Immobilization of glucoamylase onto spacer-arm attached magnetic poly(methylmethacrylate) microspheres: characterization and application to a continuous flow reactor

Magnetic poly(methylmethacrylate) microspheres (MPMMA) were prepared by the solvent evaporation method and a 6-carbon spacer-arm (i.e. hexamethylene diamine, HMDA) was covalently attached by the reaction of carbonyl groups of poly(methylmethacrylate). Glucoamylase was then covalently immobilized through the spacer-arm of the MPMMA microspheres by using either carbodiimide (CDI) or cyanogen bromide (CNBr) as a coupling agent. The activity yield of the immobilized glucoamylase was 57% for CDI coupling and 73% for CNBr coupling. Kinetic parameters were determined for both immobilized glucoamylase preparations as well as for the free enzyme. The Km values for immobilized glucoamylases CDI coupling (12.5 g l−1 dextrin) and CNBr coupling (9.3 g l−1 dextrin) were higher than that of the free enzyme (2.1 g l−1 dextrin) whereas Vmax values were smaller for the immobilized glucoamylase preparations. The optimum operational temperature was 5°C higher for both immobilized preparations than that of the free enzyme. Operational, thermal and storage stabilities were found to be increased with immobilization.