Stabilization of immobilized enzymes against water-soluble organic cosolvents and generation of hyper-hydrophilic micro-environments surrounding enzyme molecules

Enzymes usually undergo rapid inactivation in the presence of organic media . In some cases, the mechanism is quite simple. For example, when an enzyme , fully dispersed and immobilized inside porous supports, is inactivated, a t neutral pH and moderate temperature, in the presence of medium-high conce ntrations of water-miscible organic cosolvents, the unique cause of inactiv ation is the interaction of the enzyme with cosolvent molecules and the onl y inactivating effect is the promotion of conformational changes on enzyme structure. On this basis, two distinct strategies for stabilization of enzymes against organic solvents are proposed: a. reduction of the causes of inactivation: generation of hyper-hydrophilic micro-environments having a very open structure and fully surrounding ever y enzyme molecule; b. reduction of the effects of inactivation: "rigidification of enzymes" vi a multipoint covalent immobilization. By using penicillin G acylase (PGA) as a model enzyme, both strategies have been evaluated and compared. Both stabilizing strategies had significant e ffects. In this case, hydrophilization of the enzyme nano-environment was f ound to be more effective than rigidification of the enzyme via multipoint covalent attachment. The combined effect of both stabilizing strategies was also tested: multipoint covalently immobilized enzyme molecules were compl etely surrounded by hyper-hydrophilic microenvironments. In this way, nativ e PGA that was unstable against organic cosolvents (completely inactivated in less than 3 min in 95% dioxane) was transformed into a very stable immob ilized derivative (preserving more than 80% of activity after 40 days under the same conditions).