COMPARISON OF THE PROPERTIES OF TRYPSIN IMMOBILIZED ON 2 CELITE(TM) DERIVATIVES

Trypsin was immobilized on 2 Celite(TM) derivatives and the kinetic pr operties of trypsin immobilized on these derivatives were determined a nd compared. Celite(TM) was derivatized with organosilane to give amin opropyl-Celite (APC) and a portion of this derivative was then succiny lated to give succinamidopropyl-Celite (SAPC). Trypsin was covalently immobilized on APC using glutaraldehyde to activate amino groups and o n SAPC using water-soluble carbodiimide to activate surface carboxyl g roups. Enzyme loadings were 13.9 and 17.8 mg ml(-1) of beads on APC an d SAPC, respectively. Using p-tosyl-L-arginine methyl ester as substra te, 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, fo r trypsin-APC as compared with 24.5 U mi(-1) of beads, 3.77 and 20.3 m M(-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-S APC, respectively. The pH range for optimal activity was much larger f or both immobilized forms as compared with the soluble enzyme. The opt imal temperature ranges were 40-50 degrees C for trypsin-APC and 50-60 degrees C for trypsin-SAPC. The two methods of immobilization on Celi te(TM) gave biocataysts with similar kinetic properties but immobiliza tion on SAPC yielded slightly higher loadings and higher specific acti vities. (C) 1997 Elsevier Science B.V.