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Efficient Regeneration of NADPH in a 3‐Enzyme Cascade Reaction by in situ Generation of Glucose 6‐Phosphate from Glucose and Pyrophosphate
Author(s) -
Hartog Aloysius F.,
van Herk Teunie,
Wever Ron
Publication year - 2011
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201100198
Subject(s) - chemistry , cofactor , pyrophosphate , alcohol dehydrogenase , dehydrogenase , nicotinamide adenine dinucleotide phosphate , enzyme , glucose 6 phosphate , nicotinamide adenine dinucleotide , biochemistry , nicotinamide mononucleotide , nad+ kinase , stereochemistry , oxidase test
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 (PP i ) catalyzed by the acid phosphatase from Shigella flexneri (PhoN‐Sf). The G6P formed is used in turn by glucose 6‐phosphate dehydrogenase (G6P DH) 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 PP i and 0.4 mM NADP + . The substoichiometric amount of PP i needed demonstrates that phosphate cycling occurs. Under optimal conditions a total turnover number for NADPH higher than 3000 was reached.