JPH027631B2 - - Google Patents

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 found that the culture filtrate of the bacteria with isolation number AT-25 and its bacterial cell extract 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 inventor's literature research revealed the following report. can only be seen. Namely, Clostridium of Darby et al.
Report on the existence of dermatan sulfate-like polymer as a capsular component of M. welchii (J.Bacteriology, 103 ,
159 (1970)) or Steber et al.
A report on sulfated polysaccharides consisting of glucose, mannose, and galactose as cell wall components of Halococcus morrhuae (Archives of Microbiology)
105 , 173 (1975)). but,
These previously reported producing bacteria and produced substances are clearly different from the AT-25 bacteria and DF-4639 related to the present invention, as described below. sulfated polysaccharide
DF-4639 can be produced by culturing DF-4639-producing bacteria, but the most typical method is to culture AT-25 bacteria or its mutants in a medium in which the bacteria can grow, and then add the culture solution. Alternatively, it can be separated and purified from bacterial cells. The AT-25 bacterium is a bacterium newly isolated by the present inventor from a soil sample, and as a result of taxonomic examination, it is estimated that it is a new bacterial species, and tentatively it belongs to the genus Micrococcus (Micrococcus sp. AT-25). It has been deposited and preserved at the National Institute of Microbial Technology as Microbiological Research Institute No. 5255, and as Arthrobacter sp. AT-25, Microbiological Research Institute No. 1357. Hereinafter, this bacterium may be abbreviated as AT-25 bacterium. The mycological properties of AT-25 bacteria are as follows. (a) Morphology: spherical to short rod-like (spherical at late stage of culture), diameter 0.5
~0.7 x 0.7-1.5 μm, pleomorphic, non-motile, no sporulation, positive Gram staining,
No acid-fastness (b) Growth status in each medium Broth agar plate culture Moderate growth in dotted or circular shapes, with a shiny golden brown color, but no diffusivity. Juicy agar slant culture The growth was similar to that of the plate culture except that it grew in a filamentous manner. Meat juice agar liquid culture No bacterial film formation, turbidity and sediment throughout. Meat juice gelatin puncture culture (culture at 22°C) Grows along the puncture line and liquefies into a turnip. Litmus milk medium Turns litmus a little red. Coagulation is also observed in long-term culture (14 days or more). (c) Physiological properties Nitrate reduction negative, denitrification reaction negative,
MR reaction negative, VP reaction negative, indole formation none, hydrogen sulfide generation none, starch hydrolysis ability none, citric acid utilization none, inorganic nitrogen source utilization yes, water-insoluble pigment formation yes, urease action none , no oxidase action,
Catalase action, growth range PH6.0
~8.5 (optimum 7~8), temperature 10~37℃ (optimum 27~
30℃), aerobic, OF reaction negative, below 15
No acid and gas production from seed sugars. L-arabinose D-galactose D-xylose Epidermis D-glucose Sucrose D-mannose Lactose D-fructose Trehalose D-sorbitol D-mannite Glycerin Inosit Starch (d) Other properties Phosphatase action None, ability to decompose arginine None , grows in 7.5% sodium chloride, but 10
%, the minimum inhibitory concentrations for novobiocin and lysozyme are both 3.1 μg/ml, and the guanine and cytosine content (GC content)
72.9%, intracellular pigment (yellow) production, diaminopimelic acid (DAP) as a cell wall component
can be. The above mycological properties are described in Bergey's Manual of Determinative.
Bacteriology, 8th Ed. RE Buchaman and N.
E. Gibbons (Williams and Wilkins Co., 1974))
According to the description in It is strictly aerobic, and is non-fermentable in the OF test. Due to its various properties such as negative VP reaction, 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, DF−4639
All that is required is for DF-4639 to accumulate in the culture medium and the bacterial cells of the bacteria used to obtain the bacteria.For this purpose, any medium in which the bacteria used can grow can be used; In addition to carbon sources and nitrogen sources that can be converted into carbon, inorganic salts and vitamins can be used alone or in combination in appropriate proportions. Carbon sources include glucose and glycerin; nitrogen sources include yeast extract, meat extract, peptone, soybean flour,
Inorganic ammonium salts such as corn staple liquor, ammonium hydrogen phosphate, and ammonium nitrate are used, and in addition, appropriate amounts of metal salts such as magnesium, potassium, iron, and manganese can be added as inorganic salts. Furthermore, since DF-4639 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 can be within the range where the bacteria used can grow, but the temperature should be 25
It is preferable to culture at ~37°C and pH between 6.5 and 8.5. 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 preferably 50 to 200 hours. 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. Regarding bacterial cells,
If the amount is small, it can be ignored, but if it is large, it will be processed separately according to the method described later. 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, for example, 3 molar saline containing 10% ethanol, is added to the obtained precipitate and dissolved with stirring. Ethanol is added until no new precipitate is formed, and the resulting precipitate is collected, washed with ethanol and acetone, and dried to obtain a coarse powder. The coarse powder obtained here was dissolved in water again, and diluted hydrochloric acid was added to bring the pH to approx.
After adjusting to 1.0, centrifuge at low temperature (approximately 5°C) to remove insoluble matter. The supernatant is collected, neutralized with dilute caustic soda solution, and treated with a quaternary ammonium salt, such as cetyltrimethylammonium bromide (CTAB).
The precipitate formed by adding the aqueous solution is collected by centrifugation. The obtained precipitate is added to 1 molar saline solution, and impurities such as nucleic acids are sufficiently stirred and solubilized, followed by centrifugation to collect insoluble matter. This insoluble matter is added to 3 molar saline containing 10% 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 again in water with stirring at room temperature and centrifuged at about 5°C to remove traces of insoluble matter. . Ethanol was added to the solution until no new precipitate was formed, and the resulting precipitate was collected and washed sequentially with ethanol, acetone, and ether.
Drying under reduced pressure at °C yields DF-4639 as a white powder. On the other hand, 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 internal components of the bacterial cells are transferred to water. Ethanol is added to this suspension until the concentration reaches 40%, precipitated proteins, nucleic acids, etc. are removed by filtration along with bacterial cell residue, and the filtrate is concentrated to remove ethanol. Add CPC solution to this and collect the resulting precipitate.
The following treatment is performed in the same manner as for 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 Table 1 shows the values of 5 lots and the average value.

[Table] (b) Molar ratio of glucose, galactose, sulfate groups, and phosphorus content DF-4639 was hydrolyzed in 1N sulfuric acid at 100°C for 5 hours, then neutralized with barium carbonate, and Dowex
Using the effluent obtained by applying 50W (H type),
The method of Khim et al. (J.Am.Chem.Soc., 74 , 2090
(1952) was applied to separate and quantify glucose and galactose. Furthermore, the molar ratio of sulfate groups and phosphorus was calculated from the S and P contents (%). Table 2 shows an example of the molar ratio of each component when glucose is 1.0 mol.

[Table] (c) Content molar ratio of amino acids and amino sugars An example of the results of hydrolyzing DF-4639 in 3N hydrochloric acid at 100℃ for 16 hours, distilling off the hydrochloric acid, and quantifying it using an amino acid analyzer is 1.0% of glutamic acid. It is shown in Table 3 as moles.

[Table] (d) Optical rotation [α] ²⁵ _D −36±1° (C = 1.0, water) (e) Molecular weight 23000 (main peak in gel filtration method using dextran as a standard substance) (f) Ultraviolet absorption In a 2 mg/ml aqueous solution, no maximum absorption is observed between 220 and 340 nm. (g) Infrared absorption spectrum (KBr tablet) As shown in Figure 1, 1240, 840 (shoulder) and 810
It shows an absorption characteristic of sulfated polysaccharide in cm ^-1 . The biological properties of DF-4639 are shown below. (a) In vitro biological activity

[Table] (b) Fibrinolysis induction activity in rats Using rats, the euglobulin lysis time (ELT) was measured over time before and after administration of the sample (intravenous injection), and was calculated as the fibrinolytic activity increase rate (%) using the following formula. expressed.

[Table] (c) Acute toxicity LD ₅₀ 1500 mg/Kg or more (mouse, intravenous injection) As described above, DF-4639 has fibrinolytic inducing activity equivalent to or greater than heparin in in vitro and rat experiments. It was confirmed that Moreover, DF-4639 has a characteristic that its undesirable anticoagulant activity is significantly weaker than that of heparin. Furthermore, no hemagglutinating effect was observed, and the acute toxicity value was over 1500 mg/Kg (intravenous injection), so it has very few side effects and is promising as an antithrombotic drug. In addition, the present inventors have discovered that DF-
Animal experiments have shown that 4639 has useful effects such as lipoprotein lipase activation, anticancer, and infection prevention effects. Next, manufacturing examples will be described, in which % is w/v % unless otherwise specified. Example 1 Glucose 2%, peptone 0.5%, cornstarch liquor 0.5%, yeast extract 0.3%, salt 0.5%
Liquid medium 100 consisting of and 0.3% calcium carbonate
One platinum loop of AT-25 bacteria grown on an agar slant was inoculated into a 500 ml shake flask containing 30 ml of
A seed culture solution was obtained by culturing with shaking at ℃ for 3 days.
Next, 2% glycerin, 0.5% ammonium sulfate, 0.1% potassium phosphate, 0.05% sodium sulfate, and 0.2 yeast extract.
After adding 20% medium to 30 volumes of jar fermenter and sterilizing it at 120℃ for 20 minutes, the pH was adjusted to 7.5.
Adjust to. This was inoculated with 600 ml of seed culture solution and cultured for 159 hours at a temperature of 30°C, an aeration rate of 10 per minute, and a stirring rate of 250 revolutions per minute. The obtained culture solution was centrifuged to remove a small amount of bacterial cells, and 500 ml of 10% cetylpyridinium chloride aqueous solution was added to the supernatant liquid 18.
After standing overnight, the precipitate was centrifuged. This is 3M
Add to 600 ml of common salt-10% (v/v) ethanol solution, stir thoroughly to dissolve, and then add ethanol.
A precipitate formed by adding 1.6 was filtered out using a glass filter. This precipitate was washed with ethanol, then with acetone, and dried to obtain 37.0 g of crude powder.
Dissolve this in 500ml of water, adjust the pH to approximately 1.0 with 1N hydrochloric acid at 0°C, remove the resulting precipitate by centrifugation,
After neutralizing the supernatant, 500 ml of 10% aqueous cetyltrimethylammonium bromide solution was added, and the resulting precipitate was centrifuged. After thoroughly washing the precipitate with 1M saline,
Add 150ml of 3M salt-10% (v/v) ethanol solution, stir thoroughly to dissolve, and add ethanol.
After adding 450 ml and leaving it overnight, the precipitate was collected by centrifugation. After washing it with ethanol, water it again.
Dissolve in 150ml, filter through a glass filter,
A small amount of residue was washed with 50 ml of water. Combine the filtrate and washing liquid and pour into ethanol 2 with stirring,
A white precipitate was formed. 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 exhibits the above-mentioned physical properties and has a 50% reduction in thrombin activity.
The % inhibitory concentration was 0.7 μg/ml. Example 2 Medium 20 consisting of 2% glycerin, 1% soluble starch, 1% meat extract, 1% peptone and 0.2% salt was added to a 30 volume jar fermenter and sterilized at 120°C for 20 minutes. Culture was carried out for 65 hours in the same manner as in 1. After the culture was completed, the culture solution was centrifuged and separated into bacterial cells and supernatant. Add 150ml of 10% cetylpyridinium chloride aqueous solution to the supernatant,
Following separation and purification according to Example 1, DF-4639
3.2 g of white powder was obtained. On the other hand, the bacterial cells were suspended in a mixture of 1200 ml of water and 300 ml of toluene, and left at room temperature for 3 days with occasional stirring. After toluene was distilled off under reduced pressure, ethanol was added to the solution to a concentration of about 40% (v/v), and precipitated proteins and nucleic acids were removed by filtration along with the bacterial cells. The obtained filtrate is about 1
After concentration under reduced pressure, 30 ml of 10% saline and 100 ml of 10% aqueous cetylpyridinium chloride solution were added, and the resulting precipitate was centrifuged. Hereinafter, 2.4 g of white powder DF-4639 was isolated and purified according to Example 1.
Obtained.

[Brief explanation of drawings]

Figure 1 is an infrared absorption spectrum.

Claims (1)

[Claims] 1. Arthrobacter sp.AT-25 capable of producing sulfated polysaccharide DF-4639.