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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 (xyll) 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 microM and 161.9 microM, respectively, which demonstrated that this XR had dual coenzyme specificity. Moreover, this XR showed the highest catalytic efficiency (kcat =1.44 x 10(4) min(-1)) for xylose among the characterized aldose reductases. Batch fermentation was performed with Saccharomyces serivisiae W303-lA:pYES2XR, and resulted in 7.63 g/L cell mass, 93.67 g/L xylitol, and 2.34 g/L x h 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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His(354) and His(358), two highly conserved histidines in Xenopus laevis (6-4) photolyase [equivalent to His(401) and His(405), in Dunaliella salina (6-4) photolyase], are critical for photoreactivation. They act as a base and an acid, respectively. However, the remaining high repair activity when the pH value is higher than the pKa of histidine suggests the involvement of other basic amino acids in photoreactivation. According to the results of in vivo enzyme assay and three-dimension structural model of Dunaliella salina (6-4) photolyase we hypothesized that Lys(281) might be involved in the photoreactivation over the pH range from 10.0 to 11.0. To test this, we generated two mutant forms of the (6-4) photolyase, K281G and K281R mutant, by overlap extension polymerase chain reaction, and performed the enzyme assay with these mutants. From these results we conclude that the Lys(281), which is highly conserved in (6-4) photolyases, participates in the photoreactivation and acts as an acid to donate a proton to His(401) when the environmental pH is higher than the pKa value of histidine.

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