CS258637B1 - Insoluble derivatives of pectolytic enzymes and method of their preparation - Google Patents

Description

The invention relates to insoluble derivatives of pecytic enzymes and to a process for their preparation. Insoluble derivatives have hitherto been prepared from pectolytic enzymes by binding the enzyme to insoluble polymeric carriers based on agarose, cellulose, hydroxyalkyl methacrylate upon activation with cyanogen bromide [e.g. Carbohydr. Res. 32, 233 (1974); Nahrung 22: 185 (1979); Czech. Chem. Commun. 45, 163 (1980)] and on poly (6-caprolactam) or poles (2,6-dimethyl-p-phenylene oxide) after activation with glutaraldehyde [CS AO 230 892; Biotechnol. Lett. 7, 99 (1985)). Another method of binding is physical adsorption to polyethylene terephthalate (CS AO 211 694).

Disadvantages of the derivatives based on the above-mentioned carriers are insufficient mechanical stability, unsuitable hydrodynamic properties and often high cost, and therefore these derivatives have found only limited use in practice. Some progress in the preparation of insoluble derivatives has been the immobilization of pectolytic enzymes on ceramic supports containing a chemically bonded 3-aminopropyl group. However, this method of binding required a further technological operation, namely the treatment of the carrier with glutaraldehyde (CS AO 256 857). These drawbacks are solved by the novel compounds according to the invention, which are based on the insoluble derivatives of the pecytic enzymes of the general formula E-L-A and a process for their preparation. In the above formula E, the pectolytic enzyme is endopolygalacturonase, or a pectinase complex comprising endopolygalacturonase, pectin esterase and exopolygalacturonase, L is a spacer linker formed by the interaction of the 3- (2 ', 3'-epoxypropoxy) carboxy-propylsilyl-carboxy-propylsilyl , a hydroxyl, imidazole, or indole group of the enzyme, A is a porous or non-porous carrier selected from the group of ceramic, silica and glass. The essence of the preparation of these insoluble pectolytic enzyme derivatives is to suspend the carrier to which the 3- (2 ', 3'-epoxypropoxy) propyl groups are surface-suspended in a buffer solution of pH 3.0 to 6.0, to add a mixed preparation of pytolytic enzymes. of enzymes containing endopolygalacturonase, exopolygalacturonase and pectin esterase, or any of its components in an amount of 0.03 to 0.1 parts by weight per 1 part by weight of the carrier, the resulting suspension is incubated with stirring for 24 to 240 hours and the immobilized enzyme is finally recovered.

The starting reactants for the preparation of these substances are readily available, since soluble pectolytic enzymes are commonly manufactured for use in the food industry, and also said carriers are produced in a wide range of grain sizes, shapes, porosity and pore size. However, for the binding of enzymes, it is desirable to adapt these carriers, i. j. introduce a 3- (2 ', 3'-epoxypropoxy) propyl group, which forms the linker through which the enzymes bind to the carrier, and this epoxy group can be readily introduced to the carrier surface by reaction with 3- (2', 3'-epoxypropoxy) propyltrialkoxysilane [e.g., Chemické listy 77, 843 (1983)].

The advantage of the enzyme derivatives prepared in this way is, besides the low cost and simple preparation, in particular the fact that the carrier used has a rigid structure and is mechanically and thermally stable, and that there is a wide choice of particle size and shape as well as pore sizes. carrier texture that is suitable for the application.

The insoluble enzyme derivatives of the invention can be used to cleave α-1,4-glycosidic bonds in the pectan molecule and to degrade pectin substances. Their stability allows long-term use.

The following examples characterize the insoluble enzyme derivatives of the invention without limiting or limiting it.

Example 1 g of a ceramic porous support containing surface-bound 3- (2 ', 3'-epoxypropoxypropyl) was suspended in 50 ml of pH 3.8 buffer and 30 mg of endopolygalacturonase dissolved in 1 ml of the same buffer was added. The resulting enzyme derivative was filtered off and washed with a buffer of pH 3.8.A carrier was not lost during the reaction and the activity of the thus prepared endopolygalacturonase derivative in cleavage of glycosidic bonds of sodium pectan showed 2.9% of the activity of the soluble form. The stability of the preparation was such that it could be used for several months.

Example 2

Example 1 was repeated except that 10 g of silica containing surface-bound 3- (2 ‘, 3‘-epoxypropoxy) propyl groups was used in place of ceramics, and the immobilized endopolygalacturonase activity showed 16% soluble enzyme activity.

Example 3

Example 1 was repeated except that glass with surface-bound 3- (2 ‘, 3‘-epoxypropoxy) propyl group was used instead of ceramics The immobilized endopolygalacturonase activity showed 4.5% soluble enzyme activity.

EXAMPLE 4 4 g of silica containing surface-bound 3- (2 ‘, 3‘-epoxypropoxy) propyl groups were suspended in 50 ml of pH buffer.

4.2 and 30 mg of a mixed preparation of pectinase containing endopolygalacturonase, exopolygalacturonase and pectin esterase were added thereto. The suspension was incubated with stirring for 72 hours. The immobilized enzyme was filtered off and washed with pH 5.6 buffer. The pectinase activity of the mobilized preparation showed 6% of the activity of the soluble preparation.

Example 5

Example 4 was repeated except that incubation of a mixed pectinase preparation containing endopolygalacturonase, exopolygalacturonase, and pectin esterase with surface-bound 3- (2 ', 3-epoxypropoxy) propyl groups was performed in buffer pH 6.0 immobilized pectinate. 6% soluble enzyme activity Example 6

The insoluble silica-based endopolygalacturonase derivative prepared according to the invention was suspended in pH buffer.

3.8 and filled into a column (1.4 X 5 cm). A 0.5% sodium pectane solution in 0.1 M acetate buffer pH 3.8 was percolated through the enzyme column at a rate of 0.8 ml per minute. The degradation products of sodium pectane formed in the enzyme reaction in the column were isolated from the effluent, after concentration, by gel chromatography.

Claims (2)

SUBJECT Insoluble derivatives of the pectolytic enzymes of the general formula E-L-A, wherein E represents the pectolytic enzyme endopolygalacturonase, or a pectinase complex comprising endopolygalacturonase, exopolygalacturonase and pectin esterase, L is a 3-propylene-3-spacer spacer, modified carrier with amino; a carboxyl, thiol, hydroxyl, imidazole, or indole group of the enzyme, A is a porous or non-porous carrier selected from the group of ceramic, silica and glass. 2. A process for the preparation of insoluble derivatives of pectolytic enzyme according to claim 1, characterized in that the carrier selected from the group of ceramics, silica and glass to which the 3- (2 ', 3'-epoxypropoxy) propylscins are surface-suspended is suspended in a buffer solution of pH 3.0-6.0, a mixed pectinase preparation containing endopolygalacturonase, exopolygalacturonase and pectin esterase, or one of its components, is added in an amount of 0.03 to 0.1 parts by weight per part by weight of the carrier, and the resulting suspension is incubated with stirring for 24 to 240 hours and the immobilized enzyme is isolated.