Stability of immobilized thermolysin in organic solvents

Various factors affecting the stability of thermolysin immobilized by cross -linking with glutaraldehyde were elucidated, particularly in the water-imm iscible organic solvents such as ethyl acetate and tert-amyl alcohol. The m ain reason for enzyme inactivation in water-immiscible organic solvents was found to be autolysis in the water phase, which may surround the enzyme im mobilized inside the support. By contrast, in water-miscible organic solven ts thermal denaturation was the predominant cause of enzyme inactivation. C ourses of inactivation were expressed by second-order kinetics in the initi al stage, after which inactivation proceeded at a slower rate. The extent o f autolysis was found to strongly depend on the kind of organic solvent, th e water content, and type of support and these dependencies were explained by the difference in the amount and state of water inside the support. Ther molysin 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 wit h the logP value for water-miscible organic solvents and with the solubilit y of water for water- immiscible organic solvents.