Covalent immobilisation of invertase onto a reactive film composed of 2-hydroxyethyl methacrylate and glycidyl methacrylate: properties and application in a continuous flow system

Abstract

Invertase was covalently immobilised on the poly(hydroxyethyl methacrylate-co-glycidyl methacrylate) (poly(HEMA-GMA)) film. The invertase immobilisation capacity of the films was increased as the GMA ratio increased in the film structure. The immobilised invertase on the poly(HEMA-GMA-3) composition exhibited an activity of 32.7 U cm(-2) film. The optimum temperature of the immobilised invertase increased by 5 degreesC, and the optimal pH values for the free and the immobilised enzymes were determined as 5.0. The retained activity of the immobilised invertase was between 53 and 85%. Kinetic parameters were determined for immobilised invertase as well as for the free enzyme. The values of the Michael's constant K-m of invertase were significantly larger, ca. 2.7 times upon immobilisation, indicating decreased affinity by the enzyme for its substrate, whereas V-max was smaller for immobilised invertase. Activity of the immobilised invertase was quite stable with respect to free counterpart. After 168 h reaction, only 8% of immobilised invertase activity was lost. The operational inactivation rate constant (k(opi)) of the immobilised invertase at 35 degreesC with 200 mM sucrose was 8.23 x 10(-6) min(-1). (C) 2002 Elsevier Science B.V. All rights reserved.