CS234607B1 - A method for determining endo-1,4-β-xylanase activity - Google Patents

Abstract

The invention relates to a method for determining the activity of endo-1,4-β-xylanases. The essence of the determination method is that 4-O-methyl-D-glucurono-D-xylan with 4 to 20% of covalently bound dye disodium salt of 1-amino-2-sulfo-4-[3-(2- -sulfatoethylsulfonylanilino)] anthraquinone is dissolved in water or in a buffer with pH 4 to 6 and at a concentration of 0.2 to 2% is used as a substrate in incubation mixtures with endo-1,4-β-xylanases, in which the enzyme reaction carried out at a temperature of 20 to 50 °C is stopped by adding two volumes of 96% vol. ethyl alcohol, thereby precipitating the portion of unhydrolyzed substrate, which is thus separated from the soluble fragments released by the enzyme action, which are determined photometrically at 580 to 610 nm. The invention has application in biochemistry.

Description

1,234,607

The invention relates to a method for determining endo-1,4-xylanase (EC 3.2.1.8) activity. Conventional methods for assaying microbial endo-1,4-oxylanase activity are based on the quantitative determination of reducing carbohydrate released from plant xylans by the action of enzyme preparations. These methods do not allow the selective determination of acto-1,4- (1-xylanase) activity. in the presence of β-xylosidases (exo-xylanase, EC 3.2, 7.37), which contribute to the formation of reducing carbohydrates by hydrolyzing xylooligosaccharides to xylose, these methods are also not suitable for determining endo- 1,4 - (L-xylanase in solutions with a high concentration of reducing carbohydrates that need to be removed by dialysis. Alternatively, methods of determining endo-1,4-xylanase activity utilize the measurement of viscosity decrease of solutions of polymeric substrates such as carboxymethylxylan. Viscosimetric method may be considered the most sensitive and very specific method of determining endo-1,4- (b-xylanase) activity, but In the last two decades, so-called chromogenic substrates, corresponding to polysaccharides with covalently bonded CO, A.Stamm, have been introduced to determine the activity of various endoglycanases; Helv, Chira. Acta 46, 3008 (1963); P. Hagen, E. T. Reese, O., A. Stamm; Helv, Chim, Acta 49, 2278 (1966); M. Leisola, M. Linko: Anal Biochem, 70, 592 (1976) 3. In all cases, water-insoluble polysaccharide is used as a substrate and in buffers. The amount of dye released into solution in the form of soluble fragments of a polysaccharide with a covalently bound dye is then considered as a measure of enzyme activity. A method for determining the activity of endo-1,4-β-xylanase on a xylan gel with covalently bound dye, 2,234,807, whose water insolubility was achieved by crosslinking the stained polysaccharide with epichlorohydrin Css was also proposed. AO 192 202] The use of insoluble polysaccharides or derivatives thereof with covalently bound dye as substrates for the determination of the activity of polysaccharide hydrolases with endogenous attack of the substrate allows the enzyme reaction to be visually visualized by transitioning the dye into an aqueous colorless solution (filtrate or supernatant). This method of monitoring the enzyme reaction can be applied when the enzyme reaction is carried out in volumes greater than the swelling volume of the substrate in the incubation mixture. In this case, however, it is necessary to mix the reaction mixtures to ensure the same likelihood of forming the enzyme-substrate complex throughout the volume. It is known that when determining the activity of cellulases on dyed microcrystalline cellulose, the amount of dye released is dependent on the mixing of the mixture M. Leisola, M. Linko: Biochemical Laboratory Report 13, Centreof Finland Technical Research, Helsinki (1976)]. In large series analysis, the requirement for mixing is a disadvantage which is eliminated by the use of a colored polysaccharide substrate which is perfectly soluble in water and in buffers but which can be quantitatively precipitated from the aqueous solution by organic solvent under conditions where shorter color fragments the polysaccharide released by the enzyme remains in solution. On this principle, the proposed method of determining endopectinase activity (endopoly-galacturonanase, EC 3.2.1.15) by D. R. Friend, G. W. 3. Chang: □. Agr. Food Chem. 30, 892 (1982), which measures the rate of release of colored fragments from stained pectin that are soluble in 63% ethyl alcohol.

The present invention is based on the fact that 4-O-methyl-D-glucoro-no-D-xylan with 4 to 20% of covalently bound 1-amino-2-sulfo-4-O- (2-sulfatoethylsulfonylanilino)] antra salt the quinone is dissolved in a buffer of pH 4.0 to 6.0 at a concentration of 0.2 to 2.0% by weight. at a temperature of 50 to 90 ° C, and this cooling solution to 20 to 50 ° C is used as a substrate in a mixture of endo-1,4-fl-oxalazate in which the enzyme reaction occurs at 20 to 50 ° C for 5 to 5 hours. 180 min is stopped by the addition of two volumes of ethyl alcohol, resulting in a proportion of the unhydrolyzed substrate which is separated from the soluble color fragments released by the enzyme-234-227, the amount of which is proportional to the enzyme concentration in the incubation mixture and determined photometrically 580-610 nm · The advantage of the proposed method of determining endo-1,4-β-xylanase activity is that it utilizes a chromogenic substrate dissolved in water and in buffers, guaranteeing a homogeneous composition of enzyme incubation mixtures and eliminating mixing requirements such as jetome in the case of insoluble chromogenic substrates and that the enzyme reaction stops very simply by adding two oatbates of denat the ethyl alcohol, which leads to the precipitation of the substrate, with the exception of the colored fragments released by the enzyme from the initially complete clotting substrate, the further advantage being that the test is specific for endo-1,4-β-xylanase and is amalgamable by the β-xylosidase activity (exo- (phenoxylanase)) that allows the determination of endo-1,4- {λ-xylanase activity in the presence of reducing carbohydrates and other reducing agents if these do not have enzyme inhibitory activity and are suitable for analysis a large number of samples obtained, for example, after fractionation of proteins on columns by chromatography, chromatofocusing, or electrophoresis. Example 1

Dissolve xylan containing 4.8% covalently bound disodium salt of 1-amino-2-sulfo-4-3- (2-sulfatoethylsulfonylanilino) 3-anthraquinone in 0.05 M acetate buffer (decomposition). : acetic acid and sodium acetate), pH 5.4, with heating at 60 ° C and stirring so that 1 ml of the solution contains 5 mg of modified polysaccharide. After cooling to 40 ° C, 1 ml aliquots are mixed with 0.1 ml volumes of solution of n-1,4-p-oxylanase. After mixing, the mixtures are incubated at 40 ° C for 10 to 180 min. The reaction is stopped by adding two volumes of ethyl alcohol (2.2 mL). After thorough stirring, the mixtures are allowed to stand at 20-25 ° C for 30 min, mixed and centrifuged. Absorbance of clear supernatants is measured against a sample not containing enzyme at 580 nm. The color intensity of the supernatants is proportional to the amount of enzymes in the incubation mixture and the duration of incubation. The unit of activity is considered to be the amount of fb-xylanase that is released from the substrate present in the incubation mixture in 1 min of 4 234 607 xylan fragments dissolved in a 2: 1 mixture of ethyl alcohol and water containing 1% of the total amount of xylan. the dye of the 1-amino-2-sulfo-4- (β-sulfatoethylsulfonylanilino)] anthraquinone disodium salt bound to the polymer substrate. Example 2 0.3 ml volumes of a solution of 5 mg xylan with 13.6% covalent unsaturated dye of 1-amino-2-sulfo-4-N- (2-a-sulfatoethylsulfonylanilino)} anthraquinone disodium salt in 0.1 mol. "Citrate buffer (compositions: citric acid and sodium citrate) pH 4.8, are mixed with 0.2 ml volumes of an endo-1,4-β-oxy-lane solution. After incubation at 30 ° C for 5 to 180 min, the reaction is stopped by adding 1 mL of 96% ethyl alcohol. Subsequent mixing is allowed to stand for 10 min at 25 ° C, stirred again, and the precipitate centrifuged. Supernatants are photometrically measured at 595 nm against the supernatant recovered after precipitation of the enzyme-free mixture. Example 3

Xylan containing 18.0% covalently bound 1-amino-2-sulfo-4- [E - (2-sulfatoethylsulfonylamino) 3] anthraquinone covalently bound dye salt was dissolved in 0.05 M acetate buffer, pH 5.4 at 0 ° C. %, 4 wt. To 0.5 ml of this solution, 5-50 µl volumes of an endo-1,4-xylanase solution are added, mixed and incubated at 40 ° C for 5 to 180 min. After the reactions were stopped by the addition of 1 ml of 96% by volume of ethanol, the procedure was continued as in Example 2.

The invention has utility in biochemistry in characterizing enzyme preparations and determining endo-1,4-xylanase activity.

Claims (1)

OBJECT OF THE INVENTION 234 807 Method for determining the activity of endo-1,4-β-oxylanase, characterized in that 4-O-methyl-D-glucurono-D-xylan containing 4 to 20% of covalently bound dye of 1-amino-2-sulfo-4- The anthraquinone [4- (2-sulfatoethylsulfonylanilino)] - is dissolved at a concentration of 0.2-2.0% by weight. at 50-90 ° C in water or in a buffer of pH 4-6, and incubated in a volume of 0.5-2.0 ml with endo-1,4-xylanase solution at 20-50 ° C for a period of time. 5 to 180 min, at which time the enzyme reaction is stopped by adding two volumes of ethanol to precipitate a non-hydrolyzed portion of the substrate which is thus separated from the soluble colored fragments released by the enzyme, the amount of which is proportional to the enzyme concentration in the reaction mixture and were determined photometrically at 580 to 610 nm.