Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae
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...
| Main Authors: | , , , , , |
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| Format: | Journal Article |
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Wiley-VCH Verlag
2015
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| Online Access: | http://hdl.handle.net/20.500.11937/7280 |
| _version_ | 1848745324509659136 |
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| author | Zhang, M. Jiang, S. Zheng, Z. Li, Xingjiang Luo, S. Wu, X. |
| author_facet | Zhang, M. Jiang, S. Zheng, Z. Li, Xingjiang Luo, S. Wu, X. |
| author_sort | Zhang, M. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | 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 [RIGHTWARDS ARROW] Glu226 and Val274 [RIGHTWARDS ARROW] 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. |
| first_indexed | 2025-11-14T06:15:33Z |
| format | Journal Article |
| id | curtin-20.500.11937-7280 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T06:15:33Z |
| publishDate | 2015 |
| publisher | Wiley-VCH Verlag |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-72802017-09-13T14:40:29Z Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae Zhang, M. Jiang, S. Zheng, Z. Li, Xingjiang Luo, S. Wu, X. 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 [RIGHTWARDS ARROW] Glu226 and Val274 [RIGHTWARDS ARROW] 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. 2015 Journal Article http://hdl.handle.net/20.500.11937/7280 10.1002/jobm.201400786 Wiley-VCH Verlag restricted |
| spellingShingle | Zhang, M. Jiang, S. Zheng, Z. Li, Xingjiang Luo, S. Wu, X. Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae |
| title | Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae |
| title_full | Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae |
| title_fullStr | Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae |
| title_full_unstemmed | Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae |
| title_short | Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae |
| title_sort | cloning, expression, and characterization of a novel xylose reductase from rhizopus oryzae |
| url | http://hdl.handle.net/20.500.11937/7280 |