CS254162B1 - Method of soluble chromolytic substrate preparation for beta(1-3)gluconase determination - Google Patents

Description

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

Photocolourimetric methods have been proven and very effective methods for the determination of polysaccharide hydrolase enzyme activities. They consist in reacting the enzyme with an insoluble chromolytic substrate, whereby the enzyme becomes soluble and stains the reaction solution. The color intensity of the solution is then proportional to the activity of the enzyme being measured [J. Kennedyc Advances in Carbohydrate Chemistry and Biochemistry 29, 350 (1974)]. Methods are also known using water-soluble chromolytic substrates, in which a soluble enzymatic unattracted fraction of a chromolytic polysaccharide is precipitated with an organic solvent and the hydrolyzed fraction remains soluble, thereby coloring the reaction solution proportionally to the activity of the enzyme present [Κ. K. Mákinen,

I.K. Paunio: Anal. Biochem. 39, 202 (1971)]. At the present time, insoluble or soluble chromolytic polysaccharide substrates cannot be unambiguously recommended since both have their advantages and disadvantages. It depends on the specific analysis conditions, which type of substrate we prefer. The preparation of many water-soluble polysaccharide chromolytic substrates is the subject of Cs. AO 237 101 and U.S. Pat. No. 4,321,364. However, it does not include less known polysaccharides such as β (1-3) glucan isolated from the cell walls of baker's yeast (Saccharomyces cerevisiae), which is characterized by very low reactivity in subsequent alkylation modification reactions, by which the insoluble polysaccharide is converted to soluble.

SUMMARY OF THE INVENTION In the first step, the β (1-3) glucan of alkyl is sodium ethylene oxide or β-chloroethyl sulfonate or monochloroacetic acid in an alkaline environment to a degree of substitution of 0.35 to 0.8, and the alkyl polysaccharide binds color; dvojsodná so! 8-amino-5- [3- (sulfoethylsulfonyl janilino) 6-anthraquinone-sulfonic acid (Remazol brilliant blue R) in an amount of 6 to 10 percent by weight of the polysaccharide at a temperature of 15 to 25 ° C and a sodium hydroxide concentration of 1 0 to 3.0% by weight for 2 to 4 hours and the colored polysaccharide is isolated from the solution in a known manner.

The prepared chromolytic / 3 (1-3) glucan is well soluble in water and is a suitable substrate for measuring β (1-3) glucanase enzyme activity, EC 3.2.1.6. The hydroxyethyl derivative is more suitable for dye binding since it does not block the hydroxyl groups of the polysaccharide. Carboxymethyl and sulfoethyl derivatives of β (1-3) glucan are poorly colored, but they are easier to prepare than the hydroxyethyl derivative.

An advantage of the novel process for the preparation of a water-soluble chromolytic gel / 3 (1-3) glucan is that it makes it possible to prepare from this low reactive polysaccharide a sufficiently sensitive water-soluble enzyme substrate suitable for photocolourimetric determination of the β (1-3) glucanase activity, EC 3.2.1.6. is expanding the range of water-soluble polysaccharide hydrolase enzyme substrates.

EXAMPLE

To 200 g of air-dry powder / 3 (1-3) glucan isolated from baker's yeast cell walls (Saccharomyces cerevisiae) is alkalinized with 200 ml of a 20% w / w aqueous solution of sodium hydroxide and allowed to activate for 1 hour at 20 ° C. The alkali-glucan is then alkylated in a reactor from which the air is aspirated by sequentially adding 35 g of ethylene oxide at 45 DEG C. and allowed to react for 2 hours after the addition of the last portion of ethylene oxide. The reaction mixture was allowed to react for a total of 2 hours after the addition of the last portion of the dye. After binding of the dye to the polysaccharide, the reaction solution was neutralized with acetic acid and then neutral. the solution is poured into an excess of alcohol with stirring to precipitate the chromolytic / 3 (1-3) glucan, filter on a filter and The product contains 8 to 10% by weight of methanol until the wash solution is colorless. is a good water-soluble substrate and is therefore a suitable water-soluble substrate for determining the β (1-3) glucanase enzyme activity.

Example 2

200 g of air-dry powdered β (1-3) glucan isolated from baker's yeast cell walls are alkylated in a suspension of 2 liters of isopropanol and 200 ml of an aqueous solution of 20% by weight sodium hydroxide. The reaction mixture was gradually heated to 55 ° C, when 70 g of solid sodium 3-chloroethylsulfonate was added portionwise over 30 minutes. The reaction mixture is maintained at 55 ° C with stirring for 3 hours, then the solid sulfoethyl glucan is filtered off, washed with 96% by weight. The solid product is dissolved in 3 liters of water, the sodium hydroxide concentration is adjusted to 3% by weight. and stained with Remazol briliant bleu R at 25 ° C for 4 hours. The finished product is well soluble in water and is a suitable substrate for the determination of β (1-3) glucanase enzyme activity. Contains 6 wt. bound dye.

Example 3

200 g of air-dry powdered (3- (1-3) -glucan isolated from baker's yeast cell walls are suspended in 2 liters of 96% by weight. ethanol, to which 100 ml of 600 g aqueous sodium hydroxide solution had previously been added. . I ^-1 . The reaction mixture is stirred at 20 ° C for 1 hour, then 100 g of chloroacetic acid are added and heated to 55 degrees Celsius for 30 to 45 minutes, and the reaction mixture is maintained at this temperature for a total of 3 hours. Then the solid carboxymethyl-glucan product is filtered off, the ethanol is filtered off well and the solid product is dissolved in 2 liters of water, the sodium hydroxide concentration is adjusted to 3% by weight. and stained with dye (Remazol briliant blue R) as in Example 2. The finished product is well soluble in water, and is a suitable substrate for determining β (1-3) glucanase enzyme activity.

The invention has applications in enzyme analysis, biotechnology and wherever β (1-3) glucanase enzyme activity is readily measured.

Claims (2)

SUBJECT Process for preparing a soluble chromolytic substrate for the determination of β (1-3) glucanase, EC 3.2.1.6 by modification of β (1-3) glucan, characterized in that in the first step, β (1-3) glucan is alkylated with ethylene oxide or β -chloroethylsulfonate or monochloroacetic acid in an alkaline medium to a degree of substitution of 0.35 to 0.8 and in the second step a dye, a disodium salt of 8-amino-5- [3- (sulfoethylsulfonyl Janilino) 6-anthraquinone, is bound to the alkyl polysaccharide % sulfonic acid in an amount of 6 to 10% by weight of the polysaccharide at a temperature of 15 to 25 ° C and a sodium hydroxide concentration of 1.0 to 3.0% by weight for 2 to 4 hours and the colored polysaccharide is isolated from the solution.