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 K-m 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 V-max values were smaller for the immobilized glucoamylase preparations. The optimum operational temperature was 5 degreesC higher for both immobilized preparations than that of the free enzyme. Operational, thermal and storage stabilities were found to be increased with immobilization. (C) 2000 Elsevier Science B.V. All rights reserved.