Co-immobilized glucose oxidase and β-galactosidase on bovine serum albumin coated allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymer as a biosensor for lactose determination in milk

• Epoxy activated copolymer coated with BSA.
• SH and COOH groups used for glucose oxidase and β-galactosidase immobilization.
• The coupled enzyme system is used for lactose assay in milk and milk products.

Bovine serum albumin (BSA) was adsorbed on allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymer with 25% crosslink density (AGE-25) at pH 8.0 (16% w/w). The amino, thiol and carboxylic acid functional groups available on protein coated surface were utilized for covalent immobilization of glucose oxidase and β-galactosidase, both independently, and in a step-wise manner on the same matrix, with no more than 10% loss of enzyme activity during immobilization. Glutaraldehyde cross-linking after immobilization provided stable enzyme preparations. The pH-optima of the immobilized enzymes were similar to those for free enzyme but their thermal stability was vastly improved. The co-immobilized enzyme support was used as a biosensor for determination of lactose in milk with excellent reproducibility and reusability.

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