Thermal stabilization of freeze-dried enzymes by sugars

The thermal stabilization effect of sugars on freeze-dried proteins is studied, particularly the relation between stabilizing effect and the degree of sugar crystallinity. Three kinds of enzymes, alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH), were used as model proteins. Aqueous solutions of enzymes with sugars were freeze-dried and stored in dry air at 65°C. The stabilities of freeze-dried enzymes improve remarkably by addition of trehalose or raffinose. By measurement of X-ray diffractometry, the sugars are found to form fully amorphous matrix in freeze-dried samples. Furthermore, sucrose stabilizes enzymes to a great extent when it is amorphous in samples, though it shows little stabilizing effect when it is crystalline. These results indicate that the stabilizing effect of sugars closely relates to the amorphous matrix formed by the sugars.