title: Efficient regeneration of NADPH using an engineered phosphite dehydrogenase.
abstract: 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Tyler W
authors.initials: TW
authors.lastname: Johannes
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Woodyer
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D004789:Enzyme Activation; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D009249:NADP / Q000737:chemistry* / Q000235:genetics; D010626:Phenylethyl Alcohol / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D044223:Pseudomonas stutzeri / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D014993:Xylitol / Q000138:chemical synthesis*
chemical_list: D011994:Recombinant Proteins; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase; D010626:Phenylethyl Alcohol; D014993:Xylitol