Preparation of artificial hyper-hydrophilic micro-environments (polymeric salts) surrounding enzyme molecules: New enzyme derivatives to be used in any reaction medium

Although enzymes usually undergo rapid inactivations in the presence of organic media, the mechanism of these inactivations is often quite simple. An immobilized enzyme, fully dispersed inside porous supports, incubated in the presence of medium-high concentrations of water-miscible organic cosolvents under mild conditions, is mainly inactivated by the interaction of the enzyme with cosolvent molecules. Thus, the only inactivating effect is the promotion of conformational changes on enzyme structure. In this paper, we propose an optimized strategy to stabilize immobilized enzymes against the presence of organic solvent: the generation of a hyper-hydrophilic shell surrounding each individual protein molecule by using several layers of different polymers. We have optimized different variables, such as the size of the polymers, the number of polymer layers, the correct assembly of the hydrophilization protocol, etc. After building a shell formed by different layers of polyethylenimine and dextran aldehyde, the addition of dextran sulfate promoted a qualitative increase in the enzyme stability. As an example, penicillin G acylase (PGA) has been immobilized-stabilized on Sepabeads (a rigid support that does not swell when changed from aqueous to anhydrous media), and the protocol to hydrophilize the protein nano-environment has been applied. This protocol originates derivatives able to stand even 90% of dioxane without significant losses of activity after several days, while conventional derivatives were readily inactivated under these conditions.