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 glycoenzymes in calcium alginate hydrogel is proposed in this paper. As a proof of principle, three glycoenzymes acting in series (trehalase, glucose oxidase, and horseradish peroxidase) have been coimmobilized in calcium alginate beads. The release of the enzymes from the hydrogel mesh (leakage) is avoided by exploiting the enzyme's 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 been quantified spectrophotometrically. 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 bioassays exploiting the enzyme-containing beads have been developed for the optical determination of glucose and trehalose, and limit of detection values of 0.2 and of 40 μM, respectively, have been obtained.
CNR-IPCF, Istituto per i Processi Chimico-Fisici, Consiglio Nazionale delle Ricerche , Via Orabona 4, 70126 Bari, Italy.
Antonia
A
Mallardi
Dipartimento di Chimica and CSGI, Università degli studi di Bari Aldo Moro , Via Orabona 4, 70126 Bari, Italy.
Valeria
V
Angarano
Dipartimento di Chimica and CSGI, Università degli studi di Bari Aldo Moro , Via Orabona 4, 70126 Bari, Italy.
Maria
M
Magliulo
Dipartimento di Chimica and CSGI, Università degli studi di Bari Aldo Moro , Via Orabona 4, 70126 Bari, Italy.
Luisa
L
Torsi
Dipartimento di Chimica and CSGI, Università degli studi di Bari Aldo Moro , Via Orabona 4, 70126 Bari, Italy.
Gerardo
G
Palazzo
D000464:Alginates / Q000737:chemistry / Q000378:metabolism*; D001681:Biological Assay; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D005949:Glucose Oxidase / Q000737:chemistry / Q000378:metabolism*; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D006603:Hexuronic Acids / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D010316:Particle Size; D013499:Surface Properties; D014198:Trehalase / Q000737:chemistry / Q000378:metabolism*
D000464:Alginates; D004800:Enzymes, Immobilized; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D014198:Trehalase