Comparison of the properties of trypsin immobilized on 2 Celite(TM) derivatives

Trypsin was immobilized on 2 Celite(TM) derivatives and the kinetic properties of trypsin immobilized on these derivatives were determined and compared. Celite(TM) 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 mi(-1) of beads, 3.77 and 20.3 mM(-1) min(-1), respectively, for trypsin-SAPC. With beta-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 degrees C for trypsin-APC and 50-60 degrees C for trypsin-SAPC. The two methods of immobilization on Celite(TM) gave biocataysts with similar kinetic properties but immobilization on SAPC yielded slightly higher loadings and higher specific activities. (C) 1997 Elsevier Science B.V.