Abstract
A major problem when xylose is used for ethanol production is the intercellular redox imbalance arising from different coenzyme specificities of xylose reductase (XR) and xylitol dehydrogenase. The residue Lys21 in XR from Pichia stipitis was subjected to site-directed mutagenesis to alter its coenzyme specificity. The N272D mutant exhibited improved catalytic efficiency when NADH was the coenzyme. Both K21A and K21A/N272D preferred NADH to NADPH, their catalytic efficiencies for NADPH were almost zero. The catalytic efficiency of K21A/N272D for NADH was almost 9-fold and 2-fold that of K21A and the wild-type enzyme, respectively. Complete reversal of coenzyme specificity toward NADH and improved catalytic efficiency were achieved.
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Acknowledgements
This study was supported by grants from the National 863 Bioinformatics projects under contract no. 2007AA02Z333, the Chinese National Science Foundation under contract nos. 20773085 and 30870476, and the Guangdong Natural Science Foundation under contract no. 06300199. It was also supported by the Virtual Laboratory for Computational Chemistry of CNIC and the Supercomputing Center of CNIC, Chinese Academy of Sciences.
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Qi-Kai Zeng, Hong-Li Du, Jing-Fang Wang have contributed equally to this work.
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Zeng, QK., Du, HL., Wang, JF. et al. Reversal of coenzyme specificity and improvement of catalytic efficiency of Pichia stipitis xylose reductase by rational site-directed mutagenesis. Biotechnol Lett 31, 1025–1029 (2009). https://doi.org/10.1007/s10529-009-9980-x
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DOI: https://doi.org/10.1007/s10529-009-9980-x