CS274026B1 - Two-sodium salt of 0-(4-chloro-6-/benzenase-2-(1-hydroxy-3,6-bisulfonate-8-yl)amino/-1,3,5-triazine-2-yl)d-galacturonane with relative polymerization degree 10 and method of its preparation - Google Patents

Abstract

The invention concerns two-sodium salt of 0-/4-chloro-6-[benzenazo-2-(1-hydroxy-3,6-bisulphonate-8-yl)amino]-1,3,5-triazine-2-yl/D-galacturonane with relative polymerisation degree of 10, a coloured water soluble substrate, usable for detection and specification of the activity of polygalacturonase in preparations containing pectolytic enzymes and the method of their preparation The essence of preparation is based on the fact that 1 mass part water soluble oligo-D-galacturonic acid with relative polymerisation degree of 10 or its salt, possibly potassium salt, reacts in an aqueous solution with a content of 0.8 to 1.2 mass parts sodium or potassium carbonate in the presence of alkaline salt of acetic acid or mineral acid with 0.6 to 1 mass parts two-sodium salt of 2,4-dichloro-6-[benzenazo-2-(1-hydroxy-3,6-bis-sulphonaphth-8-yl)amino]-1,3,5-triazine, the occurring solution of coloured D-galacturonane is neutralised by acetic acid or mineral acid, 1 volume part neutralised solution is precipitated with 3.5 to 4.5 volume parts of a solution containing 2 to 3 volume parts 96% ethyl alcohol and 1 to 2 volume parts acetone and unbound dichlorotriazine dye is removed by ablation of the precipitate with a solution containing 2 to 3 volume parts 96% ethyl alcohol, 1 to 2 volume parts acetone and 1 volume part 0.6 to 1% aqueous solution of alkaline salt of acetic acid or mineral acid.<IMAGE>

Description

The present invention relates to the disodium O-β-4-chloro-6-benzenazo-2- (1-hydroxy-3,6-bissulfonat-8-yl) amino-1,3,5-triazin-2-yl D-galacturonate. about the average degree of polymerization i, and the process for its preparation.

Soluble covalently colored polysaccharides have recently been used successfully to quickly and easily detect the respective polysaccharide hydrolases and are also useful for determining their activity. Mc Cleary: Carbohydr. Res. 86, 97 (1980); P. Biely et al., Anal. Biochem. 144: 147 (1985); P. Biely et al .; 172, 176 (1988)]. The dye usually binds the polysaccharide directly to the hydroxyl group via an ether bond or to a derivatized polysaccharide. The principle of detection and determination of enzyme activity is to alter the solubility of colored polysaccharides as a result of enzyme hydrolysis and subsequent formation of low molecular weight colored fragments soluble in the presence of an organic solvent. In the case of pectin-degrading enzymes with such a substrate, the covalently stained pectin is the azo dye N- {1 - 1 - [(3,6-disulfo-1-naphthyl) azo] -naphthyl-ethylenediamine, prepared by D. R. Fried and G. W. Chang / J. Agric. Food Chem., 30 (5) (1982) 982), wherein the dye is coupled to pectin via an amide group to form a poorly available water-soluble carbodiimide catalyst. Another kind of colored substrate for pectinases is the sodium salt of 2- [3- (1-amino-2-sulfoanthraquinon-4-ylamino) phenysulfonyl] ethyl-ethylenediamine pectin derivative / J.S. Huang, J. Tang: Anal. Biochem., 73, 369 (1976)]. It is prepared in two stages. First, the pectin ethylenediamine derivative is prepared and the dye is then bound to the dye. However, this substrate is characterized by poor solubility in buffers.

Conventional dyeing techniques dye the polysaccharide which do not require its derivatization, in the case of pectic acid, do not provide a substrate with the desired properties. In this way, a very low degree of dye substitution is achieved with pectic acid. The substrate obtained does not yield, by enzymatic cleavage, low molecular weight colored fragments that would allow its use in the detection and determination of enzyme activity. Only low molecular weight uncoloured fragments are mentioned by the enzyme, while the dye remains bound to a polymer residue which is insoluble in 60% ethyl alcohol.

The above-mentioned disadvantages are substantially eliminated by the water-soluble chromogenic substrate O- {4-chloro-6- [benzenazo-2- (1-hydroxy-3,6-bissulfonaphth-8-yl) amino] -1,3,5-triazine disodium salt. 2-yl of D-galacturonan with an average degree of polymerization of 10 of formula I

It was found that said disodium salt of O- {4-chloro-6- [benzenazo-2- (1-hydroxy-3,6-bissulfonaphth-8-yl) amino] -1,3,5-triazin-2-yl The D-galacturonan with an average degree of polymerization of 10 can be prepared by the following process according to the invention, which consists in that 1 wt. % by weight of water-soluble oligo-D-galacturonic acid having an average degree of polymerization of 10 or its sodium or sodium olefin. potassium salt is reacted in an aqueous solution containing 0.8 to 1.2 wt. parts by weight of sodium or potassium carbonate in the presence of an alkali salt of acetic acid or a mineral acid with 0.6 to 1 wt. portion of 2,4-dichloro-6- [benzenazo-2- (1-hydroxy-3,6-bissulfonaphth-8-yl) amino] -1,3,5-triazine disodium salt (dichlorotriazine dye). The resulting solution stained with D-galaxy. The urethane is neutralized with acetic acid or mineral acid. 1 vol. part of the neutralized solution precipitates with 3.5 to 4.5 vol. parts of a solution containing 2 to 3 vol.

CS 274026 B1 parts of 96% ethyl alcohol and 1 to 2 vol. parts of acetone. Unbound dichlorotriazine dye is removed by washing the precipitate with a solution containing 2-3 vol. parts of 96% ethyl alcohol, 1 to 2 vol. parts of acetone and 1 vol. % of a 0.6 to 1% aqueous solution of an alkali salt of acetic acid or a mineral acid. Preference is given to using an acid whose salt is used during the reaction. For the use of disodium O-4 4-chloro-6-benzenazo-2- (1-hydroxy-3,6-bissulfonaphth-8-yl) amino] -1,3,5-triazin-2-yl] - D-galacturonan having an average degree of polymerization of 10 as a substrate for the detection and determination of pectinase activity, which is carried out in acetate buffer, is preferably used in the reaction of sodium acetate and acetic acid in neutralization.

The advantage of disodium O-β-4-chloro-6-benzenazo-2- (1-hydroxy-3,6-bissulfonaphth-8-yl) amino] -1,3,5-triazin-2-yl-β-D-galacturonate having an average degree of polymerization of 10 according to the invention is to obtain a colored substrate soluble in the buffers in which the enzyme reaction is carried out. A further advantage is that by enzymatic cleavage by polygalacturonase at the first attack, it releases colored fragments (DP 5), which remain dissolved in the supernatant after precipitation of the undigested substrate with an organic solvent.

These examples illustrate but do not limit the scope of the invention.

Example 1 g of oligo-D-galacturonic acid having an average degree of polymerization of 10 is dissolved in 40 ml of distilled water at 30 DEG C. and insoluble fractions are filtered off. 1 g of dichlorotriazine dye dissolved in 6 ml of water is then added to the filtrate and stirred for 10 minutes at 30 ° C. 0.5 g of sodium acetate is then added and the mixture is again stirred for 10 minutes at the same temperature. Finally, 12 mL of a 6% aqueous sodium carbonate solution was added and stirred at 30 ° C for 2 hours. The reaction mixture is then neutralized with 2M acetic acid to pH 7 and precipitated with a solution containing 120 ml of 96% ethyl alcohol and 120 ml of acetone. The precipitate is filtered off under suction and washed with a solution containing 1 part of a 0.6% aqueous solution of sodium acetate, 2 parts of 96% ethyl alcohol and 2 parts of acetone until a slightly colored filtrate. The precipitate is then precipitated by dissolving it in 50 ml of a 0.6% aqueous sodium acetate solution and then precipitating it with a solution containing 100 ml of 96% ethyl alcohol and 100 ml of acetone. Wash as above until colorless filtrate. The precipitate was washed with ethyl alcohol, acetone and dried.

1 g of precipitate was obtained with a dye content of 5.6%.

Example 2 g of Na-D-galacturonan having an average degree of polymerization of 10 is dissolved in 200 ml of water at 40 DEG C. and 5 g of dichlorotriazine dye dissolved in 40 ml of water are added and stirred for 10 minutes at the same temperature. Then 2.5 g of sodium acetate and 60 ml of 6% potassium carbonate are added successively and stirred for 2 hours at 40 ° C. The reaction mixture was cooled, neutralized with 2M acetic acid and precipitated with stirring with a solution containing 300 ml of 96% ethyl alcohol and 300 ml of acetone. The precipitate is filtered through suction and eluted as in Example 1. It is then precipitated, washed and dried in the same manner as in Example 1.

4.8 g of precipitate were obtained with a dye content of 6%.

The invention can be used in the food, textile and tobacco industries for the detection and determination of polygalacturonase activity in preparations containing pectolytic enzymes.

Claims (2)

OBJECT OF THE INVENTION 1. D-Galacturonan O-β-4-chloro-6- [benzenazo-2- (1-hydroxy-3,6-bissulfonaphth-8-yl) amino] -1,3,5-triazin-2-yl disodium salt with an appropriate degree of polymerization 10 of formula I 2. A process for the preparation of a compound according to claim 1, wherein 1 part by weight of water-soluble oligo-D-galacturonic acid having an average degree of polymerization of 10 or its sodium or potassium salt is reacted in an aqueous solution containing 0.8 to 1. 2 parts by weight of sodium or potassium carbonate in the presence of an alkali salt of acetic acid or a mineral acid with 0.6 to 1.0 part by weight of the disodium salt of 2,4-dichloro-6-benzenazo-2- (1-hydroxy-3,6- bissulfonaphth-8-yl) amino] -1,3,5-triazine, the resulting solution of colored D-galacturonan is neutralized with acetic acid or mineral acid, 1 volume of the neutralized solution precipitates with 3.5 to 4.5 volumes of a solution containing 2 up to 3 parts by volume of 96% ethyl alcohol, and 1 to 2 parts by volume of acetone and unbound dichlorotriazine dye are removed by washing the precipitate with a solution containing 2 to 3 parts by volume of 96% ethyl alcohol 1 to 2 parts by volume of acetone and 1 part by volume of a 0.6 to 1.0% aqueous solution of an alkali salt of acetic acid or mineral acid.