RU2716984C1 - Strain sarocladium kiliense - a producer of longolitine - a complex of fibrinolytic and thrombolytic enzymes and a method for producing longolitine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology. Disclosed is Sarocladium kiliense fungus strain, deposited in VKPM under registration number VKPM F-1502 – producer of longolitine, and method of producing longolitine. Method of producing longolitine involves culturing a strain of fungus Sarocladium kiliense VKPM F-1502 on a nutrient medium with subsequent separation of biomass, its concentration, purification on a membrane with pore size from 5 to 10 kDa and isolation of longolitine. Concentration involves washing off protein from membrane surface with concentrate solution, followed by mixing of washed protein solution with concentrate solution.

EFFECT: group of inventions increases output of longolitine.

2 cl, 3 tbl, 6 ex

Description

The invention relates to biotechnology, namely to the production of physiologically active compounds, and relates to a producer strain of Longolitin.

In the late 70's. employees of the laboratory of antibiotics and the laboratory of enzymatic fibrinolysis of Moscow State University M.V. The ability of the A. longa fungus to form fibrinolytic exoenzymes with plasminogen activator activity capable of lysing blood clots in in vitro experiments was revealed in Lomonosov. Arthrobotrys Longa Mecht strain No. 1 was obtained, and an enzymatic preparation of fungal origin with fibrinolytic, thrombolytic, esterase, proteolytic and plasminogen activating activities was isolated from the culture fluid. It was also found that proteinases of the pharmaceutical substance belong to the class of serine proteinases [USSR Author's Certificate No. 745943, publ. 07/05/1980]. However, the fibrinolytic and thrombolytic activity of this known complex of enzymes isolated from the culture fluid of this known strain of the fungus is not high enough.

Known strain of the fungus Arthrobotrys Longa Mecht No. 2 producer of longolithin - a complex of fibrinolytic and thrombolytic enzymes with a specific ability to activate plasminogen. The patent of the Russian Federation No. 2182596 for the first time recommended the use of a complex of fibrinolytic and thrombolytic enzymes as an external treatment for thrombosis [RF, patent No. 2182596, publ. 05/20/2002]. However, the fibrinolytic and thrombolytic activity of the complex of enzymes contained in the culture fluid of this strain of the fungus is not high enough to be used as a producer under industrial conditions.

Known strain of the fungus Arthobotrys Longa Mecht No. 3 VKPM F-942 [RF patent No. 2332450, publ. August 27, 2008] - producer of longolithin, a complex of fibrinolytic and thrombolytic enzymes.

This strain was obtained by treating Arthobotrys Longa Mecht strain No. 2 with gamma radiation at a dose of 200 kRad. Strain Arthobotrys Longa Mecht No. 3 VKPM F-942 provides stable biosynthesis of the target product in laboratory conditions in flasks.

The main disadvantage of the closest analogues is the lack of productivity and insufficient development of the process for producing Longolitin in industrial conditions.

The technical problem solved by the invention is to increase the synthesis of longolithin in the process of controlled continuous cultivation of a mutant strain of the fungus.

The technical result achieved by the present invention is to improve the industrial technology for the production of Longolithin based on a new mutant strain of the fungus - producer of Longolithin - a complex of fibrinolytic and thrombolytic enzymes with increased activity.

Description of the invention

With the improvement of the industrial technology for the production of Longolithin, technical problems were solved both in the field of improving the efficiency of the method of continuous cultivation of a strain of the fungus - producer of Longolithin, and in ensuring an increase in the efficiency of the strain.

To this end, the first stage was the selection of strain Arthobotrys Longa Mecht No. 2, and at the second stage, a new method for producing longolithin was developed.

In contrast to the technology for producing the Arthobotrys Longa Mecht No. 3 fungal strain, a new fungal strain was obtained from the Arthobotrys Longa Mecht No. 2 strain by the method of non-directional mutagenesis caused by ultraviolet irradiation without the use of preliminary treatment with chemical reagents. Species identification of the strain was carried out by sequencing sections of LTS1 and ITS2 amplified with genomic DNA using primers ITS4 and ITS5 [White, T.J. (1990) Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In: PCR Protocols, a Guide to Methods and Applications, 315-322].

As a result, a new strain was identified, with a species belonging to Sarocladium kiliense, which received the provisional name ALM4. The strain was deposited in the BRC VKPM SIC "Kurchatov Institute" - GosNIIgenetika under the registration number VKPM: F-1502. The following is a description of a new strain, and a new technology for the preparation of Longolithin substance. Longolitin activity was measured by hydrolysis of the chromogenic peptide substrate pGlu-Gly-Arg-pNA, as a more accurate method of measuring activity.

Example 1. Obtaining strain

An increase in the productivity of the new strain was achieved by undirected mutagenesis and selection from the initial culture of Arthrobotrys Longa Mecht No. 2 fungus. The spore suspension of the original Arthrobotrys Longa Mecht strain No. 2 was irradiated with ultraviolet light. Irradiated spores were plated on cups with Chapek's medium, cultured at 28 ° C for 4 days, and clones were tested for the production of proteolytic enzymes. Mutants with improved products were selected.

The result was a strain of the fungus, called Sarocladium kiliense ALM4 VKPM: F-1502.

The strain of the fungus Sarocladium kiliense ALM4 - producer of longolithin - is characterized by the following cultural and morphological characteristics.

On the agarized Chapek medium, the strain of the fungus Sarocladium kiliense ALM4 forms broad growing, colorless, fluffy, heavy, sometimes pinking with age, colonies with a smooth edge. The reverse side of the colony happens with radial folds and weak orange pigmentation. In the center of the colony there may be a bunch of vertical stolons (dense bundles of longer and less branching hyphae). The zoning of colonies is often noted, indicated by the height of the mycelium and its pigmentation. Also, during cultivation, polymorphism of colonies of the same age is noted. During its cultivation, on thin hyphae, unicellular microconidia 3-5 microns in size are constantly formed, which sprout with young hyphae, giving rise to new mycelial generations.

Physiological and biochemical properties of the strain.

Aerobe. The main method of storing the strain: in a 10-20% solution of glycerol at -70 ° C. Growth temperature range: + 18-45 ° С. Optimum growth temperature: + 40 ° C. It grows at pH values: 5.0-10.0. Optimum pH: 7.2-7.4.

Example 2. The cultivation of seed in the laboratory

To isolate the substance of Longolithin, seed was grown in flasks. Chapek's sterile medium is poured into about 10 ml into ten tubes, closed with stoppers and placed to solidify the medium on a sloping surface.

In prepared tubes with a beveled layer of Chapek's nutrient medium, the producer culture is reseeded. The tubes are corked with cotton plugs and incubated at 28 ° C for 7 days.

Tubes with grown culture are analyzed for compliance with the external characteristics of the colonies characteristic of the producer. The strain Sarocladium kiliense ALM4 VKPM: F-1502 forms broad-growing raised, colorless, fluffy, heavy, sometimes pinkish with age, colonies with a smooth edge. Tubes with the presence of colonies that differ from those described in the work are not used. Tubes with grown seed are opened in the sterile zone, the mycelium film is carefully removed from the surface of the agar medium with a microbiological hook and transferred to a container for inoculation containing 200 ml of sterile seed. The contents are thoroughly mixed and poured in approximately equal amounts into four containers for growing seed. The containers are closed with cotton plugs and placed in a shaker incubator. The cultivation of seed occurs at 28 ° C and a shaker rotation speed of 200-220 rpm. within 48 hours.

Example 3. The cultivation of seed on an industrial scale

The biosynthesis and cultivation of seed on an industrial scale was carried out in a standard industrial fermenter. A sterile seed medium and the contents of the flask with seed are loaded into the fermenter. The cultivation of seed occurs at 28 ° C, the speed of rotation of the mixer 150-200 rpm and air flow rates of 1 l / min. within 48-96 hours. We used a suitable volume fermenter standard “Fermenter sterilizable FS-100”.

The control of seed cultivation is carried out using optical microscopy and measuring the pH of the medium. For this, 100 ml of the contents are taken from the fermenter for analysis. Cultural traits of seed should correspond to the following description: 1) the formation of unicellular microconidia of 3-6 microns in size is observed. Microconidia are formed directly on the hyphae of the vegetative mycelium; 2) the pH of the medium at the end of the cultivation should be in the range of 8.0-8.2.

When switching from a laboratory shaker-incubator, in which aeration was carried out only by shaking the flasks, to the industrial scale, the optimal parameters of aeration are selected. The selection is carried out according to the results of changes in pH and the amount of protein accumulated in the culture fluid. The results are presented in table 1.

When growing seed, aeration conditions with a low speed (170 rpm and 1 l / min.) Are chosen, since at a lower speed, seed is obtained in which the shape of the microorganisms corresponds to their natural form.

The biosynthesis in the fermenter occurred better with more intensive mixing (210 rpm) and air supply (20 l / min.). A higher speed of rotation of the mixer and the supply of a larger volume of air are difficult due to foaming of the liquid and losses due to evaporation of water.

Example 4. The separation of the culture fluid and biomass

The culture fluid from the fermenter through a hose using a pump serves in the separator at a speed of 100 ml / min. Separation occurs at 8000 rpm (7000-7500 g). In order to increase the activity of the substance of Longolitin, the concentration of the supernatant of the culture fluid is carried out by ultrafiltration through a semipermeable filter membrane. The supernatant of the culture fluid is transferred under pressure up to 7 bar to an ultrafiltration unit and concentrated three times.

The criteria for choosing a filter element were: elimination of the passage of protein through the pores, the rate of filtration and protein loss due to protein retention on the membrane surface. Since the size of the serol proteases of Longolitin is at least 30 kDa, ultrafiltration cartridges with an average pore size of 5 kDa and 10 kDa (polysulfone filter element material) are used for concentration. To reduce the loss of protein deposited on the membrane, the protein is washed off, which has settled on the membrane surface, with distilled water or the resulting concentrate. The results of the selection of the filter element are shown in table 2:

To concentrate the culture fluid, a pore size of the filter membrane of 10 kDa and a washout of the protein deposited on the membrane surface with concentrate were chosen, since this option provides the highest degree of concentration and optimal operating time. Loss of protein due to passage through the pores was negligible when using pores of 5 kDa and 10 kDa. Washings and concentrate are subsequently combined during precipitation.

Example 5. Precipitation and drying of longolithin

The measured amount of acetone is pre-cooled in the freezer to a temperature of minus 20 ° C. With a peristaltic pump, at a rate of 100 ml / min, acetone is fed into the supernatant of the culture fluid. The mixture is left for 1 hour to form a precipitate. The precipitate was filtered under vacuum on a filter filter through filter paper. Due to the concentration of the culture fluid, it was possible to significantly reduce the consumption of acetone (from 600 to 205 kg per 1 kg of the obtained substance) and to reduce the formation of the acetone mother liquor (from 920 to 342 kg per 1 kg of the substance). Freeze drying led to a loss of enzyme activity by 15-20%, so it is not used. The precipitate is dried in a desiccator over concentrated sulfuric acid for 24 hours, after which the pharmaceutical substance is ready for packaging and packaging.

Example 6. Determination of the specific activity of Longolitin

Assessing the ability of thrombolytic drugs to activate plasminogen is the most important analytical method for studying the activity of modern thrombolytic drugs. A method for measuring activity on a urokinase substrate is a direct analytical method and allows one to determine with high accuracy the activity of a thrombolytic drug acting by the urokinase type. Analysis of the determination of the specific activity of longolithin by urokinase type was carried out by the method of hydrolysis of the chromogenic peptide substrate pGTu-Gly-Arg-pNA.

For analysis, a 0.05% solution of pGlu-Gly-Arg-pNA in distilled water is prepared. Next, prepare 0.05 M Tris-HC1-buffer, pH 8.2. Dilutions of the pharmaceutical substance are prepared in a concentration range from 1 mg / ml to 10 mg / ml in distilled water or 0.05 M Tris-HCl buffer, pH 8.2. For the reaction, 100 μl of sample, 150 μl of buffer and 100 μl of pGlu-Gly-Arg-pNA are mixed. The mixture was incubated for 5 min at 37 ° C. The reaction was stopped by adding 200 μl of 50% acetic acid. The resulting mixture is fully transferred to a spectrophotometric microcuvette with a volume of up to 1000 μl with an optical path length of 1 cm. The optical density is measured at 405 nm on a spectrophotometer. The measurement of optical density is carried out immediately after stopping the reaction.

Specific activity is expressed in international units (ME) corresponding to the number of μmol pNA cleaved in 1 minute or in units of action (IU) to the number of nmol (10 ^-3 μmol) pNA cleaved in 1 minute: 1 unit is ME × 10 ³ .

The specific activity (X), U / mg, is calculated by the formula:

где

Where

And ₄₀₅ is the absorption value; K is the coefficient calculated according to the calibration graph;

V is the sample volume, μl; t is the reaction time, min .; m is the mass of the FS sample, mg.

The arithmetic mean of the activities obtained in the analysis of three parallel samples of the drug is taken as the final test result. The absorption linearity (A405) determined by the calibration graph is observed in the range from 10 × 10 ⁻³ to 150 × 10 ⁻³ μM pNA / ml. The results of the measurement of urokinase activity of samples of Longolithin substance obtained by the method described in patents RU 2182596, and by the method described in this application are presented in table 3.

Industrial applicability

The present invention describes a new method for producing a substance of longolithin, which has increased activity. This method is based on the introduction of the concentration operation and the additional protein washing off from the membrane surface with a concentrate solution into the Longolithin production process, followed by mixing the washed protein solution with the concentrate solution. To increase the efficiency of the method, a new strain of Sarocladium kiliense ALM4 VKPM: F-1502, obtained as a result of mutation of the Arthobotrys Longa Mecht strain No. 2, was used.

Longolitin activity was measured by hydrolysis of the chromogenic peptide substrate pGlu-Gly-Arg-pNA, as a more accurate method of measuring activity. The activity of the substance obtained by the method described in this application is from 4 to 20 times higher than by the methods described previously. This is due to the following improvements: the use of a more productive strain; using industrial technology for growing seed; using an additional stage of concentration of the supernatant with flushing of the protein from the surface of the membrane.

Claims (2)

1. A method of producing longolitin, including cultivating a strain of the fungus producing Longolitin on a nutrient medium, followed by separating the biomass, concentrating it and purifying the protein on the membrane, characterized in that as the strain producing Longolitin, the fungus strain Sarocladium kiliense ALM4 VKPM F-1502 is used, where the concentration and purification of the protein is carried out on a membrane with a pore size of 5 to 10 kDa, while in the process of concentration, the protein is washed off the surface of the membrane with a solution of the concentrate, followed by mixing the solution of washed protein concentrate with a solution. 2. The strain of the fungus Sarocladium kiliense VKPM F-1502 - producer of longolitin - a complex of fibrinolytic and thrombolytic enzymes.