Takamizawa K, Uchida S, Hatsu M, Suzuki T, Kawai K
Department of Bioprocessing, Faculty of Agriculture, Gifu University, Japan.
Can J Microbiol. 2000 Apr;46(4):350-7.
In preparation for the development of a xylitol biosensor, the xylitol dehydrogenase of Candida tropicalis IFO 0618 was partially purified and characterized. The optimal pH and temperature of the xylitol dehydrogenase were pH 8.0 and 50 degrees C, respectively. Of the various alcohols tested, xylitol was the most rapidly oxidized, with sorbitol and ribitol being reduced at 65% and 58% of the xylitol rate. The enzyme was completely inactive on arabitol, xylose, glucose, glycerol, and ethanol. The enzyme's xylitol oxidation favored the use of NAD+ (7.9 U/mg) over NADP+ (0.2 U/mg) as electron acceptor, while the reverse reaction, D-xylulose reduction, favored NADPH (7.7 U/mg) over NADH (0.2 U/mg) as electron donor. The K(m) values for xylitol and NAD+ were 49.8 mM and 38.2 microM, respectively. For the generation of the xylitol biosensor, the above xylitol dehydrogenase and a diaphorase were immobilized on bromocyan-activated sephallose. The gel was then attached on a dissolved oxygen electrode. In the presence of vitamin K3, NAD+ and phosphate buffer, the biosensor recorded a linear response to xylitol concentration up to 3 mM. The reaction was stable after 15 min. When the biosensor was applied to a flow injection system, optimal operation pH and temperature were 8.0 and 30 degrees C, respectively. The strengths and limitations of the xylitol biosensor are its high affinity for NAD+, slow reaction time, narrow linear range of detection, and moderate affinity for xylitol.
为开发木糖醇生物传感器做准备，对热带假丝酵母IFO 0618的木糖醇脱氢酶进行了部分纯化和特性分析。木糖醇脱氢酶的最佳pH值和温度分别为pH 8.0和50℃。在测试的各种醇类中，木糖醇氧化速度最快，山梨醇和核糖醇的氧化速度分别为木糖醇氧化速度的65%和58%。该酶对阿拉伯糖醇、木糖、葡萄糖、甘油和乙醇完全无活性。该酶催化木糖醇氧化时，更倾向于使用NAD⁺（7.9 U/mg）作为电子受体，而非NADP⁺（0.2 U/mg）；而在逆向反应D-木酮糖还原过程中，更倾向于使用NADPH（7.7 U/mg）作为电子供体，而非NADH（0.2 U/mg）。木糖醇和NAD⁺的K(m)值分别为49.8 mM和38.2 μM。为制备木糖醇生物传感器，将上述木糖醇脱氢酶和一种心肌黄酶固定在溴化氰活化的琼脂糖上。然后将凝胶附着在溶解氧电极上。在维生素K3、NAD⁺和磷酸盐缓冲液存在的情况下，该生物传感器对高达3 mM的木糖醇浓度呈现线性响应。反应15分钟后稳定。当将该生物传感器应用于流动注射系统时，最佳操作pH值和温度分别为8.0和30℃。木糖醇生物传感器的优点和局限性在于其对NAD⁺的高亲和力、缓慢的反应时间、狭窄的检测线性范围以及对木糖醇的中等亲和力。
