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    Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae

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    Fulltext not available
    Authors
    Zhang, M.
    Jiang, S.
    Zheng, Z.
    Li, Xingjiang
    Luo, S.
    Wu, X.
    Date
    2015
    Type
    Journal Article
    
    Metadata
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    Citation
    Zhang, M. and Jiang, S. and Zheng, Z. and Li, X. and Luo, S. and Wu, X. 2015. Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae. Journal of Basic Microbiology. 55 (7): pp. 907-921.
    Source Title
    Journal of Basic Microbiology
    DOI
    10.1002/jobm.201400786
    ISSN
    0233-111X
    School
    Department of Chemical Engineering
    URI
    http://hdl.handle.net/20.500.11937/7280
    Collection
    • Curtin Research Publications
    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 [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.

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