Efficient regeneration of NADPH using an engineered phosphite dehydrogenase

Tyler W. Johannes, Ryan D. Woodyer, Huimin Zhao

Research output: Contribution to journalArticlepeer-review


The in situ regeneration of reduced nicotinamide cofactors (NAD(P)H) is necessary for practical synthesis of many important chemicals. Here, we report the engineering of a highly stable and active mutant phosphite dehydrogenase (12x-A176R PTDH) from Pseudomonas stutzeri and evaluation of its potential as an effective NADPH regeneration system in an enzyme membrane reactor. Two practically important enzymatic reactions including xylose reductase-catalyzed xylitol synthesis and alcohol dehydrogenase-catalyzed (R)-phenylethanol synthesis were used as model systems, and the mutant PTDH was directly compared to the commercially available NADP+-specific Pseudomonas sp. 101 formate dehydrogenase (mut Pse-FDH) that is widely used for NADPH regeneration. In both model reactions, the two regeneration enzymes showed similar rates of enzyme activity loss; however, the mutant PTDH showed higher substrate conversion and higher total turnover numbers for NADP+ than mut Pse-FDH. The space-time yields of the product with the mutant PTDH were also up to fourfold higher than those with mut Pse-FDH. In particular, a space-time yield of 230 g L-1 d-1 xylitol was obtained with the mutant PTDH using a charged nanofiltration membrane, representing the highest productivity compared to other existing biological processes for xylitol synthesis based on yeast D-xylose converting strains or similar in vitro enzyme membrane reactor systems.

Original languageEnglish (US)
Pages (from-to)18-26
Number of pages9
JournalBiotechnology and bioengineering
Issue number1
StatePublished - Jan 1 2007


  • Biocatalysis
  • Cofactor regeneration
  • Enzyme membrane reactor
  • NADPH regeneration

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology


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