DD141978A3 - PROCESS FOR PREPARING WATER-SOLUBLE ENZYME BY BINDING TO SILICA GEL - Google Patents

Abstract

The invention relates to a process for the preparation water-insoluble enzymes by binding to silica gel for use in the chemical, pharmaceutical and food industries as well in the medicine. The aim of the invention is to be in a simple economic Way, based on insoluble and stable enzyme-carrier complexes from kieselgei with high activity produce, which by a suitable modification of the Kieseigels before binding with the Enzyme is achieved. The modification of the silica gel is carried out by an acid treatment at 30 to 100 ° C, preferably at 90 ° C, with 1 to 6 N HCl, preferably with 3 N HCl, between 1 and 20 h, preferably 6 h. Proves to be particularly suitable a silica gel with a surface area of 200 to 400 m7 / g. The v Binding of the enzymes to the carrier can be done in a known manner become.

Description

193 985

Field of application of the invention

The water-insoluble Snzyme are suitable for use in the chemical, pharmaceutical and food industries as well as in medicine.

Characteristic of the Known Technical Solutions The binding of biologically active substances to insoluble matrices has become very important in recent years. It is particularly advantageous to produce immobilized enzymes because these preparations can either be used in continuous processes or can be used repeatedly in discontinuous processes. Their application can therefore bring about technological advantages in certain areas of medicine, the chemical and pharmaceutical industry and in the food and beverage industry and lead to cost savings. As a carrier supply materials natural or modified polysaccharides, synthetic organic polymers or inorganic compounds, such as porous glass and metal oxides IP Andrews H. Dig Uy enzymes, Vol were "previously used (GP Royer, technol... I 99-138 ßSTtfi M. Uelboeck ₅ D Jaworek Chimia 2%, 109 / T9757),.

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The natural carrier materials (polysaccharides, proteins) have disadvantages, especially with regard to a technical application, they are easily susceptible to microbial attack, and their hydrodynamic properties in packed reactors are poor. Often, an application of immobilized enzymes is also limited by the high cost of producing and purifying support materials (eg, porous glass and synthetic organic polymers).

Silica gel was also used several times as a matrix for enzyme binding. In this process, the protein is adsorbed on the surface of colloidal pyrogenically prepared silica gel particles and can subsequently be stabilized by chemical reagents. Because of its small particle size, use of the colloidal silica gel in a column reactor is not possible (R. Haynes, K.A. False, Biochem. Biophys. Res. Commum. 36, 235 / T9697).

The use of precipitated silica gels is limited by the low activity of the resulting enzyme-carrier complexes.

Object of the invention

The aim of the invention is to produce insoluble and stable enzyme-carrier complexes with high activity in a simple economic manner.

Explanation of the essence of the invention

The invention has for its object to modify silica gel so that after binding of the enzyme results in an enzyme-carrier complex with high activity.

In the present invention, this is achieved by using silica gel having a surface area of from 200 to 400 m / g as a carrier which has been previously subjected to acid treatment. The acid treatment of the silica gel is carried out at 30 to 100 ⁰ G, preferably at 90 ⁰ C, with 1 to 6 Έ HCl, preferably with 3 H HCl, between 1 and 20 Ji, preferably 6 tu The application of the enzymes on the support is carried out according to known · methods

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A carrier-enzyme complex is prepared, for example, by adsorbing the enzyme in buffered solution to the acid-treated silica gel and. after a washing process with glutaric dialdehyde is crosslinked ·

Furthermore, the carrier-enzyme complexes according to the invention can be obtained by covalent bonding, such as, for example, azo coupling with silanes, such as, for example, r -aminopropyltriethoxysilane, surface-modified acid-treated silica gels with enzymes.

The carrier-enzyme complexes based on acid-treated silica gels according to the invention show a substantially high catalytic activity in comparison with the untreated silica gels. Their preparation is simple and their particle size can be varied over a wide range, so that they can be used advantageously in packed reactors. Since commercial starting materials are used for the preparation of the carrier-enzyme complexes, the complexes according to the invention are distinguished from other support materials. such as As porous glass and organic polymers, by a low cost.

embodiments

The following is a description of the preparation of the carrier-enzyme complexes in Examples.

example 1

100 g of silica gel with a surface of 200-400 nT / g and with.

a grain diameter of 0.8-1.25 mm with 2 1 JE HCl

stirred at 9O ⁰ C. After 6 h. the acid is hot-decanted and the resulting acid-treated silica gel is washed acid-free with distilled water. Thereafter, the silica gel is dried in air.

The acid-treated silica gel is comminuted to a particle size of 0.2-0.4 inches and classified »

, .....- .; , -η- ι y $ ^ 85

Example 2

1 g acid-treated silica gel (prepared according to Example 1, 0.2-0.4 ram grain size) is stirred with 100 mg glucoamylase in 15 ml acetate buffer pH 5.0 at room temperature for 1 h, then the enzyme-silica gel complex is filtered off with suction washed with water and stirred for 2 hours with about 2.5% glutaraldehyde solution at room temperature, then it is washed with acetate buffer pH 5.0 / 1 M NaCl, the preparation is kept under water in the refrigerator.

used enzyme activity: 2000 E.

washed-out activity:. 1200 E specific activity: 250 U / g

relative activity: 31%

Example 3

A carrier-enzyme complex according to Example 2 based on a commercially available silica gel as a "comparison" has a specific activity of 65 U / g.

Example 4

1 g acid-treated silica gel, prepared according to. Example 1, is silylated with 5- / S-iger ^ -Aminopropyltriäthoxysilan solution in 'toluene, reacted with p-ITitrobenzoylchlorid, then with N ^a 2 ^S 2 ° 4 reduced and then diazotized. (HH Weetall Science 166, 615

100 mg of glucoamylase in 10 ml of phosphate buffer pH 7.0 are added to the silica gel diazonium salt and the mixture is stirred at 4 ° C. for 3 h. The mixture is washed as in Example 2. The complex has a specific activity of 200 U / g.

Example 5

An enzyme-carrier complex of acid-treated silica gel and bacterial alpha-amylase will decrease. Examples 1 and 2 prepared Specific Activity: 92 U / g.

Claims (2)

- * - 193 985 Claim for invention:. 1. A method for, producing water-insoluble enzymes by. Binding to silica gel, characterized in that silica gel subjected to acid treatment with a surface area of from 200 to 400 in / g, and then the enzymes are bound to the silica gel in a known manner. " 2. The method according to Pmürt 1, characterized in that the acid treatment of the silica gel at 30 to 100 C, preferably at 9O ⁰ C, with 1 to 6 IT HCl, preferably with 3 N HCl, between 1 and 20 h, preferably 6 h , he follows·