General Approach to the Immobilization of Glycoenzyme Chains Inside Calcium Alginate Beads for Bioassay

A general method to obtain the efficient entrapment of mixtures of glyco-enzymes in calcium alginate hydrogel is proposed in this paper. As a proof of principle, three glyco-enzymes acting in series (trehalase, glucose oxidase and horseradish peroxidase) have been co-immobilized in calcium alginate beads. The release of the enzymes from the hydrogel mesh (leakage) is avoided by exploiting the enzymes aggregation induced by the concanavalin A. The aggregation process has been monitored by dynamic light scattering technique, while both enzyme encapsulation efficiency and leakage have spectrophotometrically been quantified. Obtained data show an encapsulation efficiency above 95% and a negligible leakage from the beads when enzyme aggregates are larger than 300 nm. Operational stability of “as prepared” beads has been largely improved by a coating of alternated shells of polycation poly(diallyldimethylammonium chloride) and of alginate. As a test for the effectiveness of the overall procedure, analytical bio-assays exploiting the enzyme containing beads have been developed for the optical determination of glucose and trehalose and Limit of Detection values of 0.2 uM and of 40 uM respectively have been obtained.