstrates that phosphate cycling occurs. Under optimal conditions a total turnover number for NADPH higher than 3000 was reached.