Kienle I, Burgert M, Holzer H
Biochemisches Institut, Universität Freiburg, Germany.
Yeast. 1993 Jun;9(6):607-11. doi: 10.1002/yea.320090607.
An enzymatic end-point assay of trehalose using acid trehalase from yeast is described. After quantitative hydrolysis of trehalose by acid trehalase, the resulting glucose is assayed with the commercially available glucose oxidase/peroxidase dye system. Pre-existing glucose is determined in a control reaction from which acid trehalase is omitted. When intact cells are analysed for trehalose, pre-existing glucose can be washed out with ice-cold water without reducing the trehalose content of the cells. A convenient method for extraction of trehalose from intact yeast cells is heating for 20 min at 95 degrees C followed by centrifugation. The specificity of the assay is determined by the specificity of the acid trehalase preparation used. As described previously (Mittenbühler, K. and Holzer, H., 1988, J. Biol. Chem. 263, 8537-8543; Mittenbühler, K., 1988, Thesis, University of Freiburg), the following sugars and sugar derivatives do not form glucose when incubated with purified acid trehalase: sucrose, cellobiose, mellobiose, raffinose, maltose, lactose, glucose-6-phosphate, glucose-1-phosphate, galactose. The application of the new trehalose assay to yeast cells grown to different growth stages and at various temperatures is presented.
本文描述了一种使用酵母酸性海藻糖酶对海藻糖进行酶促终点测定的方法。在酸性海藻糖酶对海藻糖进行定量水解后，使用市售的葡萄糖氧化酶/过氧化物酶染料系统对生成的葡萄糖进行测定。在省略酸性海藻糖酶的对照反应中测定预先存在的葡萄糖。当分析完整细胞中的海藻糖时，预先存在的葡萄糖可用冰冷的水冲洗掉，而不会降低细胞中海藻糖的含量。从完整酵母细胞中提取海藻糖的一种简便方法是在95℃加热20分钟，然后离心。该测定法的特异性取决于所使用的酸性海藻糖酶制剂的特异性。如先前所述（Mittenbühler, K.和Holzer, H., 1988, J. Biol. Chem. 26, 8537 - 8543; Mittenbühler, K., 1988, 论文，弗莱堡大学），以下糖类和糖衍生物与纯化的酸性海藻糖酶一起孵育时不会形成葡萄糖：蔗糖、纤维二糖、蜜二糖、棉子糖、麦芽糖、乳糖、葡萄糖-6-磷酸、葡萄糖-1-磷酸、半乳糖。本文展示了新的海藻糖测定法在生长至不同生长阶段并在不同温度下培养的酵母细胞中的应用。
