Vinyl triazole carrying metal-chelated beads for the reversible immobilization of glucoamylase

Abstract

Poly(ethylene glycol dimethacrylate-1-vinyl-1,2,4-triazole) [poly(EGDMA-VTAZ)] beads with an average diameter of 100-200 μm were obtained by the copolymerization of ethylene glycol dimethacrylate (EGDMA) with 1-vinyl-1,2,4-triazole (VTAZ). The copolymer hydrogel bead composition was determined by elemental analysis and was found to contain 5 EGDMA monomer units for each VTAZ monomer unit. The poly(EGDMA-VTAZ) beads were characterized by swelling studies and scanning electron microscopy (SEM). The specific surface area of the poly(EGDMA-VTAZ) beads was found 65.8 m2/g. Cu 2+ ions were chelated on the poly(EGDMA-VTAZ) beads. The Cu 2+ loading was 82.6 μmol/g of support. Cu2+-chelated poly(EGDMA-VTAZ) beads with a swelling ratio of 84% were used in the immobilization of Aspergillus niger glucoamylase in a batch system. The maximum glucoamylase adsorption capacity of the poly(EGDMA-VTAZ)-Cu2+ beads was 104 mg/g at pH 6.5. The adsorption isotherm of the poly(EGDMA-VTAZ)-Cu 2+ beads fitted well with the Langmuir model. Adsorption kinetics data were tested with pseudo-first- and second-order models. The kinetic studies showed that the adsorption followed a pseudo-second-order reaction model. The Michaelis constant value for the immobilized glucoamylase (1.15 mg/mL) was higher than that for free glucoamylase (1.00 mg/mL). The maximum initial rate of the reaction values were 42.9 U/mg for the free enzyme and 33.3 U/mg for the immobilized enzyme. The optimum temperature for the immobilized preparation of poly(EGDMA-VTAZ)-Cu2+-glucoamylase was 65°C; this was 5°C higher than that of the free enzyme at 60°C. The glucoamylase adsorption capacity and adsorbed enzyme activity slightly decreased after 10 batch successive reactions; this demonstrated the usefulness of the enzyme-loaded beads in biocatalytic applications. The storage stability was found to increase with immobilization.