title: A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis.
abstract: Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD(+) to NADP(+). In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.
authors.affiliation: Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors.forename: Sadat Mohammad Rezq
authors.initials: SM
authors.lastname: Khattab
authors.forename: Seiya
authors.initials: S
authors.lastname: Watanabe
authors.forename: Masayuki
authors.initials: M
authors.lastname: Saimura
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kodaki
mesh_terms: D000449:Aldehyde Reductase / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D050542:D-Xylulose Reductase / Q000378:metabolism; D005285:Fermentation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010843:Pichia / Q000201:enzymology*; D015202:Protein Engineering; D014994:Xylose / Q000378:metabolism*
chemical_list: D014994:Xylose; D000449:Aldehyde Reductase; D050542:D-Xylulose Reductase