Activity of Enzyme Trypsin Immobilized onto Macroporous Poly(Epoxy-Acrylamide) Cryogel

Abstract

Bovine pancreatic trypsin has been covalently immobilized onto monolithic polyacrylamide cryogels with epoxy functionality (Epoxy-MPAAGs). The covalent immobilization of trypsin onto Epoxy-MPAAGs was achieved through incorporation of ethylenediamine-glutaraldehyde spacers on the cryogels’ surfaces. The immobilization yield and the influence of pH and temperature conditions on the activity of the immobilized trypsin were assessed by using the hydrolysis of a low molecular weight substrate, N-α-benzoyl-D, L-arginine-p-nitroanilide (BAPNA). Optimal pH and temperature values of 9 and 50 °C, respectively, were established for both the immobilized trypsin and the free trypsin. Activity measurements from the native trypsin before immobilization and the residual free trypsin after immobilization by hydrolysis of BAPNA demonstrated activity recovery (activity based immobilization yield) of about 60 – 70% for the Epoxy-MPAAGs cryogel immobilized trypsin. The Epoxy-MPAAGs cryogels immobilized trypsin was stable up to 65 days in aqueous storage conditions, at 4 °C. Apparently the Epoxy-MPAAGs cryogels immobilized trypsin showed improved catalytic activity above 50 °C.

Key words: Enzyme trypsin; immobilized trypsin; polyacrylamide cryogels