Gel entrapped enzymes: Kinetic studies of immobilized β‐galactosidase

β-galactosidase from E. coli (β-D-galactose galactohydrolase, EC 3.2.1.23) has been entrapped in a crosslinked 2-hydroxyethyl methacrylate gel with a 35% retention of activity. The kinetic behavior of the gel-entrapped enzyme has been studied in a recirculation reactor system, the substrate being o-nitrophenyl-βhyphen;D- galactopyranoside. Kinetic constants were determined for particle sizes ranging from 69 to 231 μm in diameter and compared to those of the free enzyme. External diffusion effects were eliminated by operating at high recirculation flow rates. A fourfold increase in Km(app) was observed for the 231 μm particles, consistent with existing theoretical treatments for internal diffusion effects. An Arrhenius plot of rate data showed significant curvature at higher temperatures, which was attributed to the effects of internal diffusion. The pH–activity profile of the gel-entrapped enzyme was bell-shaped at high substrate concentration and, in contrast to the free enzyme, could be fitted to the titration curve of two ionizable groups, a basic group having a pK of 8.6. The gel-entrapped enzyme had a higher pH optimum and retained a larger percentage of its maximal activity at alkaline pH than the free enzyme; its pH stability at high pH was also much better. The thermal stability of the gel-entrapped enzyme was studied and found to be 14 days at 22°C and 65 min at 45°C.