Kondakova L. Galactose oxidase immobilized on silica: preparation, properties, application.

The present work was intended to analyze the experience acquired in developing of preparation methods of samples of galactose oxidase from Fusarium graminearum, immobilized on the parent and modified silica matrices. Adsorption of galactose oxidase on silicas is proceeding moderately fast and we can not remove enzyme completely with a buffer solutions with pH 4.5 and 8.2. It was observed that at the definite ratios of the enzyme and carrier an activity of the immobilized preparations can lay over an activity of the solutions of the native biocatalyst. Covalent immobilization of galactose oxidase was carried out on the aminopropylsilicas activated with tolylene 2,4 diisocyanate and cyanuric chloride. In the both cases the sufficiently stable preparations are formed and their activity was held constant for some months. Covalent attachment of the biocatalyst with the carrier surface takes place at the adsorption galactose oxidase on amine-containing silica matrices from solutions including enzyme and galactose. Preparation activity is conserved during two months in the conditions of flow-reaction. In the absence of galactose the enzyme desorption takes place at a washing with 0.05 M phosphate buffer (pH=7.0). It is anticipated that when a solution contains galactose oxidase and its substrate, a peculiar process of autoimmobilization of the enzyme on the aminoorganosilicas surface takes place. In this solution, under the action of a biocatalyst galactose is transformed into 2,4-galactohexodialdose that serves as a crosslinking agent and provides bonding of the enzyme with the amino-containing silica matrix surface. When using galactose oxidase from Fusarium graminearum this method enables one to produce immobilized preparations with an activity of up to 90 units per 1 g of a carrier. Such preparations were successfully applied for analytical determination of galactose-containing carbohydrates (galactose, lactose, raffinose) in complex mixtures (the linear region for analysis: 0.1-2.0 mmol/L for raffinose, 2-20mmol/L for galactose, 5 100 mmol/L for lactose). Keywords: galactose oxidase, silica, aminoorganosilica, immobilization, heterogeneous biocatalyst, galactose-containing carbohydrates, Clark oxygen electrode, flow-type reactor.