Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes

Abstract

The present study describes the preparation of poly(HEMA-co-GMA) reactive membranes that were grafted with polyethylenimine (PEI) following UV photo-polymerization. The immobilization of tyrosinase was carried out via multi-point ionic interactions based on -NH2 groups of PEI and Cu(II) ions. Tyrosinase is a copper-dependent enzyme, which should show a binding affinity for the chelated Cu(II) ions on the membrane surfaces. The tyrosinase immobilization was positively correlated with the input enzyme amount in the immobilization medium. The maximum tyrosinase immobilization capacities of the poly(HEMA-co-GMA)-PEI and poly(HEMA-co-GMA)-PEI-Cu(II) membranes were 19.3 and 24.6 mg/m(2), respectively. The enzyme activity when assessed at various pH and temperatures gave broader range for immobilized preparations when compared to free enzyme. The poly (HEMA-co-GMA)-PEI-Cu(II) tyrosinase membranes retained 82% of their initial activity at the end of 120 h of continuous reaction. Moreover, upon storage for 3 months the activity of the immobilized membranes retained 46% of their initial levels. After deactivation of the enzyme, the poly(HEMA-co-GMA)-PEI membrane was easily regenerated, re-chelated with the Cu(II) ions and reloaded with the enzyme for repeated use. The mild immobilization conditions, easy and rapid membrane preparation, one-step enzyme adsorption at substantially higher levels and membrane reusability are the beneficial properties of such systems and offers promising potential in several biochemical processes. (C) 2003 Elsevier B.V. All rights reserved.