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

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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.