RU2758788C1 - Method for producing a collagenolytic enzyme - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: proposed is a method for producing a collagenolytic enzyme, including cultivation of basidiomycota of the species Correna unicolor, Ganoderma lucidum, Trametes ochracea, Coriolus versicolor, Pleurotus ostreatus, Hypsizygus ulmarius, Lentinus edodes, Funalia trogii, Fomes fomentarius or Coprinus lagopides on a liquid nutrient medium containing 6.0 to 10.0 g/l of lactose or 10 to 15 g/l of dry whey containing 70% lactose as a carbon source, and 1.5 to 5.0 g/l of urea as a nitrogen source, followed by separation of the mycelium by filtration, concentration of the native solution by means of ultrafiltration, freeze-drying.

EFFECT: invention provides an increase in the yield of the target product.

2 cl, 1 tbl, 1 ex

Description

The invention relates to biotechnology, in particular, to methods for producing enzyme preparations, in particular, collagenase, which is promising for use in medicine, cosmetology, dermatology, food and leather and fur industries.

Collagenase is a unique enzyme capable of selectively hydrolyzing collagen, which is part of the connective tissue of a living organism. By cleaving collagen fibers extremely resistant to the action of other proteases, collagenase performs the most important functions in humans and animals, participates in the transformation of connective tissue during growth and morphogenesis, as well as in the elimination of certain pathological processes.

Research of the IA Credinform company "Enzyme preparations based on collagenase and their application" [https://marketing.rbc.ru/articles/710/] showed that nowadays enzyme preparations, including those based on collagenase, are widely used in various industries and areas - from medicine to the meat industry and brewing.

In particular, collagenase-based drugs are known, such as, for example, Iruksol ointment for the treatment of skin integuments. In cosmetology, collagenase is used in cosmetic preparations - creams, serums, etc., in dietary supplements and in various procedures, in particular, in electrophoresis to reduce and dissolve scars, scars, etc.

Currently, collagenases are obtained, as a rule, by processing biological material. Collagenases are widespread in invertebrates, in which, unlike higher mammals, they are used in the process of digestion. For example, two collagenolytic proteinases have been isolated from the starfish Pycnopodia helianthoides. One of them belongs to the class of metalloproteinases, and the other with a lower molecular weight is of the chymotrypsin type. Both enzymes degraded labeled guinea pig skin collagen, but were virtually inactive on the PZ-Pro-Leu-Gly-Pro-D-Arg Clostridiopeptidase substrate [M.E. Alexander, M.N. Dresden, Comp. Biochem. Physiol. 1980, V. 67 B, p. 505-509]. Invertebrate collagenases have been found in leeches, various insects, shrimps, and various types of crabs. However, not all of them are promising sources of collagenases used in medicine and other industries, which, as a rule, is associated with a limited amount of starting material [RU 2236460, 2004].

Methods for obtaining a proteolytic complex, including collagenase activity, from crushed pancreas-containing terrestrial or aquatic animals have found wide application. Pancreas-containing organs of fish are used as raw materials: pyloric appendages of greenling, flounder, cod, herring, mackerel, horse mackerel, capelin, halibut, saury, anchovy, salmon, hepatopancreas of crab, krill, squid, large pancreas of sea pinnipeds and whales, and small ruminants, pigs, deer, chickens. [RU 2034028, 1995].

The most widespread technology is the isolation of collagenase from the hepatopancreas of commercial species of crabs [RU 2008353, 1995; RU 2096456, 1997; RU 2112036, 1998; RU 2265052, 2005], in particular, from the king crab Paralithodes camtshatica, which is associated with the solution of the problem of disposal of crab fishing waste. In addition, an important advantage of this collagenase is the relative harmlessness of crab collagenase preparations for humans. So, there is a known method of obtaining a collagenolytic preparation, which provides for the homogenization of hepatopancreas tissues in a buffer solution with a pH of 6.0-9.5 containing a non-ionic detergent and NaCl, followed by purification of the solution by microfiltration on membranes with a pore diameter of 0.45 μm, by concentrating ultrafiltration on a membrane with pore diameter 5 kDa and freeze drying. Specific proteolytic activity is not less than 10,000 U / mg [EP 0402321, 1990].

Preparations derived from hepatopancreas vary considerably in purity and potency, as well as in composition. The disadvantages of these methods can also be attributed to the duration and multistage process of collagenase isolation, which significantly reduces the yield of the product.

A known method of obtaining collagenase from the head section of various types of leeches [RU 2160280, 2000; WO 87/00860, 1987]. For this, the frozen head section of the leech is homogenized in distilled water, the suspension is repeatedly centrifuged for 10 min and the supernatant is collected. The enzyme is salted out with sulfate and dialyzed against distilled water. Collagenases of leeches have no side effects and act directly on one of the main stimulants of platelet aggregation. In addition, collagenases of leeches destroy collagen in only one bond, which makes it possible to use this enzyme together with other active factors in microsurgery. The disadvantage of this method is the length and complexity of such a method for producing collagenase.

Currently, collagenases of microbial origin are widespread, among which the most studied are collagenases synthesized by the bacterium Clostridium histolyticum, which are characterized by a high specificity of action on collagen fibers. To date, a number of collagenase-based pharmaceutical compositions have already been developed [RU 2166950, 2001; RU 2174389, 2001; RU 2139044, 1999; RU 2149644, 2000, etc.]. So, the known drug "Iruksol" (Germany), which contains collagenase in a mixture with proteases isolated from the bacterial culture of Clostridium histolyticum. "Iruksol" is a combined preparation with proteolytic and antimicrobial action for external use, used for enzymatic cleansing of wounds, including varicose ulcers, bedsores, gangrene of the extremities, especially in patients with diabetes mellitus, as well as frostbite, with long-term non-healing postoperative wounds, after radiation therapy, in preparation for skin transplantation [Susagawara R., Harper E. Publication and characterization of three forms of collagenase from Clostridium histolyticum II Biochemistry. - 1984. - V. 23. - P. 5175-5182].

The disadvantage of drugs from this microorganism, which is the causative agent of gas gangrene, is the need for thorough cleaning in special conditions, high cost and multistage, which leads to a decrease in the activity of the drug obtained at the exit and an increase in its cost.

There is also known a method of obtaining microbial collagenase from an ultrafiltrate culture fluid of Streptomyces lavendulae INMI-36-SV [RU 95104309, 1996]. The drug is obtained from an ultraconcentrate of the culture fluid of the strain Streptomyces lavendulae INMI-36-SV using ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and lyophilization. This drug has a higher collagenase activity than the drug from Clostridium histolyticum, however, the method has a number of disadvantages, in particular, multistage and relatively low yield.

Among the sources of collagenase, the use of fungi is of considerable interest due to their lower toxicity to humans and a simpler cultivation technology.

A known method of obtaining a proteolytic preparation for the hydrolysis of structural proteins, containing collagenase, keratinase and elastase from a pre-activated culture of a producer strain from among dermatophyte fungi of the species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Microsporum canis and Microsporum 2003. After isolation, purification and drying, a preparation is obtained with a total proteolytic activity of at least 0.7 PE / mg.

The disadvantage of the drug is the presence of a mixture of enzymes in its composition and insufficient activity.

The problem solved by the authors of the invention is to expand the range of raw materials used to obtain collagenase. This problem was solved by using basidiomycetes for these purposes.

Basidiomycetes (basidiomycetes), mostly saprotrophs, play an important role in the mineralization of organic compounds, especially those that are difficult to destroy (cellulose, lignin). For example, aphyllophoric basidiomycetes are xylotrophic fungi that naturally cause wood decay like white or brown rot and are able to grow on all kinds of wood substrates and their waste. Basidiomycetes are most widespread in forest soils and litter.

Currently, active research is underway aimed at creating a technology for cultivating basidiomycetes, in particular, fungi that produce valuable biologically active substances (BAS), in particular, proteins, vitamins, essential amino acids, lipids, polysaccharides, etc. Therefore, much attention is paid to the development of technologies artificial cultivation (cultivation) of basidiomycetes both in submerged conditions (submerged cultivation) and on the surface of various substrates.

The formation of valuable stable biologically active substances by basidiomycetes depends on the type of basidiomycetes used and the cultivation technology.

In particular, it is proposed to cultivate higher basidiomycetes on a nutrient medium based on beer wort [Alexandrova EA, Zavyalova LA, Tereshina VM, Garibova LV, Feofilova EP. Obtaining fruit bodies and deep mycelium of Lentinus edodes (Berk.) Sing .; Lentinula edodes (Berk.) Pegler] // Microbiology, 1998. T. 67. No. 5. S. 649-654; (http://selhozproduct.narod.ru/miselia.html]. A known method of preparing mycelium of basidiomycetes by cultivating them on a sterile nutrient medium containing 20% potato decoction, glucose or sucrose and agar at 24 ° C for 14 days [W0 2004/097007, 2004].

The disadvantage of this technology is the low biomass yield of the mycelium of higher basidiomycetes due to the lack of a sufficient amount of minerals and energy substrate (carbohydrates) in the nutrient medium, the duration of the process of preparation of inoculum.

Known methods of obtaining mycelium when growing the fruit bodies of the edible basidiomycete Pleurotus ostreatus, which consists in inoculating the grain substrate with mycelium grown by submerged cultivation on liquid nutrient media containing corn starch, corn or barley-malt extract and mineral additives [(UA 36655] , or Pleurotus djamor on a liquid nutrient medium containing carbon sources - glucose, sucrose, vegetable oil, nitrogen sources - soy flour, peptone, sodium nitrate, ammonium sulfate, as well as potassium di-hydrogen phosphate, magnesium and potassium sulfate [Krasnopolskaya et al. ., Pleurotus djamor. Methods of cultivation and antimicrobial properties // Mikology and phytopathology, 2001. T. 35. Issue 1. P. 62-67].

The disadvantage of these methods is the duration of the preparation process of the seed mycelium - up to 16 days, as well as the narrow scope of its application - only for one species of the genus Pleurotus. In addition, during their cultivation, the possibility of obtaining collagenase was not noted.

Closest to the claimed invention is the technology previously developed by the authors for obtaining collagenolytic enzymes by submerged cultivation of basidiomycetes using glucose and peptone as carbon and nitrogen sources [RU 2018100382, 2018]. The cultivation is carried out using a semi-synthetic medium of the following composition, g / l: peptone - 1.08; glucose - 10; KH ₂ PO ₄ 0.6; K ₂ HPO ₄ - 0.4; MgSO ₄ * 7H ₂ O - 0.5; FeSO ₄ 0.005; ZnSO ₄ 0.001; CaCl ₂ - 0.05; NaCl - 0.5; dry yeast extract - 2.0. Upon reaching the maximum collagenolytic activity, the mycelial biomass is separated from the native solution by filtration under vacuum, then the native solution is concentrated and purified by ultrafiltration on a semi-permeable membrane of the "MIFIL PA-20" brand with a nominal molecular weight retention limit of 20 Sha. The ultrafiltrate is lyophilized.

The specific collagenase activity of the enzyme preparation ranges from 2000 to 9000 U / mg protein, depending on the fungus used.

The technical problem solved by the authors is the creation of a technology that allows you to increase the enzymatic activity.

It was found that in order to achieve the maximum yield of collagenase during the cultivation of basidiomycetes, an optimal formulation of the nutrient medium is required.

It was found that the technical result is achieved by the fact that within the framework of the technology, including the cultivation of basidiomycetes by the method of submerged cultivation of macromycetes in a liquid nutrient medium, separation of the mycelial biomass from the native solution and concentration by ultrafiltration followed by drying the ultrafiltrate, urea is used as a nitrogen source. and as a carbon source - lactose-containing products with their content in the nutrient medium - urea 1.5-5.0 g / l and lactose 6.0-10.0 g / l.

The use of urea and lactose in lower concentrations leads to a decrease in the yield of collagenase, and at high concentrations does not lead to an increase in its yield and is not economically feasible.

As a source of lactose, lactose or products containing it, such as dry milk whey (CMC), are used.

The liquid nutrient medium used, as a rule, contains urea 1.5-5 g / l, lactose-containing product 6-15 g / l, yeast extract 2 g / l, mineral additives (KH ₂ PO ₄ , K ₂ HPO ₄ , MgSO ₄ * 7H ₂ O, FeSO ₄ , ZnSO ₄ , CaCl ₂ , NaCl) - 2-2.5 g / l, water is the rest.

Industrial applicability is illustrated by the example of the cultivation of higher mushroom cultures, which were isolated from fruit bodies collected in the northeastern suburban forest zone of St. Petersburg. Fruiting bodies (basidiomas) were collected and induced into a pure culture at the Department of Microbiological Synthesis Technology, St. Petersburg State Technical University.

Example 1. The mycelium of the fungus was placed in a nutrient medium containing carbon sources (peptone or urea), carbohydrates (in particular CMC containing 70% lactose), mineral additives: KH ₂ PO ₄ - 0.6; K ₂ HPO ₄ - 0.4; MgSO ₄ * 7H ₂ O - 0.5; FeSO ₄ 0.005; ZnSO ₄ 0.001; CaCl ₂ - 0.05; NaCl - 0.5; as well as dry yeast extract - 2.0. pH of the medium before sterilization 6.8-6.9. The medium was sterilized at 0.75⋅10 ⁵ Pa for 30 minutes. Upon reaching the maximum collagenolytic activity, the mycelial biomass was separated from the native solution by vacuum filtration, after which the native solution was concentrated and purified by ultrafiltration in a non-flowing cell of the FM 02-200 brand with a volume of 200 ml on a semi-permeable membrane of the MIFIL PA-20 brand with the value of the nominal molecular weight limit of retention of 20 kDa. The ultrafiltrate was freeze-dried in an Hego unit (Denmark) at a material freezing temperature of -40 ° C, a condenser temperature of -30 ° C, and the collagenolytic activity of the dry powder was determined. The results are shown in Table 1.

The results obtained showed that the use of the claimed invention, in comparison with analogues, can increase the yield of collagenase by 1.4-1.7 times.

Claims (3)

1. A method of obtaining a collagenolytic enzyme by cultivating basidiomycetes of the species Correna unicolor, Ganoderma lucidum, Trametes ochracea, Coriolus versicolor, Pleurotus ostreatus, Hypsizygus ulmarius, Lentinus edodes, Funalia trogii, Fomes fomentariop or Coprinus nutrient media containing liquid nutrient media containing nutrient media, carbon or Coprinus and mineral salts with subsequent separation of the mycelium by filtration, concentration using ultrafiltration and freeze drying, characterized in that lactose or dry milk whey containing 70% lactose is used as a carbon source, and urea is used as a nitrogen source with the following content in the nutrient environment: urea 1.5-5.0 g / l lactose 6.0-10.0 g / l or milk serum 10-15 g / l 2. The method according to claim 1, characterized in that urea 1.5-5.0 g / l, lactose 6.0-10.0 g / l or milk whey 10-15 g / l are used as the nutrient medium, yeast extract 2.0 g / l, mineral salts 2.056 g / l, water - the rest ”.