CS219575B1 - A method of preparing a chromogenic substrate for testing a C 1 component of cellulase activity - Google Patents

Abstract

The invention provides for the preparation of a chromogenic cellulose substrate suitable for testing cellulase Ci activity. The essence of the invention lies in the fact that microcrystalline cellulose is dyed in an alkaline medium with a suitable dye, after dyeing the chemically bound dye is washed out and the dyed substrate is dried. Optimal reaction parameters for dye binding as well as an effective method for washing out unreacted dye are presented. The invention is useful in research in determining cellulase Ci activity and in the production of cellulase enzymes.

Description

219 57S (54) Preparation of Chromogenic Substrate to Test CT Fractional Ciasase Activity 1

The invention relates to a process for preparing a chromogenic substrate for testing a C 1 component of a cellulase enzyme activity.

To date, the determination of the C 1 component of cellular activity has been done according to a gradual gravimetric method (Halliwell G .: Biochem. J.79, 1985/1961), wherein the reaction medium containing the Cx component of the cellular activity is left behind incubate with the filter paper for a sufficient period of time under standard conditions and then wash the filter paper from hydrolyzed soluble products in enzyme hydrolysis, dry the papers and weigh and calculate the enzyme activity of C 1 cellulose present in the test medium or dissolve the cellulose to dissipate determined in the filtrate by oxidation with dichromate (Selby K., Maitland CC: Biochem. J. 104, 716/1967). This method is mainly very time-consuming and is not suitable for serial analyzes. In recent years, the polysaccharide-hydrolase enzyme activity has been associated with a chromogenic polysaccharide substrate, which, after enzymatic hydrolysis, produces a colorate filtrate, the intensity of which is proportional to the enzyme activity present.

On this principle are known foreign MS. the chromogenic substrates Phadebas Amy-lase Test, (Clinical and Technical Information, Almquist, Wiksells Boktryckeri (1979), AO No. 192 202, AO No. 192 210), which are successfully used in clinical practice for the determination of alpha-amylase activity in biological fluids for diagnostic purposes.

So far, the preparation of the chromogenic substrate for cellulase activity has been prepared by a lengthy and energy intensive procedure by phosphoric acid-prepared hydrocellucose being stained at 50 ° C with Remazolbriliant blue using 200% sodium sulphate to a weight of hydrocelulose at pH 11 a the use of sodium phosphate (AOStamm: Helv. Chim. Acta 46, 3008 1963).

However, the substrate thus prepared is particularly suited for the Cx component of the cellulase activity and not a component of the C.

A similar process based on the dyeing of Whatman 1 filter paper (Polnolot R. P., Day P. R .: Appl. Microblol. 23.5, 875/1972) is also energy intensive since it operates at 100 ° C.

In the selection of microorganisms producing the Cx component of cellular activity, it is desirable to have a very precise and rapid C! components of cellulase activity, in order to precisely monitor the culture conditions and resp. to evaluate reliably high-producing mutants. In pre-operation, this is important in the management of industrial culture for maximum production of Ciceiulase activity.

Therefore, we have developed conditions for the preparation of a chromogenic substrate with the desired high-sensitivity properties of cellulase activity, which is a subject of the present invention. /

The essence of the invention is that the wood-cellulose microcrystalline cellulose, preferably made of cotton, is reacted in a first step for 30 minutes at 15-30 ° C in a 5-7 weight% aqueous solution of 0.7-1.5. % sodium hydroxide and 5 to 10 wt. sodium sulfate or sodium sulfate under constant stirring, and in the second stage adds 5 to 10% by weight. dye with the preferred sodium-8-amino-5 - (- 3 / sulphomethylsulphonyl / anilino) -6-anthraquinone sulphonic acid salt and react for 1 to 3 hours at 15 to 30 ° C, and at the third stage the chromogenic microcrystalline cellulose is washed out of the unreacted dye first with water, then sequentially with dilute ethanol to concentrated ethanol or acetone, and dried in air and otherwise at 60 to 70 ° C.

The most suitable substrate for C 1 cell activity is cellulose with a maximum crystalline fraction and therefore microcrystalline cellulose prepared by partial acidic heterogeneous hydrolysis of cotton is most suitable. It represents a purer cellulose with microflora-extractive substances, ash and proportion of mamorphic cellulose at a maximum of 10 to 15%. However, it is also possible to use microcrystalline celluloses prepared from hardened wood-cells, preferably with a lignin content (not higher than 0.1%). The optimum polymerization degree of the microcrystalline celluloses used is 150 to 250. The polymerization degree below 100 is not suitable for worse filtration in enzyme analysis, in fact, the preparation of a colored substrate.

Optimally, the amount of dye applied to the reaction is about 10% by weight of dry mlcrystalline cellulose. In lower amounts, the substrate is inadequate in the enzymatic reaction and, at higher levels, the substrate is poorly filtered and also the crystalline portion of the cellulose decreases so that it is a good substrate for the Cscelulase components. The efficiency of the dye binding reaction to microcrystalline cellulose is 50-70% and the reaction proceeds sufficiently rapidly at room temperature, which does not cause cellulosic substance to be degraded in an alkaline medium.

As an electrolyte enhancing binding efficiency

Claims (1)

Cellulose dye 219 575 is preferred to use sodium acetate, which is better eluted from the product than sodium sulfate, especially when the ethanol gradient is graded. acetone. An advantage of the process according to the invention is that it allows the first time to prepare a chromogenic substrate suitable for the serial measurement of the enzyme activity of the C 1 cellulose component. a trophotometric method, a method of preparing a chromogenic substrate is a technologically and energy-saving, chromngene substrate is stable and can store unlimited time in a dry state, 50-100 mg of chromogenic substrate is sufficient for one determination of the Cicellulase component activity, the microcrystalline chromogenic substrate may be also determine the low Ci of the cellulose activity within 2 hours. EXAMPLE 1 100 g of microcrystalline cellulose from cotton are reacted in 500 ml of water for 30 minutes at room temperature in which 5 g of sodium hydroxide and 50 g of sodium sodium goctane have been pre-dissolved. Then, 10 g of dye 8-amino-5 - (- (3) sulfoethylsulfonyl / aniline] -6-anthraquinone sulfonic acid dye salt is added in portions of the reaction mixture to complete the addition up to 30 minutes. The mixture is stirred at room temperature for 2 hours, then 7 ml of conc. acetic acid are added and the reaction mixture is filtered off and washed on the filter first with distilled water, followed by successive addition of water and ethanol with increasing concentration gradient of ethanol or The washed chromogenic substrate is dried free of air or under vacuum at 60-70 ° C. The resulting product contains 5-7% bound dye and is a good substrate for the cellulase activity. Example 1, except that 5 g of dye is applied to the reaction, and the resulting product contains 3 to 3.5% bound dye, ie almost half-sensitive sensitive to the same C 1 cellulose activity. EXAMPLE 3 The procedure of Example 1 with the rozdielomže as auxiliary electrolyte used is 50 £ sodium sulfate. The resulting product has a similar bound dye content as in Example 1. EXAMPLE 4 The procedure of Example 1 except that the cellulose is cellulose microcrystalline cellulose cellulose used for viscose fibers. The resulting product contained 5.5% bound dye. EXAMPLE 5 The procedure of Example 1 except that microcrystalline cellulose zei sputtered sulphite cellulose is used as cellulose s-a with a grading degree of 220 which is very good to filter during processing. The dye coating in the finished substrate is 6 ° / o. The invention has utility in the serial analysis of the precise and abundant determination of cellulase activity in the breeding of C 1 -cellulose-producing microorganisms and in the industrial cultivation of microorganisms producing the claimed enzymes. OBJECT OF THE INVENTION A method of preparing a chromogenic substrate for testing the Ch cellulase activity component, characterized in that the wood cellulose microcrystalline cellulose with the advantage of cotton is reacted in the first stage for 30 minutes at a temperature of 15 to 30 ° C in 5 to 7- % by weight of the aqueous composition of the composition at 0.7 to 1.5% by weight. sodium hydroxide 1 and 5 to 10 wt. % sodium or sodium sulphate while stirring, whereupon 5 to 10 wt. dye, preferably 8-amino-5 - (- 3 / sulfoethylsulfonyl) anilino) -6-anthraquinone sulfonic acid dibasic salt, by weight of microcrystalline cellulose and does not react for 1 to 3 hours at 15 to 30 ° C; in the third stage, the sachromogenic microcrystalline cellulose is continuously washed away from the unreacted dye by water, then successively diluted with ethanol to concentrated ethanol or acetone, and finally washed with product-free air-drying or vacuum pressure of 60-70 ° C. SOUP 1194-84