Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto mod ified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene met hacrylate), (poly(EGDMA/HEMA)). microbeads prepared by suspension copolymer ization for the potential use in hemoperfusion columns, not previously repo rted. The conditions of immobilization; enzyme concentration, medium pH, su bstrate and ethylene diamine tetra acetic acid (EDTA) presence in the immob ilization medium in different concentrations, enzyme loading ratio, process ing time and immobilization temperature were investigated for highest appar ent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant k(d) = 3.72 x 10(-3) d ay(-1). A canned non-linear regression program was used to estimate the int rinsic kinetic parameters of immobilized enzyme with a low value of observa ble Thiele modulus (phi < 0.3) and these parameters were compared with thos e of free urease. The best-fit kinetic parameters of a Michaelis-Menten mod el were estimated as V-m = 3.318 x 10(-4) mu mol/s mg bound enzyme proteins K-m = 15.94 mM for immobilized, and V-m = 1.074 mu mol NH3/s mg enzyme pro tein, K-m = 14.49 mM for free urease. The drastic decrease in V-m value was attributed to steric effects, conformational changes in enzyme structure o r denaturation of the enzyme during immobilization. Nevertheless, the chang e in K-m value was insignificant for the unchanged affinity of the substrat e with immobilization. For higher immobilized urease activity, smaller part icle size and concentrated urease with higher specific activity could be us ed in the immobilization process. (C) 2001 Elsevier Science Ltd. All rights reserved.