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

Abstract

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. Therelative 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 (HMDI)method gave an almost constant activity in marked contrast with the immobilized enzymes by thecyanogen bromide (CNBr) method whose activity monotonous decreased with the decreasing amountof immobilized enzymes. The values of the Michaelis constant Km and maximum reactionvelocity Vm for free and immobilized enzymes on the porous PVA beads are estimated.Apparent Km values were larger for immobilized enzymes than for the free ones, while Vm values were smaller for the immobilized enzymes. The pH, thermal, and storage stabilities of the immobilized enzymes were higher than those of the free ones. The initial enzymatic activity of the immobilized enzymes remained almost unchanged without any elimination and inactivation of the enzymes, when the batch enzymatic reaction was performed repeatedly, indicating excellent durability. © 1993, The Society of Polymer Science, Japan. All rights reserved.