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Abstract
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, at neutral pH and moderate temperature, in the presence of medium-high concentrations of water-miscible organic cosolvents, the unique cause of inactivation is the interaction of the enzyme with cosolvent molecules and the only 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 every enzyme molecule;
b. reduction of the effects of inactivation: “rigidification of enzymes” via 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 effects. In this case, hydrophilization of the enzyme nano-environment was found 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 completely surrounded by hyper-hydrophilic microenvironments. In this way, native PGA that was unstable against organic cosolvents (completely inactivated in less than 3 min in 95% dioxane) was transformed into a very stable immobilized derivative (preserving more than 80% of activity after 40 days under the same conditions).