CS240462B1 - Method of endo-1,4-beta-s-gluconases activity determination - Google Patents

Abstract

The solution relates to the method of determining the activity endo-l, 4-TJS-glucanases. The essence of the process the determination is that hydroxyethyl cellulose % comprising 2.5 to 20 wt. covalently bound red dye, trisodium salts of 2-chloro-4- (N-ethyl-N-phenylamino- 6- [4-methyl-2-sulphobenzene-l-azo-2- (l-hydroxy-3,6-bissulfonaft-yl amino] -1,3,5-triazine is dissolved in water or in pH 4 buffers to 6 and at a concentration of 0.1 to 0.5 10 / wt. the used as a substrate in incubation mixtures with endo> -1,4-β-glucanases in which it is enzyme reaction carried out at temperature 20 to 50 ° C is stopped by adding two up to four volumes of ethyl alcohol - Acetone 1: 1 to 4: 1 vol. containing 0 to 0.1 mol. Sodium acetate, thereby precipitating the high molecular weight portion of the substrate is thus separated from the soluble color fragments released by the enzyme, which was determined spectrophotometrically at 550 mn. The invention has utility in biochemistry, molecular biology and modern biotechnology in characterization, identification and determining endo-1,4-β-glucanase activities.

Description

The invention relates to a method for determining the activity of endo-1,4-β-glucanases (EC 3.2.1.4).

Conventional methods for determining the activity of endo-1,4-β-glucanases as essential constituents of cellulolytic enzyme systems are based on the quantitative determination of reducing saccharides released from insoluble cellulose or soluble derivatives thereof. However, these methods are not selective because they are involved in the formation of reducing saccharides. and other components of colulolytic enzyme systems such as e.g. exo -? - glucanase or β-glucosidase (celobiase). They cannot be used to monitor the activity of endo-1,4-glucanases in solutions containing higher levels of reducing substances, mainly sugar, nor do they allow the determination of the activity of said living cell-bound glucanases, possibly in the presence of cells when they use released low molecular weight substrate fragments as a carbon source.

The viscosimetric method for the determination of endo-1,4- / 3-glucanase activity using soluble cellulose derivatives can be described as a very sensitive and specific method for the determination of endo-1,4- / 3-glucanase activity, but due to time constraints it is not suitable for analysis more samples. Another principle for determining the activity of endo-1,4-, 3-glucanases is to measure the amount of dye released into solution in the form of soluble cellulose fragments with a covalently bound dye. Except in one case [Β. V. McCleary: Carbohydr. Res. 86, 97 (1980)] all chromogenic substrates previously proposed for the determination of endo-1,4- / 3-glucanase activity are water insoluble. They are blue derivatives of microcrystalline cellulose [M. Linko, M. Leisola: Anal. Biochem. 70, 592 (1976)], celluloses with a partially reduced degree of crystallinity (Calbiochem AG 1980, Catalog No. 219 481) or insoluble cross-linked hydroxyethylcellulose (U.S. Pat. No. AO 192 202).

The use of insoluble substrates is associated with the difficulty of their precise dosing and the need to mix the incubation mixtures, as evidenced by the results of the Finnish authors [M. Leisola, M. Linko: Biochemical Laboratory Report 13, Technical Research Center of Finland, Helsinki (1976)]. Insoluble chromogenic substrates are not suitable for the determination of enzymes bound to cell surfaces or to different cell membranes. Water-soluble substrates allow the formation of enzyme-substrate complexes throughout the mixture. Such a chromogenic substrate for the determination of endo-1,4-, 3-glucanase activity was prepared on the basis of carboxymethylcellulose, which, however, had to be partially hydrolyzed by cellulases prior to binding the dye (Australian Patent 48 831/79).

Determination of activity on a soluble chromogenic substrate consists in measuring the release rate of the stained fragments soluble upon addition of an organic solvent that quantitatively precipitates the starting polymer substrate and its high molecular weight fractions upon partial enzyme degradation. Since the assay is based on substrate depolyraerization resulting in the formation of colored fragments soluble in the presence of organic solvents, the accuracy of the assay is significantly influenced by the ionic strength of the environment [Β. V. McCleary: Carbohydr. Res. 86, 97 (1976)]. Similar substrate properties have a chromogenic substrate for the determination of endo-1,4-, 3-glucanase activity prepared by the present invention - trisodium salt of 4- (N-ethyl-N-phenylamino) -6- [4-methyl- -2-sulfobenzene-1-azo-2- (1-hydroxy-3,6-bissulfonaphth-1-yl) amino] -1,3,5-triazin-2-ylhydroxyethylcellulose.

The present invention is characterized in that the hydroxyethylcellulose contains from 2.5 to 20% by weight of hydroxyethylcellulose. covalently bonded red dye of the trisodium salt of 2-chloro-4- (N-ethyl-N-phenylamino) -6- [4-methyl-2-sulfobenzene-1-azo-2- (1-hydroxy-3,6-bissulfulfate) onaft-1-yl) amino] -1,3,5-triazine are dissolved in a buffer of pH 4.0-6.0 at a concentration of 0.1-1.5% by weight. with vigorous stirring at 20 to 30 ° C and the resulting solution is used as a substrate in admixture with a solution or suspension of endo-1,4- / 3-glucanase-bound particles in which the enzyme reaction takes place at 20 to 50 ° C. ° C for 5 to 180 min. by adding two to four volumes of a 1: 1 to 1: 4 to 1 by volume mixture of ethyl alcohol. containing 0 to 0.1 mol. . i ^-1 sodium acetate, thereby precipitating a proportion of unhydrolyzed substrate which is separated from centrifuged color fragments released by the enzyme after centrifugation, the amount of which is proportional to the enzyme concentration in the mixture as well as the duration of incubation and which becomes offotometrically at 550 nm.

An advantage of the proposed method of determining the activity of the endo-1,4-, 3-glucanases is that it uses a chromogenic substrate soluble in water and in buffers, respectively. in more concentrated buffers, it is homogeneously dispersed in the m-part and ensures uniform distribution of the substrate in the enzyme incubation mixtures and removes mixing requirements, and that the enzyme reaction is stopped simply by the addition of an organic solvent, resulting in precipitation of the substrate except low-molecular fragments released by the enzyme from the flood-precipitation substrate. Another advantage is that the method of activity determination is specific for endo-1,4-, 3-glucanases, that it is exceptionally sensitive, that it is not affected by the presence of exo- / 3-glucanases and β-glucosidases, that it allows the determination of endo- 1,4-β-glucanases also in the presence of reducing agents, e.g. of carbohydrates, if these do not have an inhibitory effect on enzymes, that it is suitable for analysis of the enzyme in the presence of live bu240462 som as well as an enzyme bound to cellular structures and that it is particularly suitable for analyzing the large number of samples obtained after protein fractionation by chromatography, chromatofocusing , electrophoresis and electrofocusing.

Example 1

8 mg of hydroxyethylcellulose containing 10 wt. Trisodium salt of 4- (N-ethyl-N-phenylamino) -6- [4-methyl-2-sulfobenzene-1-azo-2- (1-hydroxy-2,6-bisulfonaphthyl) amino] -1,3,5-triazin-2-yl (Ostazine brilliant red H-3B) was dissolved with stirring in 10 ml of citrate-phosphate buffer composition: 1 part 0.1 mol / ^l of citric acid and 1 ml 0.2 mole part. I ^-1 phosphate monohydrate and 2 parts of water, about 4.8 pM. 0.5 ml aliquots of a solution preheated to 30 DEG C. are mixed with 5 to 50 μ \ undiluted, diluted ten times and fifty times the cellulose solution and endo-1,4-β-glucanases, respectively, and the mixtures were incubated at 30 ° C for 10 min and quenched by the addition of three volumes of 96% ethyl alcohol-acetone 2: 1 v / v (1.5 mL), thereby precipitating the high molecular weight portion of the substrate, while the low molecular weight fragments released by the enzyme remain in solution after addition of the organic precipitant After standing at 22 ° C for 10 minutes, the mixtures are mixed and centrifuged. 1 min at 2,000 g.

Absorbance of the clear supernatants is measured at 550 nm against a sample containing no enzyme solution. The color intensity of the supernatants is to some extent proportional to the amount of enzyme in the incubation mixture as well as the duration of incubation. The sensitivity of the assay is determined by the precipitation temperature of the non-hydrolyzed substrate as well as the ionic strength of the solution. The unit of activity of endo-1,4-β-glucanase is the amount of colored fragments dissolved in a 1: 2: 1 by volume citrate-phosphate buffer-ethyl alcohol-acetone mixture containing 1 wt. of the total amount of dye bound in the substrate present in the incubation mixture. The conversion of the absorbance values at 550 nm into the cuticles or international units of activity is possible after determining the amount of reducing saccharides released under the same conditions from an equivalent amount of uncoloured hydroxyethylcellulose.

Example 2

Hydroxyethylcellulose containing 15 wt. 4 (N-ethyl-N-phenylamino) -6- [4-methyl-2-sulfobenzene-1-azo-2- (1-hydroxy-3,6-bissulfonaphth-1-yl) amino] covalently bonded trisodium salt -l, 3,5-triazin-2-yl was stirred at 25 ° C dissolved in 0.1 mol. .I ^-1 acetate buffer, pH 5.0, 0.1 M 7diel composition. I ^{- 1 part} of sodium acetate and 3 parts of 0.1 ml of ¹ acetic acid to a substrate concentration of 16 mg / ml, under these conditions a small part of the substrate remains undissolved but homogeneously dispersed as a fine precipitate 0.25 ml of this substrate solution heated to 30 ° C is mixed with 0.25 ml of a suitable diluted enzyme solution and incubated at 30 ^° C. The enzyme reaction is stopped and evaluated as in Example 1, but taking into account the higher content of hydroxyethylcellulose-bound dye.

Example 3

The procedure was as in Example 1 except that hydroxyethylcellulose containing 2.5 wt. of a covalently bound dye, that the enzyme reaction is carried out at a temperature of 50 ° C and is stopped by adding four volumes of ethyl alcohol acetone 4: 1 v / v.

Example 4

With increased accuracy requirements for the determination of endo-1,4- [beta] -glucanase activity according to Examples 1 to 3, the assay was measured for each sample. time dependence of release of colored substrate fragments. The graphs of absorbance at the $ 50nm duration of the enzyme reaction determine the slope of the reaction for the initial reaction time, which is proportional to the concentration of the enzyme in the incubation mixture and thus its activity.

Example 5

The method of determining the activity of endo-1,4- [beta] -glucanases on color hydroxyethylcellulose given in Examples 1-4 provides reproducible results if the parameters that affect the solubility of the color fragments released by the enzyme in the mixture after addition of the organic precipitant are maintained in all cases. temperature, precipitation and centrifugation, and ionic strength of the precipitation mixture. The effect of minor changes in salt concentration can be eliminated by adding an internal electrolyte, e.g. 0.2 mol / ^l sodium chloride into the substrate solution. An increase in ionic strength reduces the solubility of colored hydroxyethylcellulose fragments in the presence of an organic precipitant, resulting in an overall decrease in assay sensitivity.

example

The effect of varying the concentration of salts in the enzyme solution on the reproducibility of the endo-1,4-d-glucanase assay as set forth in Example 5 is removed by adding an internal electrolyte to the precipitation mixture. The enzymatic reactions carried out according to Examples 1 to 4 are stopped by adding three volumes of a 2: 1 volume of ethyl alcohol-acetone containing 0.1 mol. I ^-1 sodium acetate.

240162

The invention has utility in biochemistry, molecular biology and modern biotechnology in the characterization, identification and determination of endo-1,4-, β-glucanase activities.

Claims (1)

Method for determining the activity of endo-1,4- [beta] -glucanases, characterized in that hydroxyethylcellulose containing from 2.5 to 20% by weight is used. covalently bonded red dye, trisodium salt of 2-chloro-4 (N-ethyl-N-phenylamino-6- [4-methyl-2-sulfobenzene-1-azo-2- (1-hydroxy-3,6-bissulfonate) 1-yl-amino] -1,3,5-triazine is dissolved in a concentration of 0.1 to 1.5% by weight at 20 to 30 ° C in water or in a buffer of pH 4 to 6 and incubated. in a volume of 0.5 to 2.0 ml with a solution of endo-1,4-β-glucanases at a temperature of 20 to 50 ° C after INVENTED for 5 to 180 min, when the enzyme reaction is stopped by adding two to four volumes of ethyl alcohol-acetone 1: 1 to 4: 1 v / v. containing 0 to 0.1 mol. I ¹ sodium acetate to precipitate high molecular proportion of the substrate to be separated by centrifugation from the soluble fragments of color released by enzyme activity, the quantity of which is proportional to the enzyme concentration in the reaction mixture and the duration of incubation, and are determined spectrophotometrically at 550 nm.