CS257830B1 - A method for preparing immobilized exopolygalacturonase - Google Patents

Abstract

The aim of the solution is a method for preparing immobilized exopolygalacturonase. The essence of the method is that the enzyme is anchored at pH 4.2 to 6.4 and a temperature between 5 and 30 °C on polyethylene terephthalate. Immobilized exopolygalacturonase is used in the complete degradation of pectin substances, e.g. in the canning and winemaking industries and in the preparation of D-galacturonic acid.

Description

257830 2

The present invention relates to a process for preparing an immobilized exopolygalactononase pectolytic enzyme which specifically catalyzes the sequential cleavage of terminal alpha-1,4-glycosidic bonds of pectic acid and oligogalacturonic acids by monomeric units.

Exopolygalacturonase was immobilized only by covalent binding to an insoluble polyacrylamide-based carrier after its activation with water-soluble carbodiimides (Collection Czech, *

Chem. Commun. 50, (1985) in print). A disadvantage of the above-mentioned process based on cervical immobilization is the operational complexity associated with the activation of the carrier, as well as the disadvantageous hydrodynamic properties of the biogel, which are manifested in the column application of the immobilized enzyme. Another drawback is the severe availability and relatively high cost of both the carrier and the activating agent.

These drawbacks are solved by the process of the present invention, wherein the enzyme is anchored at a pH of 4.2 to 6.4 and a temperature of 5 to 30 ° C to polyethylene terephthalate, which is prepared from ethylene glycol and terephthalic acid by a process which guarantees the high hydrophobic adsorption of the enzyme to the carrier. {Chem. Prum. 26, 598 (1976). The basic method of preparing immobilized exopolygalacturonase consists in the operational simplicity of the procedure and the permanent anchoring, which allows repeated immobilized enzymes to be used in long-term continuous processes, while maintaining optimal enzyme reaction conditions. Another advantage of the preparation prepared according to the method is that the method of its action does not change the polymer substrates as compared to the effect of the free enzyme. The starting reactants for the preparation of immobilized exopolygalacturonase are readily available because exopolygalacturonase is present in commercial pectinase preparations manufactured by the food industry. The carrier used is polyethylene terephthalate. production and barley accessible.

Similarly, the immobilization itself is very simple, easily convertible and technically inexpensive. It consists in reacting the soluble enzyme with the carrier in a buffer solution of pH 4.2-6.4, typically 5-30 ° C, lower temperature adversely affecting the rate of reaction, higher, on the other hand, may cause enzyme inactivation. The reaction time with the predominantly desired conversion rate, temperature and amount of enzyme. Usually it ranges from 4 to 16 hours. Upon completion of the reaction, the immobilized enzyme is separated from the supernatant by decantation, frit filtration, or centrifugation, washed with buffer and stored in suspension at 4 ° C.

The method of the invention is explained in the following examples. Example 1

Exopolygalacturonase present in available technical preparations of pectinases, respectively of them and higher plants, isolated by any method, e.g., goal and ion exchange chromatography Biochim. Biophys. Acta 422, 349 (1976); Collection Czech. Chem. Commun 42, 2114 (1977) Biologia 36, 197 (1981), was immobilized on polyethylene terephthalate as follows: To 1 g of polyethylene terephthalate washed with 0.1 mol / L acetate buffer pH 4.8 suspended in 10 ml of the same buffer was 10 mg of the preparation ofexopolygalacturonase is added. After 4 hours of stirring at room temperature, the gel is separated appropriately. Example .2

The polyethylene terephthalate suspended in water or in buffers at pH 4.2-6.4 is charged to the column through which the solution containing the exopolygalacturonase in the pH buffer corresponding to the equilibration solution is filtered until the affluent shows enzyme activity. The column is then washed with pH 4.8 buffer solution.

Claims (1)

OBJECT OF THE INVENTION Process for the preparation of an immobilized exopolygalacturonase characterized in that the enzyme is anchored at polyethylene terephthalate at a pH of 4.2 to 6.4 and a temperature of between 5 to 30 ° C.