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PMID:25709086
| Citation |
'Zhang, M, Jiang, ST, Zheng, Z, Li, XJ, Luo, SZ and Wu, XF (2015) Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae. J. Basic Microbiol. ' |
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| 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. J. Basic Microbiol. 2015, 55, 1-15. |
| Links |
PubMed Online version:10.1002/jobm.201400786 |
| Keywords |
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| edit table |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
|
enables |
GO:0032866: D-xylose:NADP reductase activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
| GO:0032866: D-xylose:NADP reductase activity |
ECO:0000314: |
F |
Table 4 shows the substrate specificity and kinetic parameters for the activity of RoXR. Table 6 shows that wild-type RoxR uses NADPH as a cofactor |
complete | ||||
Notes
See also
References
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