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

本课题研究了无定形糖基质对冻干酶的热稳定作用。主要内容如下：1.糖类的热稳定作用通过对几种糖类（葡萄糖、蔗糖、海藻糖等）的热稳定作用进行了研究。三种酶（ADH、LDH和MDH）。对于每种酶和每种糖，制备溶液，并将溶液在液氮中冷冻。然后将冷冻的样品在-15 ° C下冷冻干燥。由此获得的样品在干燥空气中在65 ° C下储存，并研究酶活性的变化。2.糖的结晶度与稳定效果的关系通过对冷冻干燥样品的X射线衍射测定，发现稳定酶的上级糖形成完全无定形结构。相反，发现在干燥状态下结晶的糖在稳定酶方面较差。对于蔗糖样品，低结晶度样品的稳定效果好于高结晶度样品。这些结果表明，糖的热稳定作用与糖所形成的无定形结构密切相关。3.糖热稳定作用的微观机理糖的热稳定作用是由于糖分子与酶之间的氢键作用维持了酶的三维结构而产生的。为了确定糖分子是否与酶发生了氢键作用，我们进行了FT-IR光谱的测量。对于水含量远小于LDH在水中的偶联水量的样品，观察到由于氢键的峰位移。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.