Reversible immobilization of catalase by metal chelate affinity interaction on a new generation matrix

p(AAm-AGE) cryogel was prepared by radical polymerization of acylamide and allyl glycidyl ether. Cibacron Blue F3GA is covalently attached on p(AAm-AGE) cryogel, via the reaction between the chloride groups of the reactive dyes and the epoxide groups of the AGE. Cibacron Blue F3GA attached p(AAm-AGE) cryogel was chelated with Fe(III) ions. p(AAm-AGE)-CB-Fe(III) cryogel was characterized by FTIR, SEM and swelling degree analysis. The IMAC cryogel carrying 25.8µmol Fe(III) ions was used in adsorption studies under different conditions (i.e., pH, protein initial concentration, flow rate, temperature and ionic strength). Maximum adsorption capacities were found to be 75.7 mg/g for p(AAm-AGE)-CB-Fe(III) cryogel and 60.6 mg/g for p(AAm-AGE)-CB cryogel, respectively, and the adsorbed amounts per unit mass of cryogel reached to a plateau value at about 1.5mg/mL at pH 6.0. Michealis-Menten constant and Vmax values were significantly affected by adsorption of catalase onto the p(AAm-AGE)-CB-Fe(III) cyyogel. The Km values were found to be 0.73 g/L for the free catalase and 0.18 g/L for the immobilized catalase. The Vmax value of free catalase (2.0x103 U/mg enzyme) was found to be lower than that of the immobilized catalase (2.5x103 U/mg enzyme). Activity of immobilized catalase was determined higher in a wider temperature range than the free enzyme. It was also observed that enzyme could be repeatedly adsorbed and desorbed on the p(AAm-AGE)-CB-Fe(III) cryogel.