Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a nonporous solid support

In order to eliminate the kinetic limitation of chymotryptic hydrolysis of proteins due to diffusion, nonporous hydroxyalkyl methacrylate solid suppor t was developed and used for oriented immobilization of chymotrypsin by mea ns of suitable polyclonal antibodies. Nonporous microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in an alcohol-toluene mixture stabilized with cellulose acet ate butyrate. The resulting particles were 1.2 mu m in diameter and possess ed narrow size distribution. After modification with adipic acid dihydrazid e they contained 2 mu mol of reactive groups available for coupling of anti -chymotrypsin antibodies. Prepared immunosorbent adsorbed 166.7 mu g of chy motrypsin per 1 g of dry carrier. Immobilized chymotrypsin retained practic ally 100% of its native proteolytic activity. Kinetic parameters of catalys is by chymotrypsin immobilized via this way were improved due to the good s teric accessibility of the enzyme active site for high-molecular-mass subst rates, when digestion of proteins in batch experiments was used. (C) 1999 E lsevier Science B.V. All lights reserved.