CS254164B1 - A method of preparing test tablets for determining β (1 → 3) glucanase - Google Patents

Abstract

The purpose of the solution is to prepare test tablets for the determination of beta(l->3) glucanase enzyme activity. The stated purpose is achieved by mixing a powdered chromolytic substrate of beta(l->3) glucan isolated from the cell walls of the yeast Saccharomyces cerevisiae containing 6 to 8 wt. % of covalently bound dye with a swelling volume in water of 12 to 20 ml. g ' 1 with microcrystalline cellulose cross-linked with epichlorohydrin in a weight ratio of 1 : : 2 to 1:3 and finally, after adding 2 wt. % of talc and homogenization, it is tableted on a tabletting machine into tablets weighing 100 mg + 3 mg. The method of preparing test tablets for the determination of beta-glucanase has applications in biochemistry.

Description

The invention relates to a method for preparing test tablets for the determination of (3 (1- + 3) glucanase; EC 3.2.1.6.

In recent years, an appropriate chromolytic polysaccharide substrate has been successfully used to determine polysaccharide hydrolase enzyme activities, which, after enzymatic hydrolysis, yields a colored titrate, the intensity of which is proportional to the enzyme activity present. On this principle are known abroad and Czechoslovak. chromogenic substrates such as the Phadebas Amylase Test [Clinical and Technical Information Almwuist, Wiksells Boktryckeri (1979)], or Cs. AO 192 202 and 192 210, which are used, in particular, in clinical practice to determine alpha amylase activity in biological fluids for diagnostic purposes. Similar methods are known for the preparation of cellulosic chromolytic substrate, whether for measuring the C _t component of cellulase activity [AO Stamm: Helv. Clin. Chim. Acta 46, 3008 (1963)] or the C 1 components of cellulase activity [RP Poincelot, PR Day: Appl. Microbiol. 23, 5, 875 (1972)]. In serial analyzes, the powder form of the chromolytic substrate is disadvantageous because it is necessary to weigh the chromolytic substrate separately for each analysis, which reduces labor productivity. To this end, the substrates are tabletted in such a way that the test tablet disintegrates readily in contact with an aqueous enzyme solution, the substrate swells several times rapidly, thereby creating the conditions for the enzyme reaction to proceed.

However, we have found that polysaccharide substrates with a covalently bound dye are practically not tabletable, excipients must be added which provide both the appropriate rheological properties of the granulate during tabletting and also do not inhibit the enzymatic reaction itself. In amylase test tablets, this problem is solved by mixing the amylase substrate in the dry state with microcrystalline cellulose, which is inert to amylase enzymes and provides the desired physical properties of both the granulate and the finished test tablet.

However, this method cannot be used successfully in cases of tabletting of β (1 + 3) -linked polysaccharides substrates (3 (1- + 4) respectively), since microcrystalline cellulose is a β (3-1-4) polysaccharide , ie the respective enzyme would react not only with the chromogenic substrate but also with the colorless substrate, thereby obtaining incorrect values of the measured enzyme activity.We have found that the fused microcrystalline cellulose prepared, for example, according to AO 136 062 does not show any substrate properties for? Glucanase and is therefore suitable as an excipient in the preparation of tablet test forms for the determination of β (1- + 3) glucanase.

SUMMARY OF THE INVENTION The powder chromolytic substrate / 3 (1- + 3) glucan isolated from yeast cell walls of Saccharomyces cerevisiae contains 6-8% by weight of the invention. a covalently bound dye of 8-amino-5- [3- (sulfoethylsulfonyl) anilino] 6-anthraquinonesulfonic acid disodium salt and a swelling volume in water of 12 to 20 ml. g- ¹ is mixed with microcrystalline cellulose cross-linked with 10 to 20 wt. epichlorohydrin (2-chloromethyloxirane) in a weight ratio of 1: 2 to 1: 3, and finally 2% by weight of epichlorohydrin is added. talc and after homogenization, it is tabletted on a tabletting machine for tablets weighing 100 mg, with an accuracy of + 3%.

The optimum particle size of both chromolytic substrate / 3 (1- + 3) glucanate and cross-linked microcrystalline cellulose should be in the range of 80 to 250 μτα. In order to sufficiently suppress the substrate affinity of microcrystalline cellulose to β (1- + 3) glucanase, it is necessary for the cellulose to be cross-linked with epichlorohydrin such that it contains 10 to 15 percent bound epichlorohydrin by weight of dry microcrystalline cellulose. The optimal weight of the test tablet is about 100 mg for sufficient accuracy to determine (3 (1- + 3) glucanase activity).

The advantage of a new way of preparing a tablet test to determine the β (1- + 3) glucanase activity is that it allows to increase labor productivity by multiple analyzes of the enzyme activity in question, with high precision of the photocolourimetric method used and without reducing the sensitivity of the method. by using non-crosslinked microcrystalline cellulose as an auxiliary additive to achieve optimal physico-mechanical properties of the test tablets.

Example 1

100 g of chromolytic substrate / 3 (1- + 3) glucan isolated from yeast cell walls of Saccharomyces cerevisiae containing 6% w / w. bound dye disodium 8-amino-5- [3- (sulfoetylsulfonyljanilíno] 6-anthraquinone and vetkosťou particles of 80-250 and a swelling volume of 12 ml water. g ^_1 is homogenized with 200 g of cross-linked microcrystalline cellulose with 20 wt.% epichlorohydrin with the addition of 6 g of talc, whereupon the homogenized dry granulate is tabletted on a tableting machine for tablets weighing 100 mg + 3 mg.The finished tablets are suitable for serial measurement of β (1- + 3) glucanase activity.

Example 2

The procedure of Example 1 is followed except that 300 g of crosslinked microcrystalline cellulose crosslinked with 10 wt. with epichlorohydrin and chromolytic substrate / 3 (1- + 3) glucan containing 8 wt.

5 4 of the bound dye and the swelling volume ml. gi.

The invention can be widely used in biochemistry in the determination of β (1- * 3) glucanase activity, whether for research purposes or in industrial applications.

Claims (1)

Process for preparing test tablets for the determination of β (1 → 3 µg glucanase, EC 3.2.1.6) characterized in that the powdered chromolytic substrate β (1 → 3 µg) glucan isolated from yeast cell walls of Saccharomyces cervisiae containing 6 to 8 wt. covalently: bound dye of 8-amino-5- [3- (suit oethylsulfonyl) anilino] 6-anthraquinone sulfonic acid disodium salt and a swelling volume in water of 20 to 20 ml. epichlorohydrin in a weight ratio of 1: 2 to 1: 3 and then, after adding 2% by weight of talc and homogenizing, it is tabletted on a tabletting machine.