In situ entrapment of alpha-chymotrypsin in the network of acrylamide and 2-hydroxyethyl methacrylate copolymers

A mild and reproducible method has been developed for the entrapment of alp ha-chymotrypsin into a crosslinked copolymer. A porous copolymer was synthe sized at 293 K by solution copolymerization of acrylamide and 2-hydroxyethy l methacrylate, alpha-Chymotrypsin was entrapped during copolymerization at different polymerization stages. The effect of crosslinking on enzyme load ing and retention of its activity was examined. Copolymer with 2% crosslink ing could entrap >90% of the enzyme. The activity of free and immobilized a lpha-chymotrypsin was determined by using N-benzoyl-L-tyrosine ethyl ester and casein as low and high molecular weight substrates respectively. Storag e as well as thermal stability of the immobilized enzyme was superior to th at of the free one. Effect of calcium and heavy metal ions was studied on i mmobilized enzyme activity. The immobilized enzyme showed little variation in activity with pH and retained 50% activity after nine cycles. The Michae lis constant K-m of the free and immobilized enzyme was estimated to be 2.7 and 4.2 x 10(-3) mM, respectively, indicating no conformational changes du ring entrapment. (C) 2000 John Wiley & Sons, Inc.