- Cloning, Expression, and Characterization of Xylose Reductase with Higher Activity from Candida tropicalis
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Feiwei Zhang , Dairong Qiao , Hui Xu , Chong Liao , Shilin Li , Yi Cao
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J. Microbiol. 2009;47(3):351-357. Published online June 26, 2009
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DOI: https://doi.org/10.1007/s12275-008-0225-9
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Xylose reductase (XR) is a key enzyme in xylose metabolism because it catalyzes the reduction of xylose to xylitol. In order to study the characteristics of XR from Candida tropicalis SCTCC 300249, its XR gene(xyl1) was cloned and expressed in Escherichia coli BL21 (DE3). The fusion protein was purified effectively by Ni2+-chelating chromatography, and the kinetics of the recombinant XR was investigated. The Km values of the C. tropicalis XR for NADPH and NADH were 45.5 uM and 161.9 uM, respectively, which demonstrated that this XR had dual coenzyme specificity. Moreover, this XR showed the highest catalytic efficiency (kcat=1.44x04 min-1) for xylose among the characterized aldose reductases. Batch fermentation was performed with Saccharomyces serivisiae W303-1A:pYES2XR, and resulted in 7.63 g/L cell mass, 93.67 g/L xylitol, and 2.34 g/Lh xylitol productivity. This XR coupled with its dual coenzyme specificity, high activity, and catalytic efficiency proved its utility in in vitro xylitol production.
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Citations
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- Cloning, Expression, and Functional Characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate Synthase Gene in Escherichia coli
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Yi Yi , Dairong Qiao , Linhan Bai , Hui Xu , Ya Li , Xiaolin Wang , Yi Cao
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J. Microbiol. 2007;45(2):153-157.
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DOI: https://doi.org/2519 [pii]
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Abstract
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5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.
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