JPS63273471A - Microbial strain at-25 - Google Patents

Abstract

PURPOSE:To provide a microbial strain AT-25 useful for the production of sulfated polysaccharide DF-4639 having antithrombotic action, lipoprotein lipase activating action, carcinostatic action, etc. CONSTITUTION:The objective Arthrobacter AT-25 (FERM P-1357) is a Gram-positive brevi-type bacillus positive to catalase and negative to oxidase, free from acid resistance and sporulation property, having salt resistance, unable to grow in an environment having a salt concentration of >=10%, having absolute aerobic property, non-fermentative by OF-test, negative to VP reaction, arginine decomposition property and phosphatase activity, etc. The At-25 strain is cultured in a proper medium at 25-37 deg.C and pH6.5-8.5 under aerobic condition and a sulfated polysaccharide DF-4639 having the following physical and chemical properties is separated from cultured liquid and microbial cells. Molecular weight, 23,000; specific rotation, [alpha]D<25>=-36+ or -1 deg. (C=1.0, water); molar ratios of glucose:galactose:sulfate base:phosphorus are 1.0:6.3:7.5:0.7; etc.

Description

[Detailed description of the invention] The present invention relates to AT-25 bacteria.

As a result of searching for microbial products that have antithrombotic effects, the present inventor discovered that the culture filtrate and bacterial cell extract of the bacteria with isolation number AT-25 exhibited excellent antithrombotic effects. Ta. As a result of subsequent research, the present inventor succeeded in separating and purifying its active substance, and confirmed that it is a new substance whose main component is a sulfated polysaccharide. Conventionally, animal-derived heparin is well known as a sulfated polysaccharide, but the fact that sulfated polysaccharides are directly produced by microorganisms is extremely rare, and the following reports were found in the literature research conducted by the present inventor. The only thing that can be done is That is, Derby (
Clostridium walch by Darby et al.
A report on the presence of dermatan sulfate-like polymer as a capsular component of B. ii (J, Bacteriology, 103.
159 (1970)) or Stebe
(Archives o
f Microbiology 5.173 (197
5)) is limited to the two sides. However, these previously reported production bacteria and production substances are clearly different from the AT-25 bacteria and DF-4639 related to the present invention (as described later). is DF
-4839-producing bacteria may be cultured, but the most typical method is to culture AT-25 bacteria or its mutant strain in a medium in which the bacteria can grow, and then purify it from the culture solution or bacterial cells. It is.

The AT-25 bacterium is a bacterium that was newly isolated by the present inventor from a soil sample, and as a result of taxonomic examination, it was presumed to be a new bacterial species, and was tentatively classified into the genus Micrococcus.
Cus sp.
robacter sp.)^T-25 and has been deposited and preserved at the Microbial Technology Research Institute as Chokokenjo Bacteria Collection No. 1357. Hereinafter, "Virus" may be abbreviated as AT-25 bacteria.

The mycological properties of AT-25 bacteria are as follows.

a) Morphology 0 spherical to short culm-like (spherical in late stage of culture), diameter 0.5-0.
7. It has a medium growth in shape and shows a shiny golden brown color, but it is not diffusive.

■ Meat juice agar slant culture The results were the same as in the case of plate culture in (■) except that they grew in the form of threads.

■Meat juice agar liquid culture There was no fungal film formation, and there was turbidity and sediment throughout.

■Meat juice gelatin puncture culture (culture at 22°C) Grows along the puncture line and becomes a turnip-shaped vessel.

0 lidmus milk medium lidmus turns slightly red. Also, long-term culture (14 days or more)
Coagulation can also be seen.

C) Physiological properties ■Nitrate reduction negative, ■Denitrification reaction negative, ■MR reaction negative, ■VP reaction negative, ■Indole formation none, ■Hydrogen sulfide formation none, ■Starch hydrolysis ability
None, ■ Ability to utilize citric acid None, ■ Ability to utilize inorganic nitrogen sources, ■ Formation of water-insoluble pigments, ■ No urease action, ■ No oxidase action, [Phase] catalase action, ■ Growth range 9) 16.0-8.5
(optimum 7-8), temperature 10-37℃ (optimum 27-30℃)
), ■Aerobic, ■OF reaction negative, ■No acid or gas generation from the following 15 types of sugars.

Shi-Arabinose D-Galactose D-Sorvit D-jP ShiO-s Maltose D-Mannitol-glucose Sucrose Inojito-Romannose Lactose Glycerol Low-fructose Trehalose Starch d) Other properties ■Phosphatase action None, ■Arginine Resolution None, ■ Grows in sodium chloride 7.5* but not in sodium chloride, ■ Minimum inhibitory concentration for novobiocin and lysozyme are both 361 μg/ml, ■ Guanine and cytosine content (GC content) 72. !
n.

■Intracellular pigment (yellow) production, ■Contains diaminopimelic acid (DAP) as a cell wall component.

The above mycological properties are explained in Bergy's manual (Berci's manual).
ey's Manual of Determinat
ive Bacteriol-ogy, 8th Ed,
R, E, Buchaman and N, E, Glbb
ons(Williams and Wilkins
According to the description by Co., 1974)), the Orchard is a Durham-positive short bacillus that is non-motile, catalase-positive, oxidase-negative, has no acid fasting properties and no spore formation, and is salt tolerant, but It cannot grow if it contains 10% or more of salt, is absolutely aerobic, and is non-fermentable in the OF test.

Due to its various properties such as negative vP reaction, negative arginine decomposition ability, and negative phosphatase activity, it is most appropriate to assign it to the genus Arthrobacter.

Next, the basic method for obtaining sulfated polysaccharide DF-4639 will be described. That is, 0F-
All you need is one pile of 4639, and any medium that can grow the bacteria can be used, and in addition to carbon and nitrogen sources that can be used by the bacteria, inorganic salts and vitamins can be used. They can be used alone or in appropriate proportions or combinations. Carbon sources include glucose and glycerin, nitrogen sources include yeast extract, meat extract, peptone, soybean flour, cornstarch liquor, and inorganic ammonium salts such as ammonium hydrogen phosphate and ammonium nitrate.Other inorganic salts include magnesium, Appropriate amounts of metal salts such as potassium, iron, manganese, etc. can be added. In addition, (IF-41339 is a sulfated polysaccharide, it is extremely meaningful to add an appropriate amount of sulfate such as sodium sulfate or ammonium sulfate. The culture temperature and pH of the medium should be within the range where the bacteria used can grow. Temperature: 25-37℃, pH: 6.5-8.5
It is preferable to culture between Cultivation is preferably carried out under aerobic conditions, for example by shaking culture or aerated agitation culture in a culture tank.

The culture time may be 24 hours or more, but the culture time may be 50 to 200 hours.
time is preferable.

When the culture is completed, the culture is separated into bacterial cells and a filtrate using a centrifuge, and the filtrate is treated as described below. Note that if the amount of bacterial cells is small, they can be ignored, but if the amount is large, they should be treated separately according to the method described below.

That is, a solution of a quaternary ammonium salt, such as cetylpyridinium chloride (CPC), is added to the filtrate and stirred, and the resulting precipitate is separated and collected using a high-speed centrifuge. An electrolyte solution of an appropriate concentration, e.g. 10
Add 3 molar saline containing zero ethanol and stir to dissolve. Add ethanol until no new precipitate forms,
The resulting precipitate is collected, washed with ethanol and acetone, and dried to obtain a coarse powder. The resulting crude powder is dissolved in water again, diluted hydrochloric acid is added to adjust the pH to about 1.0, and then centrifuged at a low temperature (about 5° C.) to remove insoluble matter. The supernatant is collected and neutralized with a dilute caustic soda solution, and a quaternary ammonium salt such as cetyltrimethylammonium bromide (CTAB) aqueous solution is added thereto, and the resulting precipitate is collected by centrifugation. The resulting precipitate is added to 1 molar saline solution to sufficiently stir and solubilize contaminants such as nucleic acids, and then centrifuged to collect insoluble matter. This insoluble matter is added to a 3 molar saline solution containing one ring of ethanol and dissolved while heating to about 50°C. Ethanol is added to this solution to precipitate it, and the resulting precipitate is collected and washed with ethanol and then acetone, then dissolved in water again with stirring at room temperature and centrifuged at about 5°C to remove traces of insoluble matter. .

Add ethanol until no new precipitate forms in the solution,
The resulting precipitate is collected, washed sequentially with ethanol, acetone, and ether, and dried under reduced pressure at about 70°C to form DF-4.
639 is obtained as a white powder.

On the other hand, in order to obtain DF-4639 from bacterial cells, toluene or the like is added to an aqueous suspension of bacterial cells, and the suspension is left at room temperature to allow the bacterial cells to self-lyse, and the components within the bacterial cells are transferred to water.

Ethanol is added to this suspension until the suspension reaches 4 ml, precipitated proteins, nucleic acids, etc. are removed by filtration together with bacterial cell residue, and the filtrate is concentrated to remove ethanol. A CPC solution is added to this, and the resulting precipitate is collected and treated in the same manner as the culture filtrate.

According to the above method, DF-4639 is obtained as a sodium salt of a sulfated polysaccharide, and has the following physicochemical properties.

a) Elemental analysis values, sugar and protein content The 50-point values and average values are shown in Table 1.

Table 1 Note 1) (:, A, Antonopoulos, Acta
Chem, 5cand, 16, 1521 (1962)
According to the method described in.

Note 2) P, S, Chen et al., Anal, Chem,
According to the method described in 28.1756 (195B).

Note 3) Phenol-sulfuric acid method (galactose conversion) Note 4)
Lowry-Forin method (converted to bovine serum albumin) b) Glucose, galactose, sulfate groups, and phosphorus content molar ratio 0F-4639 was hydrolyzed in 1N sulfuric acid at 100°C for 5 hours, then neutralized with barium carbonate, and then dissolved in Dowex 50W.
The method of Khim et al. (J, ^m, chem, soc, 74.209
0 (1952) was applied to separate and quantify glucose and galactose. In addition, the molar ratio of sulfate groups and phosphorus is the content of S and P (calculated from leopard. Table 2 shows an example of the molar ratio of each component when glucose is 1.0 mol.

C) Amino acid and amino sugar content molar ratio 0F-463
9 was hydrolyzed in 3N hydrochloric acid at 100° C. for 16 hours, the hydrochloric acid was distilled off, and the results were determined using an amino acid analyzer. An example of the results is shown in Table 3, assuming 1.0 mol of glutamic acid.

d) Optical rotation [α] -36±1° (C-1,0, water) e) Molecular weight 23.000 (main peak in gel filtration method using dextran as a standard substance) f) Ultraviolet absorption 2 rng/m 1 220-340 nm in aqueous solution
No maximum absorption is observed.

g) Infrared absorption spectrum (Br tablet) As shown in Figure 1, it shows absorption specific to sulfated polysaccharides at 1240.840 (shoulder) and 810 cl'.

The biological properties of DF-4639 are shown below.

a) In vitro biological activity Note 1) Using Daiichi Chemical ■ product Note 2) Niglobulin dissolution time was measured using rat blood, and the sample concentration showing the maximum activity was expressed as the optimal concentration.

Note 3) Using rat plasma fraction, the sample concentration that doubled the clotting time of the control was determined.

Note 4) Clotting time was measured using bovine fibrinogen (Sigma product) and bovine thrombin (Parke Davis-Sankyo product), and the sample concentration that doubled the clotting time of the control was defined as the 50 zero inhibitory concentration.

b) Fibrinolytic induction activity in rats Using rats, the knee globulin dissolution time (ELT) before and after administration of the sample (intravenous injection) was measured over time and expressed as the rate of increase in fibrinolytic activity (96) using the following formula.

C) Acute toxicity LD5゜t, somg/kg or higher (mouse, intravenous injection), indicating that 0F-4639 exhibits fibrinolytic inducing activity equivalent to or higher than heparin in in vitro and rat experiments. was confirmed. Moreover, DF
-4639 has a characteristic that its unfavorable anticoagulant activity is significantly weaker than that of heparin. Furthermore, no hemagglutination promoting effect was observed, and the acute toxicity value was 1.
It is 500 mg/kg or more (intravenous injection), has very few side effects, and is promising as an antithrombotic drug. In addition, the present inventors have found through animal experiments that DF-4639 has useful effects such as riboprotein lipase activating activity, anticancer activity, and infection prevention effect.

Next, manufacturing examples will be described.The machine was w/v96 unless otherwise specified.

Example 1 Glucose 2*, peptone 0.5% F, cornstarch liquor 0.5, yeast extract 0.3 zero, salt 0.5% F
Liquid medium 100 consisting of and 0.3 zero of calcium carbonate
One platinum loop of AT-25 bacteria grown on an agar slant was inoculated into a 500 ml shake flask containing +nl.
A seed culture solution was obtained by culturing with shaking at ℃ for 3 days. Next, 20 glycerin, 0.5 ammonium sulfate, 0.1 k potassium phosphate,
20 fL of a culture medium consisting of 0.05 k sodium sulfate, 0 °C yeast extract, and 0° poultry was added to a 301-volume jar fermentor and sterilized at 120°C for 20 minutes, and then the pH was adjusted to 7.5.

This was inoculated with 600 ml of seed culture solution and cultured for 159 hours at a temperature of 30 t, an aeration rate of 1 i/min, and stirring at 250 revolutions/min. The obtained culture solution is centrifuged to remove a small amount of bacterial cells, and the supernatant is r! 500 ml of an aqueous cetylpyridinium chloride solution was added to the ill, and after standing for day and night, the precipitate was centrifuged. This was added to 600 ml of a 3M salt-10 zero (v/v) ethanol solution, stirred sufficiently to dissolve, and then 1.61 ml of ethanol was added and the resulting precipitate was filtered out using a glass filter. This precipitate was mixed with ethanol,
Then, it was washed with acetone and dried to obtain 37.0 g of coarse powder. This was dissolved in 500 ml of water, and at 0°C I
The resulting precipitate was removed by centrifugation after adjusting the pH to approximately 1.0 with N-hydrochloric acid, and after neutralizing the supernatant, 500 ml of an aqueous solution of cetyltrimethylammonium bromide was added, and the resulting precipitate was centrifuged. After thoroughly washing the precipitate with 1M saline, add 150ml of 3M salt-104k (v/v) ethanol solution, stir thoroughly to dissolve, add 450ml of ethanol, leave overnight, and centrifuge to remove the precipitate. collected. After washing this with ethanol, it was dissolved again in 150 ml of water, filtered through a glass filter, and a small amount of residue was removed in 50 ml of water.
Washed with.

The filtrate and washing liquid were combined and poured into ethanol at 2° C. with stirring to form a white precipitate. This precipitate was filtered through a glass filter, washed successively with ethanol, acetone, and ether, and dried under reduced pressure at 55° C. for 5 hours to obtain 13.9 g of DF-4639 as a white powder.

This substance exhibited the above-mentioned physical properties, and the five-pronged inhibitory concentration of thrombin activity was 0.7 μg/ml.

Example 2 Glycerin 29g, soluble starch 10, meat extract 10,
A medium 201 consisting of 1 part peptone and 0.2 parts salt was added to a 301 volume jar fermentor, and heated at 120°C.
The cells were sterilized for 20 minutes and cultured for 65 hours in the same manner as in Example 1. After the culture was completed, the culture solution was centrifuged and separated into bacterial cells and supernatant. 150 ml of an aqueous solution of 1096 cetylpyridinium chloride was added to the supernatant, followed by separation and purification according to Example 1 to obtain 3.2 g of white powder of 0F-4639. On the other hand, the bacterial cells were suspended in a mixture of 1,200 ml of water and 300 ml of toluene, and incubated at room temperature for 3 minutes with occasional stirring.
I left it for days. After toluene was distilled off under reduced pressure, ethanol was added to the solution until the concentration reached about 40% F (V/V), and precipitated proteins and nucleic acids were removed by filtration together with the bacterial cells.

After concentrating the obtained filtrate under reduced pressure to about 1° C., 30 ml of 10% saline and 100 ml of 10*cetylpyridinium chloride aqueous solution were added, and the resulting precipitate was centrifuged. Hereinafter, white powder DF-4 was purified in accordance with Example 1.
2.4g of 639 was obtained.

[Brief explanation of drawings]

Figure 1 is an infrared absorption spectrum.

Claims (1)

[Claims] AT-25 bacterium (Feikoken Bacteria No. 5255 or Fiber Science Laboratory No. 1357)