Kinetic Assessment of Trypsin Immobilized onto Macroporous Polyacrylamide Gels (MPAAGs)

Abstract

This paper exposes some kinetic consequences of covalent immobilization of enzyme trypsin, onto the supermacroporous polyacrylamide monolithic cryogels with epoxy functionality (Epoxy-MPAAGs), of initial total monomer concentrations 5, 10 and 15% (w/v). The specific activities of the immobilized trypsin were determined and found to be 12.6, 280 and 116 U/gprotein for 5, 10 and 15% (w/v) cryogels respectively at 30 °C. These results suggest improvement in specific activity with the increase in the initial total monomer concentrations. The kinetic parameters KM and vmax were determined at 30 °C for both the immobilized trypsin and the free trypsin by using the enzyme catalysed hydrolysis of N-α-benzoyl-D, Larginine-p-nitroanilide (BAPNA). The immobilized trypsin onto the 5% (w/v) cryogel attained KM of 0.83 mM and vmax of 63 U/g-protein. The KM values for the immobilized trypsin conjugates did not differ much from that of the free trypsin (0.56 mM) under similar conditions. Activity results from the immobilized trypsin onto the 5% (w/v) cryogel, obtained under the incubation (12.8 U/g-protein) and flow-through conditions (12.6 U/g-protein) were significantly the same. Due to their mechanical stability and good flow properties, the monolithic Epoxy-MPAAGs cryogel-trypsin conjugates have prospects for applications both in stationary and on-line flow-through bio-catalytic systems/bioreactors.