Formation of Amorphous Matrices of Sugars by Drying and their Function of Molecular Inclusion

本课题研究了无定形糖类基质对冻干酶的热稳定作用。主要内容如下：1.糖的热稳定作用。以几种糖(葡萄糖、蔗糖、海藻糖等)为研究对象，考察了糖的热稳定作用。和三种酶(ADH、LDH和MDH)。对于每一种酶和每一种糖，都准备了一种溶液，并将该溶液冷冻在液氮中。然后将冷冻样品在-15ﾟC下冷冻干燥，将所得样品在65ﾟC的干燥空气中保存，并研究其酶活性的变化。2.糖的结晶度与稳定化效果的关系通过对冷冻干燥样品的X射线衍射仪的测定，发现优于稳定酶的糖形成了完全无定形的结构。相反，发现在干燥状态下结晶的糖在稳定酶方面表现较差。对于蔗糖样品，低结晶度样品的稳定效果要好于高结晶度样品。这些结果表明，糖的稳定作用与糖形成的无定形结构密切相关。3.糖的热稳定作用的微观机理认为，糖的热稳定作用是由于糖分子与酶之间的氢键维持了酶的三级结构。为了确认糖分子是否与酶发生氢键作用，我们进行了红外光谱的测量。当样品的含水率远小于水中LDH的偶联水量时，由于氢键的作用，样品的峰发生了移动。这一结果表明糖分子与酶之间确实存在氢键。

This project has dealt with thermal stabilization effect of amorphous matrix of sugars on freeze-dried enzymes. Summary is as follows :1.Thermal stabilization effect of sugars.Thermal stabilization effect was examined by using several kinds of sugars (glucose, sucrose, trehalose, etc.) and three kinds of enzymes (ADH,LDH,and MDH) . For each enzyme and each sugar, a solution was prepared, and the solution was frozen in liquid nitrogen. Then the frozen sample was freeze-dried at-15ﾟC.The samples thus obtained was stored at 65ﾟC in dry air, and the change of the enzyme activity was investigated. The Activity of trehalose sample is preserved most for each enzyme used in the present study.2.Relation between crystallinity of sugars and stabilization effectBy the measurement of X-ray diffractmetry for freeze-dried samples, sugars which are superior to stabilize enzymes are found to form fully amorphous structure. In contrast, sugars that were found to be crystallized in the dried state, were inferior in stabilizing enzymes. Moreover, for suerose samples the stabilization effect of low crystallinity samples is better than that of high crystallinity samples. These results indicate that the stabilizing effect of sugars closely relates to the amorphous structure formed by sugars.3.Microscopic mechanism of the thermal stabilization effect of sugarsIt is considered that the thermal stabilizing effect of sugars is caused because hydrogen-bonding between sugar molecules and enzymes maintain the tertial structure of enzymes. In order to confirm whether suger molecules are hydrogen-bonding to enzymes, we have done the measurement of FT-IR spectroscopy. Peak shift due to hydrogen-bonding was observed for the samples whose water contents are quite less than the amount of coupled water for LDH in water. This result indicates that hydrogen-bond between sugar molecules and enzymes surely exists.