Spacer Effects on Enzymatic Activity of TrypsinImmobilized onto Porous Chitosan Beads

Abstract

Trypsin was covalently immobilized onto the surface of porous chitosan beads with and without alkyl chain spacers of different lengths.The relative activity (RA) of the immobilized trypsin wasfound to the high toward a small ester substrate, p-toluene sulphonyl-L-arginine methyl ester (TAME), but rather low toward casein, a high molecular weight substrate.Trypsin immobilized withspacer gave an almost constant activity by varying the surface concentration in marked contrastwith the immobilized trypsin without spacer whose activity monotonously decreased with decreasingsurface concentration.RA of the immobilized trypsin for hydrolysis of a high molecular weight substrate was a strong function of spacer length.The thermal and strage stabilities of the immobilized trypsin were higher than those of the free trypsin. The trypsin immobilized directly onto the surface of chitosan beads without any spacer gave a higher stability than those immobilized with spacers.The spacer effect on the activity could be explained interms of flexibility of the immobilized trypsinmolecule.