Α-Galactosidase loaded microparticules: synthesis, characterization and In Vitro release studies

Enzymes have been used in various industrial sectors for a long time. The growing need and interest in the use of renewable materials for biorefinery in recent years has brought more attention to the industrial application of enzymes. α-Galactosidase (α-D-galactoside galactohydrolase, EC 3.2.1.22) is a group of glycoside hydrolase enzymes that catalyze the hydrolysis of terminal α-galactosyl moieties of both simple and complex oligosaccharides. α-Galactosidases are common in nature. Immobilization of enzymes is a common practice that makes it possible to reuse the enzyme several times, mainly to minimize the share of enzyme costs on the process economy. The use of an immobilized enzyme also generally facilitates downstream processing because it can be easily removed from the medium. Considering the interaction between enzymes and support carriers, enzyme immobilization methods can be classified as physical and chemical methods. Adsorption and encapsulation are included in physical methods without covalent interactions between enzymes and support carriers, while covalent binding and cross-linking belong to chemical methods. In this project, α-galactosidase enzyme isolated from corn flour and partially purified by gradient ammonium sulfate precipitation was immobilized in calcium alginate chitosan microparticles with encapsulation method. For this purpose, synthesis, characterization and in vitro release of microparticles were carried out. Optimum conditions for the preparation of immobilized enzyme were determined as; 0.1 mg protein amount, 3% (w/v) alginate, 0.15 M CaCl2, 0.5% (w/v) chitosan, 1 hour shaking, 0.05% (v/v) glutaraldehyde and 1 hour incubation. Characterization studies of free and immobilized α-galactosidase prepared under optimized conditions were performed. The optimum temperature for the free and immobilized enzyme was determined as 40°C. In the thermal stability test, the free and immobilized enzyme maintained %50 of their initial activity at 60°C and 70°C, respectively.. The optimum pH values for free and immobilized enzymes were found to be pH 4.5 and 5.5, respectively. The free enzyme maintained more than 80% of its activity in the pH range of 3.0 - 6.5, while the immobilized enzyme maintained more than 70% of its activity between pH 4.5 - 6.0. In the reuseability assay, immobilized enzyme could be used twice. In vitro release studies were performed for the immobilized enzyme.