Comparison of the properties of trypsin immobilized on 2 Celite derivatives

Abstract Trypsin was immobilized on 2 Celite™ derivatives and the kinetic properties of trypsin immobilized on these derivatives were determined and compared. Celite™ was derivatized with organosilane to give aminopropyl-Celite (APC) and a portion of this derivative was then succinylated to give succinamidopropyl-Celite (SAPC). Trypsin was covalently immobilized on APC using glutaraldehyde to activate amino groups and on SAPC using water-soluble carbodiimide to activate surface carboxyl groups. Enzyme loadings were 13.9 and 17.8 mg ml −1 of beads on APC and SAPC, respectively. Using p -tosyl- l -arginine methyl ester as substrate, the catalyst specific activity, K M app and k cat / K M app were 17.8 U ml −1 of beads, 3.60 and 21.0 mM −1 min −1 , respectively, for trypsin-APC as compared with 24.5 U ml −1 of beads, 3.77 and 20.3 mM −1 min −1 , respectively, for trypsin-SAPC. With β -lactoglobulin as substrate, K M app and k cat / K M app were 0.36 and 1.62 mM −1 min −1 for trypsin-APC and 0.54 and 1.39 mM −1 min −1 for trypsin-SAPC, respectively. The pH range for optimal activity was much larger for both immobilized forms as compared with the soluble enzyme. The optimal temperature ranges were 40–50°C for trypsin-APC and 50–60°C for trypsin-SAPC. The two methods of immobilization on Celite™ gave biocataysts with similar kinetic properties but immobilization on SAPC yielded slightly higher loadings and higher specific activities.