CS254163B1 - A method of preparing an ohromolytic substrate for determining ((1-3) glucanase) - Google Patents

Abstract

The purpose of the solution is a method of preparing a chromolytic substrate for the determination of beta-glucanase. The stated purpose is achieved by, in the first stage, the beta (1-3) glucan is alkylated with ethylene oxide or monochloroacetic acid in an alkaline medium to a degree of substitution of 0.2 to 0.3 and, in the second stage, the dye disodium salt of 8-amino-5-[3- - (sulfoethylsulfonyl-Janilino] 6-anthraquinonesulfonic acid is bound to the alkyl polysaccharide in an amount of 10 to 12% by weight per weight of the polysaccharide at a temperature of 15 to 20 °C and a sodium hydroxide concentration of 1.0 to 2.0% by weight for a period of 2 to 4 hours. The method of preparing a chromolytic substrate for the determination of beta-glucanase has applications in enzyme analysis, biotechnology and wherever it is necessary to promptly measure beta (1-3) glucanase enzyme activity.

Description

The invention relates to a method of preparing a chromolytic substrate for the determination of β (1- + 3) glucanase, EC 3.2.1.6.

In recent years, the well-established method for determining polysaccharide hydrolase enzyme activities is the photocolourimetric method. It consists in reacting the s-a enzyme with an insoluble polysaccharide gel on which a suitable dye is covalently bound. During hydrolysis by the respective polysaccharide hydrolase enzymes present, the chromolytic gel becomes progressively soluble, with the color intensity of the supernatant being proportional to the enzyme activity. Understandably, this is true only if the polysaccharide chromolytic gel is perfectly accessible to the enzyme molecule and that the covalently bound dye to the polysaccharide has no inhibitory effect on the enzyme reaction itself [K. Kenedy: Advances in Carbohydrate Chemistry and Biochemistry, 29, (1974) p. 350-376]. Methods for the preparation of different polysaccharide chromolytic substrates of Cs. AO 192 210, 203 676, 219 575, 224 156. These are mostly polysaccharides most commonly found in nature and therefore the most widely used in research and industry. Preparation of water-soluble chromolytic substrates and rare polysaccharides is the subject of Cs. AO 237 101

It is known that the various polysaccharides differ in their chemical and supersolecular structure, which must be respected in the preparation of a chromolytic substrate suitable for the determination of the respective enzyme polysaccharide hydrolase activity.

Lowly studied microbial polysaccharides include β (1- + 3) glucan, which is part of the cell walls of yeast microorganisms from which it is isolated. It is a water-insoluble polysaccharide and very little reactive in subsequent modification reactions in both alkaline or acidic media. For these reasons, direct binding of the dye to this polysaccharide is unsuitable for the preparation of a sufficiently sensitive chromolytic substrate to determine the β (1- + 3) glucanase enzyme activity.

The principle of the invention is that in the first step the β (1- (3) glucan alkyl) is ethylene oxide or monochloroacetic acid in an alkaline medium to a degree of substitution of 0.2 to 0.3 and in the second step the dye is disodium bound to the alkyl polysaccharide. so! % of 8-amino-5- [3- (sulfoethylsulfonyl) amino] 6-anthraquinone sulfonic acid in an amount of 10-12 wt. % by weight of polysaccharide at a temperature of 15 to 25 ° C and a sodium hydroxide concentration of 1.0 to 2.0 wt. for 2 to 4 hours.

The prepared chromolytic gel has a swelling volume of 12 to 15 ml / g, which is sufficient for the desired sensitivity to determine the enzyme activity. The optimum degree of substitution is 0.2 for hydroxyethyl and 0.3 for carboxyethyl.

At these substitution levels, the chromolytic gel has maximum yields and still does not dissolve in water when eluting the free dye. At higher degrees of substitution, water solubility is already rapidly increasing, resulting in a significant loss of chromolytic gel in the wash. The hydroxyethyl derivative is better colored than the carboxymethyl derivative, but the latter is easier to prepare.

An advantage of the novel process for the preparation of the chromolytic gel / 3 (1- + 3) glucan is that it makes it possible to prepare from this low-reactive polysaccharide a sufficiently sensitive enzyme substrate suitable for the colorimetric determination of the respective enzyme activity.

Example 1

200 g of dry / 3 (1- + 3) glucan isolated from yeast cell walls of Saccharomyces cerevisiae are suspended in 200 ml of ethanol (96% by weight), in which 75 ml of 600 g of aqueous sodium hydroxide solution have previously been dissolved. 1 ^_1. the suspension was stirred at 20 ° C for 40 minutes, then added in portions 40 g of monochloroacetic acid. the mixture was then gradually heated to 55 ° C for 1 h and at a final temperature of 55 ° C, the reaction The mixture is kept under stirring for a total of 3 hours, then the free sodium hydroxide is neutralized with conc. acetic acid, the reaction mixture is filtered and the insoluble carboxymethyl- [beta] (1- + 3) glucan is filtered first with distilled water, then with alcohol and The product is suspended in 2 liters of 2% sodium hydroxide and at 15 ° C, the dye of 8-amono-5- [3- (s) sodium salt is added portionwise over 30 minutes. ulfoethylsulfonyl or amino-6-anthraquinonesulfone in an amount of 20 g. Then, 100 g of sodium acetate is added to the solution, and the reaction mixture is stirred at 15-25 ° C for 4 hours. After completion of the dyeing, the reaction solution was neutralized with conc. with acetic acid, the insoluble product is aspirated and washed on the filter with distilled water until the washing water is colorless. Finally, the product water is displaced with ethanol and finally ethanol is freely evaporated in air or in a vacuum oven at a temperature of up to 60 ° C. The dried product is sieved and a fraction with a particle size of between 80 and 250 μΐη is used. The finished gel has a swelling volume of 12 to 15 ml in water. g ^-1 and is a very good substrate for the determination of β (1- + 3) glucanase.

Example 2

To 200 g dry powdered β (1- + 3) glucan isolated from yeast cell walls of Saccharomyces cerevisiae was added, with stirring, 200 ml of a 20 wt% aqueous solution of sodium hydrogencarbonate. and allowed to activate for 1 hour. The activated glucan is then sealed in a sealed reactor with 2 liters of acetone and fed 22 ml of ethylene oxide sequentially through the valve. The reaction mixture is heated to 40 ° C and kept at this temperature for 2 hours. After completion of the reaction, free sodium hydroxide in the reactor was neutralized with conc. acetic acid, insoluble hydroxyethyl- [beta] (1-3) glucan is filtered off, washed

3, with water and finally ethanol and further dyed according to the procedure of Example 1 with 12 g of 8-amino-5- [3- (sulfethylsulfonyl) anilino] 6-anthraquinonesulfonic acid disodium salt. 3) glucan with a covalently bound dye is a very good substrate for determining the β (1-3) glucanase enzyme activity.

The invention can find wide application in enzyme analysis, biotechnology in measuring β (1-3) glucanase enzyme activity.

Claims (1)

Of sodium hydroxide of 20% by weight. it is activated for 1 hour. Thereafter, the activated glucan was quenched in a sealed reactor with 2 liters of acetone and passed through the valve successively with 22 mL of ethylene oxide. The reaction mixture was heated to 40 ° C and maintained at that temperature for 2 hours. After completion of the reaction, the free hydroxidodium in the reactor is neutralized with conc. with acetic acid, the insoluble product hydroxyethyl / 3 (1-3) glucan is filtered off, washed with 6 6 with water, finally with ethanol and further colored according to the procedure of Example 1 with 12 g of color disodium. salts of 8-amino-5- [3- (sulfoethylsulfonyl) anilino] 6-anthraquinone sulfonic acid. The resulting product (3 (1-3) glucan by covalently bound dye is a very large substrate for the determination of β (1-3) -glucanase enzyme activity. The invention can be widely used in enzyme analysis, biotechnology to measure β (1-3) glucanase enzyme activity. SUBJECT A method of preparing a chromolytic substrate for the determination of β (1-3) glucanase, EC 3.2.1, -6 characterized in that, in the first step, β (1-3) glucan isolated from the cell walls of Saccharomyces cerevisiae alkyl is ethylene oxide or by a mono-chloroacetic acid in an alkaline medium to a degree of substitution of 0.2 to 0.3, and a dye is bound to the alkyl polysaccharide in the second step; a disodium salt of 8-amino-5- [3- (sulfoethylsulfonyl) -alkyl] aniline] 6-anthraquinone sulfonate in an amount of 10 to 12 wt. to a polysaccharide weight at 15 to 25 ° C and a sodium hydroxide concentration of 1.0 to 2.0 percent by weight; for 2 to 4 hours.