Method for production of protheolytic reagent for structural protein hydrolysis

FIELD: biotechnology, in particular reagent for structural protein hydrolysis.

SUBSTANCE: method for production of protheolytic reagent includes cultivation of producer strain selected from dermatophyte of species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Microsporum canis, Microsporum gypseum, preferably from fungus strains capable to produce complex of structural protein proteinases (scleroproteases) with total protheolytic activity at least 0.7 U/mg including collagenase 0.1-4.5 U/mg; keratinase 0.1-0.5- U/mg; elastase 0.5-1.9 U/mg. Cultivation is carried out, for example, in wort agar-agar or in wort broth for 20-24 days.

EFFECT: scleroproteases with improved specific activity; method for protheolytic cleavage of specific substrates (scleroproteins) with increased rate and depth.

2 dwg, 12 ex

 

The invention relates to biotechnology, in particular to a method for proteolytic preparation for hydrolysis of structural proteins: collagen, keratin, elastin. Structural proteins - fibrillar proteins, scleroprotein is the most resistant to physico-chemical factors and are not subjected to proteolysis by digestive enzymes of humans and animals. Protease structural proteins in contrast to other proteolytic enzymes are able to hydrolyze these proteins are characterized by a kind of composition at the level of primary structure, durable molecular and supramolecular bonds secondary, tertiary and Quaternary structures.

Known proteases of microbial origin, allowing you to break down collagen and keratin, in particular, from the phytopathogenic fungi of the genera Aspergillus, Penicillium, Keratinomyces; actinomycetes (A. albus, A. griseus), streptomycete (S. fraidiospirallis, S. griseus, S. fulvoviridis), Pseudomonas, Clostridium histolyticum, and other (1). Also known collagenase fungi Trichophyton schoenleinii, keratinase Microsporum canis and other dermatophytes (2, 3). Anastasya activity installed Trichophyton verrucosum, Microsporum gypseum (4). However, if there is a relatively wide range of potential producers of these enzymes latter differ in the way they impact on breaking down proteins, different speed and productivity of the reaction with the formation of p the EIT complexes of the final products.

Despite the demand in different industries proteolytic enzyme preparations, gidroliznaya structural proteins, methods for their selection are not widely used.

Currently this purpose a method of producing collagenase from crab (5). The method has limited opportunities for industrial production, as obtained by this method, the enzyme deficient and roads.

A method of obtaining proteolytic enzymes for processing of raw hides, using as producer Streptomyces lavandulae PMBC S-910. Proteolytic activity of the culture liquid is 40-90 PE/ml for Kunitz and 3.7-8.7 u/ml on the Anson, and collagenase activity with ninhydrin is 4-6 u/ml, and Chertinskaya - 0,8-2,0 u/ml (6).

To the disadvantage of the method include a lack of specificity of the produced complex in relation to structural proteins, and the presence of a nutrient medium containing a food component.

Object of the invention is the finding of microorganisms - producers eksperimentov proteolytic complex having high collagenase and pronounced keratinase and Lactasoy activity with the aim of creating a drug gidrolizuemye structural proteins.

Technical result achieved in the implementation of izopet the tion, is to increase the number of proteases that promote the hydrolysis of structural proteins and the specific activity of the protease complex structural proteins (scleroprotein) in the culture fluid or surface culture. This is achieved by optimizing the composition of the final product by reducing the content of ballast proteins and its reduction due to the use environment of the simple composition (wort-agar wort broth).

The problem is solved by using producer of protease structural proteins (scleroprotein) from among the fungi dermatophytes. Sowing dose mushroom contribute in liquid or on solid nutrient medium, cultivated for more than 20 days at 28°C, after which the biomass of the fungus (mycelium and microconidii) are removed. The remaining culture liquid is subjected to sterilizing filtration, freeze-dried or pre-advanced cleanse conventional methods and dried purified product. When surface cultivation using dense nutrient - biomass of the fungus is removed from the surface, and the remaining agar matrix is extracted with water or weak salt solution by shaking with several portions of the extractant. The obtained extracts are combined and also dried. Get a proteolytic product with purification G3x.

DL the determination of the enzymatic activity of freeze-dried preparations (G3x) used the following methods. Definition collagenase activity of the enzyme products were drug “asacol” (manufactured by Calbiochem), keratinase activity - drug “keratin-azure (Sigma), elastase activity - Congo-mouth elastin” (Sigma). For carrying out enzymatic reactions take 1.5-10 mg of substrate, 1,5-10 mg enzyme preparation, 1 ml of Tris-buffer (pH 8.0 to 8.2). In a control sample made only by the appropriate substrate and buffer (without enzyme preparation). The reaction is performed at 37°C for 1 h (keratinase activity - 24 h), after which the samples are centrifuged at 6-7 thousand rpm for 10 minutes, and supernatant photometrate at the optimum wavelength. Per unit keratinase activity took the change in optical density at 650 nm 0.01 carried out 1 mg of enzyme in 1 h; per unit elastase activity - change of optical density at 490 nm 0.01 carried out 1 mg of enzyme in 1 h; per unit collagenase activity took the change in optical density of 0.1 under the same conditions.

To determine collagenase activity of initial samples of the culture fluid, and extracts agar matrix (enzyme purification GC) as a substrate was taken 1%suspension of collagen, the fermentation is conducted under the same conditions for 5 or 17 h in test tubes were taken in 0.1 ml of culture fluid; added is about 1.9 ml of the suspension substrate. Experienced test tube was kept in an incubator, after which each was added to 2 ml of 20%trichloroacetic acid (THU). When setting the reference sample reagents were made in reverse order: the enzyme THU, substrate. The presence collagenase activity was determined by the accumulation of amino nitrogen was determined by ninhydrin method. Per unit of activity was taken the number of amine nitrogen, equivalent to 1 µmol of leucine liberated from the substrate at the desired fermentation conditions (37°C, pH 8.0) for 1 h

As producers used strains of dermatophytes from the collection of the FGI " VGNKI".

Example No. 1. In a bottle with liquid nutrient medium, for example wort broth, bring sowing dose of pure culture of the fungus Trichophyton mentagrophytes, strain 31. The culture is grown in a thermostat at a temperature of 28°C for 20 days. Then the culture fluid is separated sterilizing filtration. Remaining on the filter culture sterilized by autoclaving. The culture fluid was dried by lyophilization method. The output of the dry drug is 10 mg/ml After drying receive the drug with the total proteolytic activity of 4.8 U/mg.

Example No. 2. In liter flask with a dense nutrient medium (wort-agar) make the culture of T. mentagrophytes strain 23, were cultured for 20 days at 28&x000B0; With, then raised lawn fungus is removed by the scraper. The remaining dense nutrient medium washed with physiological saline and sterile filtered through a Seitz filter. The remaining agar medium was added an equal volume of physiological solution of sodium chloride and extracted metabolites by shaking shuttel-apparatus for 15 min, the Extract is drained and also subjected to sterilizing filtration. The extraction was repeated two more times. All the filtrates unite freeze-dried. Get the drug with the total proteolytic activity of scleroprotein 3,45 U/mg.

Example No. 3. Liquid nutrient medium growing culture of the fungus T. mentagrophytes, strain 7400 at 28°C for 20 days, after that experience collagenase activity in the filtered culture fluid (without pre-drying). Collagenase activity of the culture liquid (GC) is 27.5 U/ml

Example No. 4. The strain of the fungus T. equinum 51 is grown on wort broth for 20 days, and processed as in example 1 without drying. The filtrate of the culture fluid is collagenase activity equal to 15.1 U/ml

Example No. 5. In a bottle with liquid nutrient medium (wort broth broth + of Hottinger) contribute sowing dose of culture of the fungus T. verrucosum strain 165 and incubated for 21 days at 28°C. the Cult of the Central fluid released from the fungus as in example 1 and tested on the content of enzymes. Collagenase activity is 39.9 U/ml

Example No. 6 and 7. Strains of T. verrucosum 149 180 and incubated as in example 1 and tested on the content of the enzymes in the culture fluid. Collagenase activity is respectively 13 and 12.4 U/ml

After freeze drying of the culture fluid of strain 180 receive the drug with a total activity of scleroprotein 7.7 u/mg.

Examples 8 and 9. Liquid nutrient medium inoculated with cultures of fungi Microsporum canis, 68 strains and 17. After 21 days of cultivation separate the growth medium (culture fluid) and dried. Get two drugs with a total activity of scleroprotein respectively of 1.05 and 0.77 U/mg.

Examples 10 and 11. Strains of Microsporum canis 3483 and 3498 incubated as in example No. 1, and then tested on the content of the enzymes in the culture fluid. Collagenase activity respectively of 16.5 and 7.5 U/ml

Example No. 13. The strain of Microsporum gypseum 6402 plated on liquid nutrient medium (wort broth) and grown for 20 days.

The culture fluid was separated and freeze dried as in example No. 1. Collagenase activity in the dry preparation is 0.7 U/mg.

The results are shown in the diagram in figure 1 and 2. Values collagenase activity for liquid enzyme preparations was found in the literary sources (Fig. 2)converted the and 1 h of reaction.

Charts confirm the presence of proteolytic activity in all studied species of dermatophytes: strains of the genus Trichophyton (T. verrucosum, T. equinum, T. mentagrophytes, and Microsporum (M. canis, M. gypseum) - collagenases, keratinases and elastases. Chart 2 illustrates the high collagenase activity of enzyme preparations derived from the studied strains, in comparison with the activity of the analogue medications. The results show the possibility of using these fungi as producers of specific proteases (e.g., collagenase) or complex of the protease structural proteins.

The list of references

1. Grachev I.M. and other Laboratory workshop on the technology of enzyme preparations. Textbook for high schools. M., “Light and food industry”, 1982, 240 pages

2. Rippon J.W. increasing interest among collagenase from Trichophyton schoenleinii. Journal of bacteriology, 1968, vol. 95, No. 1, pp.43-46.

3. Daniels G. The digestion of human hair keratin by Microsporum canis Bodin. J. Gen. Environ., 1953, vol. 8, pp.289-294.

4. V. Meevootisom, Niederpruem D.J. Control of exocellular proteases in dermatophytes and especially Trichophyton rubrum. Sabouradia, 1979, vol. 17, pp.91-106.

5. Ahiable and other culture cells using collagenase crabs. Abstracts of the VGNKI, M., 2001, n.218-219.

6. SU 1778010, 15.01.1993.

The method of obtaining proteolytic preparation for hydrolysis of structural proteins, characterized by the fact that carry out the cultivation of the pre is sustained fashion activated culture of the fungus-dermatophyte, selected from the species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton eguinum or Microsporum canis, Microsporum gypseum, selected for their ability to produce collagenase, keratinase and elastase, the separation, purification and drying to obtain a drug that has the total proteolytic activity of at least 0.7 N E/mg.



 

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FIELD: biotechnology, in particular reagent for structural protein hydrolysis.

SUBSTANCE: method for production of protheolytic reagent includes cultivation of producer strain selected from dermatophyte of species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Microsporum canis, Microsporum gypseum, preferably from fungus strains capable to produce complex of structural protein proteinases (scleroproteases) with total protheolytic activity at least 0.7 U/mg including collagenase 0.1-4.5 U/mg; keratinase 0.1-0.5- U/mg; elastase 0.5-1.9 U/mg. Cultivation is carried out, for example, in wort agar-agar or in wort broth for 20-24 days.

EFFECT: scleroproteases with improved specific activity; method for protheolytic cleavage of specific substrates (scleroproteins) with increased rate and depth.

2 dwg, 12 ex

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