Development of a xylitol biosensor composed of xylitol dehydrogenase and diaphorase

In preparation for the development of a xylitol biosensor, the xylitol dehy drogenase of Candida tropicalis IFO 0618 was partially purified and charact erized. The optimal pH and temperature of the xylitol dehydrogenase were pH 8.0 and 50%C, respectively. Of the various alcohols tested, xylitol was th e most rapidly oxidized, with sorbitol and ribitol being reduced at 65% and 58% of the xylitol rate. The enzyme was completely inactive on arabitol, x ylose, glucose, glycerol, and ethanol. The enzyme's xylitol oxidation favor ed the use of NAD(+) (7.9 U/mg) over NADP(+) (0.2 U/mg) as electron accepto r, while the reverse reaction, D-xylulose reduction, favored NADPH (7.7 U/m g) over NADH (0.2 U/mg) as electron donor. The K-m values for xylitol and N AD(+) were 49.8 mM and 38.2 mu M, respectively. For the generation of the x ylitol biosensor, the above xylitol dehydrogenase and a diaphorase were imm obilized on bromocyan-activated sephallose. The gel was then attached on a dissolved oxygen electrode. In the presence of vitamin K-3, NAD(+) and phos phate buffer, the biosensor recorded a linear response to xylitol concentra tion up to 3 mM. The reaction was stable after 15 min. When the biosensor w as applied to a flow injection system, optimal operation pH and temperature were 8.0 and 30%C, respectively. The strengths and limitations of the xyli tol biosensor are its high affinity for NAD(+), slow reaction time, narrow linear range of detection, and moderate affinity for xylitol.