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Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae.
- Source :
-
Journal of basic microbiology [J Basic Microbiol] 2015 Jul; Vol. 55 (7), pp. 907-21. Date of Electronic Publication: 2015 Feb 24. - Publication Year :
- 2015
-
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.<br /> (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Subjects :
- Aldehyde Reductase chemistry
Aldehyde Reductase isolation & purification
Arabinose metabolism
Cloning, Molecular
Coenzymes metabolism
Electrophoresis, Polyacrylamide Gel
Kinetics
Mutagenesis, Site-Directed
Pichia genetics
Recombinant Proteins metabolism
Rhizopus metabolism
Substrate Specificity
Xylose metabolism
Aldehyde Reductase genetics
Aldehyde Reductase metabolism
Rhizopus enzymology
Rhizopus genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1521-4028
- Volume :
- 55
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Journal of basic microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 25709086
- Full Text :
- https://doi.org/10.1002/jobm.201400786