AbstractWater‐insoluble proteases were prepared by immobilizing papain, ficin, and bromelain onto the surface of porous chitosan beads with any length of spacer by covalently fixation. The activity of the immobilized proteases was found to be still high toward small ester substrate,N‐benzyl‐L‐arginine ethyl ester (BAEE), but rather low toward casein, a high‐molecular‐weight substrate. The relative activity of the immobilized proteases with spacer gave an almost constant value for the substrate hydrolysis within the surface concentration region studied. The values of the Michaelis constantKmand the maximum reaction velocityVmfor free and immobilized proteases on the porous chitosan beads are estimated. The apparentKmvalues were larger for immobilized proteases than for the free ones, whileVmvalues were smaller for the immobilized proteases. The pH, thermal, and storage stability of the immobilized proteases were higher than those of the free ones. The initial enzymatic activity of the immobilized protease maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent
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