Various factors affecting the stability of
thermolysin immobilized by cross-linking with
glutaraldehyde were elucidated, particularly in the water-immiscible organic
solvents such as
ethyl acetate and
tert-amyl alcohol. The main reason for
enzyme inactivation in water-immiscible organic
solvents was found to be
autolysis in the water phase, which may surround the
enzyme immobilized inside the support. By contrast, in water-miscible organic
solvents thermal denaturation was the predominant cause of
enzyme inactivation. Courses of inactivation were expressed by second-order kinetics in the initial stage, after which inactivation proceeded at a slower rate. The extent of
autolysis was found to strongly depend on the kind of organic
solvent, the water content, and type of support and these dependencies were explained by the difference in the amount and state of water inside the support.
Thermolysin was immobilized onto
Amberlite XAD-7 as a compact aggregate inside the support which may increase the stability of the
enzyme. Finally, it was shown that the stability of the
immobilized enzyme could be correlated with the logP value for water-miscible organic
solvents and with the solubility of water for water-immiscible organic
solvents.