title: Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae.
abstract: Rhizopus oryzae is valuable as a producer of organic acids via lignocellulose catalysis. R. oryzae metabolizes xylose, which is one component of lignocellulose hydrolysate. In this study, a novel NADPH-dependent xylose reductase gene from R. oryzae AS 3.819 (Roxr) was cloned and expressed in Pichia pastoris GS115. Homology alignment suggested that the 320-residue protein contained domains and active sites belonging to the aldo/keto reductase family. SDS-PAGE demonstrated that the recombinant xylose reductase has a molecular weight of approximately 37 kDa. The optimal catalytic pH and temperature of the purified recombinant protein were 5.8 and 50 °C, respectively. The recombinant protein was stable from pH 4.4 to 6.5 and at temperatures below 42 °C. The recombinant enzyme has bias for D-xylose and L-arabinose as substrates and NADPH as its coenzyme. Real-time quantitative reverse transcription PCR tests suggested that native Roxr expression is regulated by a carbon catabolite repression mechanism. Site-directed mutagenesis at two possible key sites involved in coenzyme binding, Thr(226)  → Glu(226) and Val(274)  → Asn(274), were performed, respectively. The coenzyme specificity constants of the resulted RoXR(T226E) and RoXR(V274N) for NADH increased 18.2-fold and 2.4-fold, which suggested possibility to improve the NADH preference of this enzyme through genetic modification.
authors.affiliation: Key Laboratory for Agricultural Products Processing of Anhui Province, School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, Anhui Province, P.R. China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Key Laboratory for Agricultural Products Processing of Anhui Province, School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, Anhui Province, P.R. China.
authors.forename: Shao-tong
authors.initials: ST
authors.lastname: Jiang
authors.affiliation: Key Laboratory for Agricultural Products Processing of Anhui Province, School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, Anhui Province, P.R. China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: Key Laboratory for Agricultural Products Processing of Anhui Province, School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, Anhui Province, P.R. China.
authors.forename: Xing-jiang
authors.initials: XJ
authors.lastname: Li
authors.affiliation: Key Laboratory for Agricultural Products Processing of Anhui Province, School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, Anhui Province, P.R. China.
authors.forename: Shui-zhong
authors.initials: SZ
authors.lastname: Luo
authors.affiliation: Key Laboratory for Agricultural Products Processing of Anhui Province, School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, Anhui Province, P.R. China.
authors.forename: Xue-feng
authors.initials: XF
authors.lastname: Wu
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001089:Arabinose / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D014994:Xylose; D001089:Arabinose; D000449:Aldehyde Reductase
keywords: Pichia pastoris; Real-time qRT-PCR; Rhizopus oryzae; Site-directed mutagenesis; Xylose reductase