The strain streptomyces sp. z-11-6-producer of extracellular l - glutaminases

 

(57) Abstract:

The invention relates to the field of biotechnology and medicine. The resulting enzyme can be used in analytical chemistry, food industry and clinical biochemistry. Proposed genetically stable strain of Streptomyces sp. Z-11-6 with high L-glutamatemediated activity of 1.6-1.8 IU/ml of culture fluid. L-glutaminases from Streptomyces sp. Z-11-6 has a higher substrate specificity to L-glutamate, temperature and pH stability, resistance to the effect of metal ions, inhibitors and chelating agents. 3 tab., 6 Il.

The invention relates to the field of biotechnology and medicine, specifically to a new strain - producer of extracellular L-glutaminases with high substrate specificity for L-glutamate.

Extracellular glutaredoxins microbial origin (L-glutamate: O2oxido-reductase (deaminase) EC 1.4.3.11] is the enzyme that catalyzes the specific reaction of the oxidative deamination of L-glutamate in the presence of water and oxygen with the formation of a-Ketoglutarate, ammonia and hydrogen peroxide:

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This enzyme can be used as analytical reagent, t is transferring enzyme, which opens up opportunities in analytical chemistry (qualitative and quantitative assessment of enzymatic processes), as well as in the food industry (food quality). Especially important is the use of this enzyme in clinical biochemistry for the determination of glutamate - pyruvate-transaminase and glutamate-oxaloacetate-transaminase in biological fluids, which allows early diagnosis of diseases of the heart and liver. It is known that myocardial infarction glutamate-pyruvate-transaminase levels and glutamate - oxaloacetate-transaminase levels go from damaged myocardial cells and enter the bloodstream. Determination of serum concentrations of these two transaminases can give valuable information about the severity and stage of damage to the heart muscle. In addition, the concentration of these two transaminases in serum plays an important role in the early diagnosis of such diseases as liver disease among persons employed with various organic solvents used in chemical and other industries.

Up to this time was known glutaredoxins from snake venom, kidneys of rats and mice, the liver birds. Were found produce on seeds of Calophyllum inophyllum. (U.S. Pat. USA N 4 357 425).

Producers of glutaredoxins microbial origin are microorganisms belonging to the group of streptomycete:

Streptomyces endus, ZIMET 43772 - Pat. GDR N 240910. Pat. GDR N 272 101;

Streptomyces cattleya, ZIMET 4 378 5 - Pat. GDR N 261932. Pat. GDR N 272 101; Streptomyces sp. A 7700, FERMP-6241 and

Streptomyces sp. A 8063, FERM8-6242-Pat. USA N 4605615 and its analogues: Pat. Japan N 58 149 676, Pat. Germany N 3307607 and N 3348379;

Streptomyces violascens, H 82-N-SY7 - Pat. Japan N 057043685

Streptomyces sp. X-119-6, FERMP-6560 - Pat. USA N 4 623 626. Pat. USA N 4 614 714 and their patents equivalents: EP N 097949, Pat. Japan N 112271, N 145346, N 146707, N 59002 686, N 59034882.

Of the above links only underlined are directly related to new strains-producers of glutaredoxins. The rest - to the practical use of these strains to obtain L-glutaminases in laboratory conditions. All technical solutions contain the same number of similar features.

As the prototype was selected producing strains with the highest glutamatemediated activity described in U.S. patent N 4623626. This producer was isolated from soil samples taken in Tonosho-Masha, Katori-gun, and Shiba-Ken (Japan). According to their morpho-cultural and physiological-biochemical properties of this strain was otne the cation and fractionation of organic solvents, selective adsorption, ion exchange chromatography, gel filtration, dialysis and lyophilization. Thus purified enzyme had a specific activity of 55 u/mg protein, and its output accounted for 18.4% of the original. The purified enzyme had a relative substrate specificity to L-glutamate: if taken as 100% activity of the enzyme for L-glutamate, his activity to L-aspartate was 0.6%, in relation to other amino acids, the enzyme activity was less than 0.1%. KMby L-glutamate is 2,110-4M at a pH of 7.4. The optimum pH: 7.0 to 8.5. pH stability: stable in the pH range of 5.5 to 10.5 at 37oC for 60 minutes Temperature stability: stable up to 65oC at pH 5.5 for 15 minutes, the Enzyme is not inactivated by copper chloride in a concentration of 1 mm at a pH of 7.4, but inhibited by 45% p-chloromercuribenzoate.

At present, the urgent task is to obtain highly productive strains producing extracellular L-glutaminases with high substrate specificity. The problem was solved in the process of finding a natural producer strain and obtain from it the mutation-selection approach genetically stable mutant strain, with the full range of Oulu. M. C. University of the soil sample taken from the peatland Lipetsk region, was isolated Streptomyces sp. Z-11, which is the producer of extracellular glutamate oxidase. According to the determinant of actinomycetes Gause (1983), in their morpho-cultural and physiological-biochemical properties of the strain most similar to Streptomyces canofumeus, Krassilnikov 1970 (type strain: inmi 516 = VKM 70). As a result of mutagenesis, in which as a mutagen used nitrous acid, natural strain was obtained genetically stable mutant strain Streptomyces sp. Z-11-6, glutamatergicheskaya activity which was 40 times higher than natural and is of the order of 1.6-1.8 IU/ml of culture fluid. Partially purified enzyme has a high substrate specificity for L-glutamate. Of the 20 amino acids, including D-glutamate, glutaredoxins from Streptomyces sp. Z-11-6 showed high activity only to L-glutamate, which allows to determine the concentration of L-glutamate with other amino acids. In addition, Streptomyces sp. Z-11-6 under cultivation in liquid media containing yeast or corn extracts, produces pigments melanoides type in much smaller quantities than the natural strain, which facilitates the operation extracts is at 4oC for 4-6 months was observed maintaining productivity on 99-98%. According to their morpho-cultural and physiological-biochemical properties of the mutant strain, Streptomyces sp Z-11-6, close to nature.

Morphological and cultural properties of Streptomyces sp. Z-11-6

Vegetative hyphae of 0.6-0.8 μm in diameter, form a well-developed branching mycelium, not disintegrating into fragments. Hyphae of the aerial mycelium, 0.9 to 1.0 μm, in the Mature state are chains of spores. Chains of spores spiral, in the form of well-developed, correct, stretched helices from two to four turns. Disputes of 0.6-0.7 x 0.9 to 1.0 μm, still, elipsometry with a smooth surface. Sclerotia-, sporangia, pycnidia and Cinematone patterns not found.

The strain is gram-positive, not acid-fast. The cell wall contains L-diami-epimenidou acid (cell wall type 1).

Growth of the strain Streptomyces sp. Z-11-6 on different diagnostic media at 28oC for 14 days are presented in table 1.

With growth ovsanna agar at 28oC for 7-14 days Streptomyces sp. Z-11-6 forms a dense, growing into the agar concentric colonies with scalloped edge d ~ 1-3 mm of the Surface of the colony covered in smooth air m is UP>oC, optimum at 26-28oC. Aerobe.

Physiological biokhimicheskii properties

The use of carbon sources:

This strain was grown on ISP environment 9 environment (Gottlieb and Predjama) with different carbon sources in the 28oC for 21 days. The results were evaluated at 7, 14 and 21 days of growth when compared with the negative control (medium without added carbon source) and positive control (medium with D-glucose). Streptomyces sp. Z - 11-6 well disposes of glucose, arabinose, sucrose, xylose, mannitol, fructose, galactose, maltose, rhamnose, starch, glycerin and hydrolyzed casein. Relatively well - raffinose, do not use - Inositol. Poorly decomposes gelatine, well hydrolyzes starch.

The use of nitrogen sources:

This strain was grown on a medium of the following composition (%): glucose - 1, MgSO47H2O - 0,05, NaCl - 0,05, FeSO47H2O - 0,001, K2HPO4- 0.1, agar and 1.5, pH 7.0. The results were evaluated on day 15 compared with the negative control (medium without nitrogen source) and positive control (medium with L-Proline and the environment with L-asparagine). The strain Streptomyces sp. Z-11-6 consumes bad as the sole source of nitrogen the following substances: arginine, cysteine, qi is
The medium used to maintain the strain, consists of 2% oat flour and 1.8% agar. The liquid medium for the cultivation contains: 3% glucose, 0.6% of corn extract, 0.6% ammonium sulfate, 3% calcium carbonate, 0.3% calcium chloride, and 0.1% of magnesium chloride and potassium chloride; the pH of the medium to 7.0. Seeds were grown overnight and received by the inoculum (5% by volume) were seeded Wednesday, poured on 100 ml of 750 mm Catalogne bulb. The cultivation was carried out at 26oC on a rocking chair (190 rpm). The maximum yield of product was observed after 28 h from the beginning of cultivation and was of the order of 1.6-1.8 IU/ml of culture fluid.

Scheme has been devised to purify the enzyme. Mycelium grown culture Strepto-myces sp. Z-11-6 was separated from the culture fluid by filtration through a dense fabric. The filtrate was concentrated on separating apparatus AR - 0,1 [0,2 MPa] ("Biotest", , Kirishi). For the deposition of a protein mixture used ammonium sulfate (50% saturation). The precipitate, collected by centrifugation at 8000 g for 15 min, pererestorani in 0.05 M K-phosphate buffer (pH 7.0) with 0.6 M NaCl. The obtained enzyme solution was passed through a column (2.0 x 25 cm) with DEAE-cellulose (diethylaminoethyl), balanced 0.05 M K-phosphate buffer (pH 7,, obrazovavshijsya during dialysis was removed by centrifugation, and the supernatant again missed through a column (2.0 x 25 cm) with DEAE-cellulose, equilibrated 0.05 M K-phosphate buffer (pH 7.0). Thus purified enzyme had a specific activity of about 10 units/mg protein, and the output it was 25-30%. The purity of the enzyme was determined by native gel electrophoresis in 10% Page.

Measurement glutamatemediated activity. Measurement of enzyme activity was carried out on the basis of peroxidase chromogenic reaction. The reaction mixture contained 1 ml): 2.5 u of peroxidase from horseradish (300 u/mg, 1.0 mmol of 4-aminoantipyrine, 17.5 mmol of phenol, 10 mcmole L-glutamate in 0.1 M K-phosphate buffer, pH 7.0. The reaction mixture was incubated at 37oC for two minutes and the reaction was initiated by adding the studied enzyme. Glutamatemediated activity was measured by the change in optical density of the solution at a wavelength of 500 nm on the spectrophotometer Hitachi 200S 20 (Japan). The enzyme activity was calculated by the formula:

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where Ath- glutamatergicheskaya activity of the samples; - the change in the value of optical density of the solution in the cell for one minute at 37oC; Vabout- the total volume of solution in the cell; V is A. Per unit of enzyme activity (u) was taken such amount that catalyzes the formation of one micromole of hydrogen peroxide per minute at 37oC.

Investigated a number Entomologicheskoe properties of the investigated L - glutaminases.

Substrate specificity: the enzyme has a high substrate specificity for L-glutamate: of the 20 amino acids glutaredoxins from Streptomyces sp. Z-11-6 showed high activity only to L-glutamate (table.2).mfor L - glutamate is about 0.34 mm at pH 7.0.

The optimum pH: the activity of the enzyme at various pH values were measured at 30oC, using as substrate L-glutamate in 0.2 M acetate buffer (pH 3.5 to 6.0); 0.2 M phosphate buffer (pH 6,0 - 8,5) and 0.2 M glycine-NaOH buffer (pH 8,5-11,5). The optimum pH of the investigated enzyme wide and is observed at pH from 6.5 to 9.5 (Fig. 1).

pH stability: the enzyme is incubated under appropriate pH values from 3.5 to 11.5 at 37oC for 60 min, at 45oC and 60oC for 15 min, then quickly cooled and measured glutamatemediated activity by the method described above. When the incubation of the enzyme at 37oC for 60 min last was stable at a pH of from 5.5 to 9.5 (Fig. 2); when inquire and at 60oC for 15 min in the pH range of 5.5 to 6.5 (Fig. 4).

Operating temperature range: reaction mixture containing the substrate L-glutamate (10 µmol/ml), incubated at temperatures from 20 to 90oC for 20 minutes Glutamatemediated activity was measured by the method described above, using as the initiator of the reaction of the studied enzyme. According to the data obtained, the temperature range at which the enzyme activity is more than 50% of the original, is from 20 to 65oC, with an optimum of about 40oC (Fig. 5). Temperature stability: after incubation of the enzyme at a temperature of from 30 to 90oC and pH buffer solutions 5,5,6,5 and 9.5 for 20 min, the solution was rapidly cooled and measured glutamatemediated activity by the method described above.

According to the data obtained at pH 5.5 enzyme was stable in the temperature range from 30 to 60oC and lost 45% of its original activity at 70oC; at a pH of 6.5, the enzyme was stable up to 50oC, losing 45% of its original activity at 60oC; at a pH of 9.5 - 40oC and lost 45% of its original activity at 50oC (Fig. 6). When the incubation of the enzyme in buffer solutions with pH 5,5,6,5 and 9.5 at the 90oC residual activerow and chelating agents on enzyme activity: to study the effect of different chemicals on glutamatemediated activity of the studied enzyme in the reaction mixture was added substances in a concentration of 1 mm and after incubation for five minutes was performed measurement of enzyme activity by the method described above. The results are presented in table 3.

As can be seen from table 3, the enzyme is quite tolerant to metals and SH-reagents. The enzyme is inhibited by 18% PHMB and 10% KSCN, but is not inhibited by chloride copper diethyldithiocarbamate, while the activity of already known glutaredoxins suppressed PMB 45% (U.S. Pat. USA N 4623626), and copper chloride 50% (U.S. Pat. USA N 4623626).

The stability of the enzyme. The purified enzyme was lyophilized. When stored in lyophilized form in a sealed vials under argon for 6 months at room temperature, the enzyme retains 90% of its original activity.

Thus, relatively high temperature and pH stability of the studied enzyme, as well as its resistance to copper ions and PHMB, high substrate specificity and high affinity to L-glutamate allow us to conclude about the great prospects of the use of glutaredoxins of a new strain of Streptomyces sp. Z-11-6 as a reagent in various clinical, biochemical and diagnostic tests.

The strain Streptomyces sp. Z-11-6 deposited in culture collections of the Department of Microbiology, Moscow state University. M. C. University and for more than a TLD is>/P>The description of Fig. 1-6: Fig.1. The optimum pH of the investigated L-glutaminases from Streptomyces sp. Z-11-6. Fig.2. pH stability of extracellular L-glutaminases from Streptomyces sp. Z-11-6 during its incubation for 60 minutes at 37oC. Fig.3. pH stability of extracellular L-glutaminases from Streptomyces sp. Z-11-6 during its incubation for 15 minutes at 45oC. Fig.4. pH stability of extracellular L - glutaminases from Streptomyces sp. Z-11-6 during its incubation for 15 minutes at 60oC. Fig.5. The operating temperature range of extracellular L-glutaminases from Streptomyces sp. Z-11-6. Fig.6. Temperature stability of extracellular L-glutaminases from Streptomyces sp. Z-11-6.

The strain Streptomyces sp. Z-11-6-producer of extracellular L-glutaminases.

 

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FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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