Congo red attached monisized poly(HEMA-co-MMA) microspheres for use in reversible enzyme immobilisation

摘要

Monosize and non-porous poly(2-hydroxyethylmethacrylate-co-methylmethacrylate) (poly(HEMA-co-MAA)), microspheres were prepared by disperison polymerisation of HEMA and MMA in an ethanol-water medium in the presence of an initiator (#alpha#,#alpha#-azobisisobutyronitrile,AIBN). An affinity dye, i.e., Congo Red (CR) was attached covalently and then Fe~(3+) ions were incorporated. The poly(HEMA-co-MMA)-CR attached and poly(HEMA-co-MAA)-CR-Fe~(3+) incorporated microsphere were used in the immobilisation of glucose oxidase (GOD) via adsorption. The adsorption capacities of these microspheres were determined by varying the concentration of GOD in the adsorption medium. GOD adsorption capacities of the Fe~(3+) incorporated microspheres (165 mg g~(-1)) was greater than that of the dye-attached microspheres (126 mg g~(-1)). The non-specific adsorption of the GOD on the poly(HEMA-co-MAA) microspheres was negligible. The K_m values for both immobilised poly(HEMA-co-MMA)-CR-GOD (7.2) and poly(HEMA-co-MMA)-CR-Fe~(3+)-GOD (6.8) were higher than that of the free enzyme (6.6 mM). Optimum reaction pH was 5.0 for free and 7.0 for both immobilised preparations. Optimum reaction temperature of the adsorbed enzymes was 10 deg C higher than that of the enzyme and was significantly broader. After 10 successive uses the retained activity of the adsorbed enzyme was 93%. It was observed that enzyme coule be repeatedly adsorbed and desorbed on the CR attached poly(HEMA-co-MMA) microspheres without significant loss in adsorption capacity or enzyme activity.