CS237101B1 - Preparation method water-soluable chromolytical polasacharide ferment substrate - Google Patents

Abstract

A method of preparing water soluble chromolytic polysaccharide enzyme substrates. The purpose of the invention is to prepare water soluble chromolytic polysaccharide substrates suitable for enzyme measurements activities. This purpose is achieved by being insoluble polysaccharides are most derivatized by alkylation to water-soluble products, which in the second stage stain with dye Remazol brilliant blue R and after staining to remove the physically bound dye washing or extraction with methanol. Prepared chromolytic substrate with content covalently bound dye 3 to 8 wt. it dissolves very well in water and thus can serve as a water-soluble substrate for measuring polysaccharide hydrolase enzyme activities. The invention has utility where needed The activity of polysaccharide-hydrolase is rapidly and enzymes Interoperative control of enzyme production or when measuring enzyme activity for diagnostic purposes.

Description

The invention relates to a process for the preparation of water-soluble chromolytic polysacbaride enzyme substrates suitable for determining the respective polysaccharide hydrolase enzyme activities.

The photocolourimetric method is the most effective and therefore the most widely used method of determining polysaccharide hydrolase enzyme activities in recent years. It consists in reacting the enzyme with an insoluble polysaccharide gel to which a suitable dye is covalently bound. During hydrolysis by the respective polysaccharide hydrolase present, the colored polysaccharide gradually progresses into solution, the color intensity of the solution being proportional to the enzyme activity.

Understandably, this is only assuming that the polysaccharide ohromolytic gel is perfectly accessible to the enzyme molecule and that the co-bound dye on the polysaccharide has no inhibitory effect on the enzyme reaction itself [J. Kennedy: Advances in Carbohydrate Chemistry and Biochemistry, 29, (1974) p. 350 to 357],

The dye-binding reaction to the polysaccharide must be conducted so that the distribution of the dye molecules is homogeneous on the polysaccharide macromolecule, which can be achieved by the correct choice of the reaction conditions in the dyeing of the polysaccharide.

Methods are known in which the dye-binding reaction, in particular to the soluble polysaccharide, is carried out in solution and, after coloring, the soluble polysaccharide is converted into an insoluble form by crosslinking. in some cases it can also be used in soluble form. In this case, the polysaccharide is separated from the reaction solution by dialysis and precipitation with an organic solvent [MSkinen®]. K., Paunio I. K .: Anal. Biochem. 39, 202 (1971)]. The above method was used for staining with "Blue Dextran 2000", used as a color standard for gel testing by gel permeation chromatography.

For diagnostic purposes, a method for determining alpha amylase activity in biological fluids using a stained soluble amylopectin under the name DYAMYL is known [Babson, S. R., Babson, A.L .: Clinica Chimica Acta 44, 193 (1973)]. Reacton Rot 2M-Geigy dye is used as a dye in this chromolytic substrate, with an absorbance maximum of 546 nm.

In most cases, insoluble chromolytic substrates, screened together with different cross-linking reagents and a different degree of swelling in water, have hitherto been used to determine enzyme activities.

A certain general disadvantage of such substrates was that a significant excess of substrate was needed for each assay, ranging from 50 to 150 milligrams per assay. Of course, if a soluble substrate is used, it is possible to determine the enzyme activity of the respective enzyme using 5 mg of soluble chromolytic substrate, i. j. with a substrate amount of at least 10 times lower. This is particularly important in determining those enzymes where substrates are relatively difficult to obtain, cost-intensive, or in cases where routine diagnostic practice requires a relatively high number of enzymatic allectivity assays to be performed daily.

For soluble polysaccharides such as e.g. glycogen, α-mannan, pululan, pectic acid and others, it is usually not a problem to prepare a water-soluble chromolytic substrate, since the correct choice of degree of dye substitution will preserve the solubility of the colored polysaccharide.

This is more complicated, as our research has shown on insoluble polysaccharides such as cellulose, starch, lichenate, chitin, xylan, β-mannan and others. Even by repeated substitution of the applied dye, an ohromolytic, good soluble polysaccharide is usually not obtained. In addition, at a higher degree of coloration of the polysaccharide, the talkmer can periodically observe a significant inhibitory effect of the bound dye on enzyme hydrolysis, which would substantially reduce the sensitivity of the enzyme reaction.

The above-mentioned drawbacks of the preparation of soluble chromolytic polysaccharide substrates suitable for the determination of polysaccharide hydrolase activities are solved by the new application according to which a soluble enzyme substrate can be prepared from an insoluble polysaccharide which meets all the conditions for measuring the respective enzyme activity.

SUMMARY OF THE INVENTION In the first step, the insoluble polysaccharide is alkylated in an alkaline medium with ethylene oxide and / or monochloroacetic acid and / or propylene oxide, and / or an alkyl halide having a carbon number of 1-2 and / or sodium β-chloroethyl sulfonate to such a degree. substitution in which the polysaccharide is already water-soluble, whereupon the remazol dye Remazol briliant blue R (disodium salt of 8-amino-5- [3- (sulfoethylsulfonyl) anilino] 6-anthraquinonesulfonic acid) is bound to the alkyl polysacbarld in the second step ) in an amount of 3 to 10% by weight per weight of polysaccharide at a temperature of 15 to 25 ° C and a sodium hydroxide concentration of 0.7 to 1.0% by weight for 30 to 90 minutes, and in a third step the alkylated chromolytic polysaccharide precipitates from solution alcohol, acidified with acetic acid to neutralize the free alkali in the reaction mixture and finally the colored polysaccharide is freed from physically bound dyes and washing or extracting with methanol.

iPokial refers to the height of the degree of substitution which determines the good solubility of the polysaccharide in water, which depends on the type of polysaccharide and the alkylating reagent. So eg. carboxymethylcellulose becomes soluble in water at a degree of substitution of 0.4, while starch is already soluble at a degree of substitution of 0.2. In the alkylation of polysaccharides with ethylene oxide to achieve water solubility, it is only necessary to substitute the polysaccharides for a degree of substitution of 0.2 to 0.3, and in the alkylation of sodium β-chloroethyl sulfonate to only 0.1 to 0.2.

The alkylation is carried out in the presence of 20 to 40% by weight of sodium hydroxide. at temperatures of 50 to 60 ° C for 2 to 4 hours. After the necessary alkylation, the reaction mixture is diluted with water so that the sodium hydroxide concentration reaches 0.8 to 1.0% by weight, at which concentration the dye is added in portions so that the dye dosing is complete within 20 minutes and after addition of the total. the reaction mixture is allowed to react for a further 60 minutes at 15-20 ° C. The colored polysaccharide is then precipitated with an acidified alcohol. The amount of acid is selected such that it is only sufficient to neutralize the free sodium hydroxide. The precipitated chromolytic polysaccharide is then scrubbed or extracted preferably with methanol, optionally with a methanol-acetone mixture, until quantitative removal of the physically bound dye to the polysaccharide.

For polysaccharides that are already soluble without alkylation, the dye can be bound directly in an alkaline medium. However, it is necessary to select an amount of dye which does not reduce the solubility of the finished product, which is about 3-10% by weight. by weight of dry polysaccharide.

In particular, the advantage of the proposed method is that it also enables water-insoluble chromolytic substrates meeting all the criteria of enzyme substrates suitable for measuring the respective enzyme activities from insoluble polysaccharides by prior alkylation followed by covalent bonding of the dye to the polysaoharide. Further advantages are that the prepared substrates are more insoluble to the enzyme reactions than the insoluble substrates and therefore 10 times less substrate are required for analysis with the same assay sensitivity. An essential advantage is the possibility of determining activity in a substantially smaller sample volume than in the case of insoluble twisted substrates.

Further advantages and a more precise description of the novel process are evident in the following examples, which are not exhaustive, but merely illustrative.

EXAMPLE 1 1 g of powdered cellulose of the polymerization stage 650 is suspended in 600 ml of isopropanol, to which 20 ml of 25% by weight sodium hydroxide solution have been previously emulsified. The reaction mixture is stirred at room temperature for 1 hour, then 8 g of sodium chloroacetate is added and the reaction temperature is raised gradually to 60 ° C with stirring for 1 hour, at which time it is left to react for 3 hours. The insoluble carboxymethylcellulose is suction filtered and washed with alcohol. The well aspirated carboxymethylcellulose is poured in a beaker of 300 to 400 ml of distilled water in which the cellulose is dissolved and added, after dissolution, in portions, a total of 1.3 g of Remazol briliant blue R and allowed to react with stirring for 1 hour with gradual addition of acetate. sodium total 8 g. After completion of the reaction, the solution is slowly poured into 4 liters of alcohol (with vigorous stirring) acidified with an amount of acetic acid just enough to neutralize the free sodium hydroxide in the reaction solution of chromolytic carboxymethylcellulose. The precipitated product is filtered off and washed on the filter with methanol, respectively. extract with methanol in a Soxleth extrafector.

The finished chromolytic product, water-soluble hydroxyethylcellulose contains 5% bound dye and a degree of substitution with monochloroacetic acid of 0.42.

Example 2

As in Example 1, only chitin is used as a polysaccharide instead of cellulose.

The resulting chromolytic carboxymethylchitin product has 5.5% bound dye and a degree of substitution with monochloroacetic acid of 0.48.

Example 3

The procedure as in Example 1 except that the polysaccharide used is a lichenate and a dye amount of 1.7 g.

The resulting chromolytic carboxymethyllichenan product has 6.2% bound dye and a monochloroacetic acid substitution degree of 0.44.

Example 4

Procedure as in Example 1 except that xylan is used as the polysaccharide.

The resulting chromolytic carboxymethylxylan product contains 4 wt. of the bound dye and the degree of substitution with monochloroacetic acid 0.38.

Example 5

The process of Example 1 except that potato starch and an amount of sodium chloroacetate of only 4.5 g and a dye amount of 1.7 g were used as the polysaccharide.

The resulting chromolytic carboxymethyl starch product contains 7 wt. bound dye and degree of substitution with monochloroacetic acid 0.25.

Example 6

The procedure of Example 1 except that 2.5 g of ethylene oxide was used as the alkylating reagent and the reaction was run at 40-50 ° C for 2 hours.

The resulting hydroxyethylcellulose derivative is further modified by binding the dye of Example 1.

The rotary product, a chromolytic water-soluble hydroxyethyl cellulose derivative, with a degree of substitution of 0.3 relative to hydroxyethyl and a bound dye content of 4.3% is suitable as a soluble substrate for determining cellulose activity.

Example 7

The process of Example 6, except that 3-mannan was used as the polysaccharide.

The soluble chromolytic substrate has similar properties in terms of degree of sabstitution and amount of bound dye as in Example 6 and is suitable for measuring mannase activity.

example

The procedure of Example 6, except that a lichenate is used as the polysaccharide.

The resulting hydroxyethyllichenan product with a covalently bound dye is a suitable soluble substrate for determining lichenase activity.

Example 9

The process of Example 6 except that potato or corn starch is used as the polysaccharide.

The resulting chromolytic hydroxyethyl starch product is a suitable soluble substrate for determining alpha amylase activity.

Example 10 g of sodium pectic acid is suspended in 200 ml of isopropanol and 5 ml of 5% w / w sodium hydroxide are added with stirring. After 30 minutes add 0.8 g Remazol brilliant blue R and stir at 15-20 ° C for 1 hour.

The resulting product, water-soluble chromolytic sodium pectate, is a good substrate for determining endopolygalacturonase activity.

Example 11

The procedure as in Example 10, except that the α-mannan is used as the alcohol polysaccharide.

The resulting product, chromolytic water-soluble N-mannan is a suitable enzyme substrate for determining N-mannanase activity.

Example 12 g of methylcellulose with a methoxy content of 28% by weight. is suspended in 100 ml of ethanol and 5 ml of a 2% w / w aqueous solution of sodium hydroxide is added with stirring. and the reaction mixture is allowed to stir for 30 minutes at room temperature, then 1.0 g of Remazol briliant blue R is added in portions and allowed to react with stirring at 15 to 20 ° C for 1 hour. After 1 hour, the alkali present is neutralized with acetic acid and the unreacted dye is eluted or extracted as in Example 1. The resulting product, water-soluble chromolytic methylcellulose is a very good substrate for measuring cellulase enzyme activity.

Example 13 g of potato starch is suspended in 100 ml of isopropanol, in which 8 ml of a 40% strength by weight aqueous sodium hydroxide solution have been previously dissolved. After homogenization of the reaction mixture, 3.5 g of sodium 3-chloroethylsulfonate was added and the reaction mixture was heated to 55 ° C, where the temperature was allowed to react for 90 minutes. Cool to 20 degrees Celsius and add 1.7 g of Remazol brilliant blue R and react for 1 hour with stirring. Thereafter, the chromogenic water-soluble sulfoethyl starch is filtered and washed, or extracted with methanol from the unreacted dye.

The finished substrate is very good for determining alpha amylase enzyme activity.

Example 14

The procedure of Example 1 except that 3.0 g of propylene oxide was used as the alkylating reagent and the reaction mixture was allowed to react at 55 ° C for 2 hours.

The resulting hydroxypropylcellulose derivative is further modified by binding the dye of Example 1.

The finished product, a chromolytic water-soluble derivative of hydroxypropylcellulose, with a degree of substitution of 0.35 and a bound dye content of 5.2% by weight, is suitable as a soluble substrate for the determination of cellulose activity.

Example 15 g of water-soluble ethylcellulose with a degree of substitution of 0.58 is suspended in 100 ml of methanol, in which 5 ml of an aqueous solution of sodium hydroxide at a concentration of 2% by weight are previously dissolved.

Allow the reaction to stir for 30 minutes at room temperature, then add 1.0 g of Remazol brilliant blue R in portions and allow to react with stirring at 15 to 20 ° C for 1 hour. After 1 hour, the alkali present is neutralized with acetic acid and the unreacted dye is eluted or extracted as in Example 1.

The resulting product, water-soluble chromolytic ethylcellulose is a very good substrate for measuring cellulosic enzyme activity.

The invention has widespread application wherever it is necessary to determine enzyme polysaccharide hydrolase activities, either in the in-process control of the production of individual enzymes or in the diagnostic determination of enzyme activities in biological fluids.

Claims (1)

237101 at room temperature, then 1.0 g of Remazol brilliant blueR is added in portions and allowed to react with stirring at 15-20 ° C for 1 hour. After 1 hour, the alkali present is neutralized with acetic acid and the unreacted dye is washed out or extracted as in Example 1. The resulting product, water-soluble chromolytic ethylcellulose, is a very good substrate for measuring cellulose enzyme activity. The present invention has widespread use wherever it is necessary to determine enzyme polysaccharide-hydrolase activities, whether in the intermediate control of the production of individual enzymes, or in the diagnostic determination of enzyme activities in biological fluids. A method for the preparation of water-soluble chromolytic polysaccharide enzyme substrates, characterized in that in the first step the insoluble polysaccharide is alkylated with a alkaline medium with ethylene oxide and / or monochloroacetic acid, and / or bopropylene oxide, and / or alkyl halides, and / or sodium 5-chloro-ethylsulfonate to a degree of substitution in which the polysaccharide is already water-soluble, whereby in the second step the alkyl polysaccharide binds the remazol color Remazol brilliant blue R (disodium salt 8-amino- 5- [3- (sulfoethylsulfonyl-anilino] -6-anthraquinone sulfonic acid) in an amount of 3 to 10% by weight of polysaccharide at 15 to 25 ° C and sodium hydroxide concentration of 0.7 to 1.0% by weight for 30 to 90 minutes and, in a third step, the alkylated chromolytic polysaccharide precipitates from the solution with alcohol-acidified acetic acid to neutralize the free alkali in the reaction mixture and finally the dyed polysaccharide is freed from physically bound dye by washing or extraction with methanol.