Immobilization of thiol proteases onto porous poly(vinyl alcohol) beads

Water-insoluble enzymes were prepared by immobilizing thiol proteases such as papain, ficin, and bromelain, onto the porous poly(vinyl alcohol) (PVA) beads by covalent fixation. The relative activity (RA) of the immobilized enzymes was found to be rather high toward small ester substrates, N-benzyl-L-arginine ethyl ester (BAEE), but rather low toward casein, a high molecular weight substrate. RA of the immobilized enzymes by the hexamethylene diisocianate (HMNDI) method gave an almost constant activity in marked contrast with the immobilized enzymes by the cyanogen bromide (CNBr) method whose activity monotonous decreased with the decreasing amount of immobilized enzymes. The values of the Michaelis constant Km and maximum reaction velocity Vm for free and immobilized enzymes on the porous PVA beads are estimated.