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- Title
Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae.
- Authors
Zhang, Min; Jiang, Shao‐tong; Zheng, Zhi; Li, Xing‐jiang; Luo, Shui‐zhong; Wu, Xue‐feng
- 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, Thr226 → Glu226 and Val274 → Asn274, were performed, respectively. The coenzyme specificity constants of the resulted RoXRT226E and RoXRV274N for NADH increased 18.2-fold and 2.4-fold, which suggested possibility to improve the NADH preference of this enzyme through genetic modification. J. Basic Microbiol. 2015, 55, 1-15
- Subjects
CLONING; GENE expression; XYLOSE reductase; RHIZOPUS oryzae; ORGANIC acids; NICOTINAMIDE adenine dinucleotide phosphate; LIGNOCELLULOSE
- Publication
Journal of Basic Microbiology, 2015, Vol 55, Issue 7, p907
- ISSN
0233-111X
- Publication type
Article
- DOI
10.1002/jobm.201400786