JPS6250475B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel physiologically active substance SPF-1 and a method for producing the same.

More specifically, the present invention relates to SPF-1, a physiologically active substance that is extremely promising as an antitumor substance, and a method for producing the same.

Conventionally, attenuated viable bacterial cells of hemolytic streptococcus have been prepared into preparations and have already been used as anticancer drugs.

In addition, a method of crushing Streptococcus pyogenes bacterial cells, extracting the active ingredients with water or a salt solution, adding an organic solvent, and recovering the antitumor ingredients as precipitates (Japanese Patent Publication No. 38-1647), Known methods include lysing bacteria with a lytic enzyme, lysozyme, cellulase, or proteolytic enzyme and fractionating the active fraction as a water-soluble fraction (British Patent No. 1163865).

As described above, it is widely known that Streptococcus bacteria themselves or their bacterial cell components have antitumor activity, but what was previously known was that the Streptococcus bacteria themselves or their cell components have antitumor activity. It was nothing more than matter. If you try to isolate the active ingredient from the bacterial body or inside the bacterial body,
It was necessary to fractionate the entire bacterial body by lysing it or mechanically crushing it. Such treatment complicates purification and makes it extremely difficult to isolate the active ingredient. A protein with a molecular weight of 150,000 is known to have been actually isolated and measured as an active ingredient. (Japanese Patent Publication No. 48-43841) As a result of intensive research in search of superior anti-tumor active ingredients for hemolytic streptococci, the present inventors discovered a completely new physiologically active substance SPF-1 in the culture solution. be.

The physiologically active substance SPF-1 of the present invention is excreted outside the bacterial cells during culture and becomes present in the culture solution, so it can be removed by passing through the bacterial cells and the culture solution can be purified. becomes quite easy.

The physiologically active substance SPF-1 of the present invention is excreted into the culture medium and is characterized by its extremely small molecular weight of approximately 500 to 7,000.

Until now, there have been no physiologically active substances related to streptococcus that have accumulated in the culture solution, and no known molecular weight of less than tens of thousands of thousands of pounds.
SPF-1 is recognized as a completely new substance.

In addition, although the physiologically active substance SPF-1 of the present invention is recognized as a peptide substance based on elemental analysis and color reaction, it has a unique absorption in the ultraviolet absorption spectrum and is not considered to be a widely known antitumor active substance. It is a substance that is clearly different and is recognized as a new substance.

The present invention includes a method for producing the physiologically active substance SPF-1, which is characterized by culturing a physiologically active substance SPF-1-producing bacterium belonging to the genus Streptococcus and collecting the physiologically active substance SPF-1 from the culture. It is something.

In the present invention, bacteria that produce the physiologically active substance SPF-1 belonging to the genus Streptococcus are widely used; one example is Streptococcus pyogenes ATCC21060.
can be given.

Natural media such as meat extract medium, yeast extract medium, and brain heart infusion medium (BHI medium) are often used as the culture medium, but any medium that allows Streptococcus bacteria to grow effectively can be used as a carbon source. A general medium containing a nitrogen source, etc. can also be used.

Culture at PH6.0-8.0, preferably 6.8-7.2 and 30-30
It is common to perform static culture at 40°C, preferably 35 to 37°C, in an anaerobic manner, but other modified methods such as agitation culture may also be employed.

In the present invention, adding penicillin or its related substances at an appropriate time during culture plays an important role in obtaining the physiologically active substance SPF-1.

The timing of addition of penicillin or related substances is 37℃.
A period of 3 to 15 hours, particularly 5 to 10 hours after the culture reaches logarithmic growth phase is preferred. After that, by continuing the culture for 1 hour to 20 hours, preferably 5 to 15 hours, the physiologically active substance SPF is added to the culture solution.
-1 can be accumulated in large quantities.

Penicillin or its related substances may be any known related substances that have similar effects to penicillin, but penicillin G is commonly used. The amount added is 100~ for penicillin G.
About 3000 units/ml, preferably 1000 units/ml of culture solution is sufficient.

The resulting culture solution was centrifuged to remove bacterial cells, ammonium sulfate was added to the solution, and a fraction with a saturation level of 50 to 80% was collected. The resulting precipitate was added to a buffer containing a stabilizer. Dissolve in liquid.

A solution containing the physiologically active substance SPF-1 can be stored in a frozen state or by lyophilization. This substance can be further purified by contacting it with an ion exchanger or gelling agent, if necessary. As the ion exchanger, ion exchange resin, ion exchange cellulose, ion exchange Cephadex (manufactured by Pharmacia), etc. are used, and as the gelling agent,
Toyo Pearl HW50F or HW-50SF (Toyo Soda
Co., Ltd.), Cephadex (Pharmacia Co., Ltd.), etc. are used. Although calcium phosphate gel can be used as it is, it is convenient to use it in the form of hydroxylapatite. The aqueous solution of the substance obtained as described above is passed through a column filled with these ion exchangers or gelling agents at an appropriate speed, or it is poured into a container containing the ion exchanger or gelling agent. The aqueous solution is added all at once to bring these treatment agents and active substances into contact. Elution is performed using a buffer solution with appropriate salt concentration and pH. Two or more ion exchangers, gelling agents, or calcium phosphate gels may be used in combination. For example, contact with DEAE Cephadex and add the eluted solution to Toyopearl.
Contact with HW50F or 50SF and elution can further improve the purification effect.

The substance obtained here is a peptide substance, which becomes a white powder when freeze-dried.

Next, we will show the physicochemical properties of the physiologically active substance SPF-1.

1 Elemental analysis C: 41.26%, H: 4.91%, N: 6.52% 〓 〓 〓 36.64% 4.10% 5.40% 2 Molecular weight When measured by gel permeation method, the molecular weight is approximately 500 ~
It is 7000.

3. Decomposition point This substance turns brown at 170℃ and turns black at 200℃ and decomposes.

4 Specific rotation [α] ²⁰ _D = +63.3 to 64.3° (c = 1.04) 5 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 3.3% aqueous solution of this substance is shown in Figure 1, and the ultraviolet absorption spectrum of a 3.1% aqueous solution The absorption spectrum is shown in FIG. All have characteristic absorption at 257nm, 265nm, 280nm, and 287nm.

6 Infrared absorption spectrum It is shown in FIG.

7 Solubility in solvents Soluble in water, but insoluble in solvents such as methanol, ethanol, n-butanol, isobutanol, n-propanol, n-hexane, chloroform, acetone, methyl isobutyl ketone, and ethyl ether. .

8 Distinction between basic, acidic, and neutral The pH of a 0.85% aqueous solution of this substance is 6.5.

9 Color of matter It is a white powder.

10 Color reaction Ninhydrin reaction + Biuretz reaction + Mauritsch reaction - Decier reaction - Anthrone reaction - Cysteine sulfate reaction - 11 Stabilization This substance is L-cysteine, dithiothreitol (DTT), glycerol, albumin, globulin, (NH ₄ ) ₂ Stabilized by adding SO ₄ , salt, etc.

Next, examples of the present invention will be shown.

In the Examples, the activity unit of the physiologically active substance SPF-1 was measured using the Udaka method (Journal of Antibiotics
vol35No.10 1319-1325OCT1982). For measurement, polymer-permeable E. coli mutant strain MP-2 was used.
(FERM-P5432) (Agric.Biol.Chem.43 371
(1979) to conduct a bioassay using antibacterial activity against MP-2 as an indicator.

That is, a medium ( _M3 medium) consisting of 1.75% Bacto Antibiotic Medium 3 (product of Difco) and 1.3% agar was heat sterilized at 120°C for 15 minutes, dispensed into 20ml portions into a shear dish, and allowed to cool. Prepare plate medium.

Meanwhile, peptone 0.5%, meat extract 0.5%,
A medium consisting of 0.3% NaCl and 0.8% agar was heated at 120℃ for 15 minutes.
Sterilize by heating for a minute. Thereafter, the medium is kept in a constant temperature bath at 42°C, and when the temperature of the medium reaches 42°C, MP-2 bacteria, which have been previously cultured at 37°C for 17 hours, are added to the medium so that 10 ⁴ cells are present in 1 ml. by pipette 2
Collect ml and add it to the surface of _M3 medium prepared in advance and spread quickly and uniformly to solidify. Appropriately dilute the test solution containing the physiologically active substance SPF-1, and soak 0.05 ml of the solution into a paper disk (diameter 8 mm) (Toyo Shi). Place this paper disk on the production plate,
Culture at 37°C for 17 hours, and measure the size of the inhibition zone created by the physiologically active substance SPF-1. 1 unit (1u) concentration of SPF-1 that gives a diameter of inhibition circle of 10mm
It is defined as

Example 1 Streptococcus pyogenes ATCC21060 (BHI
Inoculate 100 ml of culture medium and pre-culture by static culture at 37℃ for 8 hours.
After culturing anaerobically at 15°C with stirring for 15 hours, 1000 units/ml of penicillin G was added and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contains 120 units of the physiologically active substance SPF-1.
It contained ml.

Example 2 Yeast extract 3.0% BHI 1.0% Polypeptone 1.0% Maltose 0.3% KH ₂ PO ₄ 0.1% MgSO ₄ 7H ₂ O 0.02% PH = 7.2 Streptococcus pyogenes ATCC21060 was added to medium B2 with the following composition as in Example 1. Inoculate 100ml of precultured culture solution into
After culturing anaerobically at 37°C for 15 hours with stirring, 1000 units/ml of penicillin G was added, and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 152 units/ml of the physiologically active substance SPF-1.

Example 3 Meat extract 1% Polypeptone 1% NaCl 0.5% Maltose 0.3% KH ₂ PO ₄ 0.1% MgSO ₄ 7H ₂ O 0.02% PH=7.1 Streptococcus pyogenes ATCC21060 was added to medium C2 with the following composition as in Example 1. Inoculate 100ml of precultured culture solution into
After culturing anaerobically at 37°C for 15 hours with stirring, 1000 units/ml of penicillin G was added, and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 240 units/ml of the physiologically active substance SPF-1.

Example 4 Meat extract 1.5% Polypeptone 1.0% NaCl 0.5% Casamino acids 0.5% Yeast extract 0.2% PH=7.1 Streptococcus pyogenes ATCC21060 was precultured in the same manner as in Example 1, and 100 ml of the culture medium D2 with the following composition was added to the medium D2. After inoculating the cells stored at 20°C and culturing them anaerobically at 37°C for 15 hours with stirring, 1000 units/ml of penicillin G was added, and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 108 units/ml of the physiologically active substance SPF-1.

Example 5 Meat extract 1.5% Polypeptone 1.0% NaCl 0.5% Casamino acids 0.25% Yeast extract 0.25% PH = 7.1 100 ml of the seed culture solution obtained by pre-cultivating Streptococcus pyogenes ATCC21060 in the same manner as in Example 1 was added to medium E2 with the following composition. After inoculation and culturing anaerobically at 37℃ for 15 hours with stirring,
Add 1000 units/ml of penicillin G and further culture.
Continues for 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 180 units/ml of the physiologically active substance SPF-1.

Example 6 Streptococcus pyogenes in the medium E2
Inoculate 100 ml of the culture solution stored at -20°C with ATCC21060 precultured in the same manner as in Example 1, and culture it anaerobically at 37°C for 15 hours with stirring. Then, add 1000 units/ml of penicillin G and continue culturing for 5 hours. continue. The resulting culture was centrifuged to remove bacterial cells.

The culture solution contained 100 units/ml of the physiologically active substance SPF-1.

Example 7 Meat extract 3.0% Polypeptone 1.0% NaCl 0.5% Casamino acids 0.5% PH = 7.1 Streptococcus pyogenes ATCC21060 was precultured in the same manner as in Example 1 in the following medium F2.
After inoculating the stock and culturing anaerobically at 37°C for 5.5 hours with stirring, 1000 units/ml of penicillin G was added and the culture was continued for an additional 15 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 120 units/ml of the physiologically active substance SPF-1.

Example 8 In the following medium G2, BHI 3.7% Polypeptone 0.5% Maltose 0.3 KH ₂ PO ₄ 0.1% MgSO ₄ 7H ₂ O 0.02% PH = 7.1 Streptococcus pyogenes ATCC 21060 was precultured in the same manner as in Example 1, and 100 ml of the culture was prepared. After inoculating and culturing anaerobically at 37°C for 15 hours with stirring, add 1000 units/ml of penicillin G and continue culturing.
Continues for 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 300 units/ml of the physiologically active substance SPF-1.

Example 9 In the medium E3, Streptococcus pyogenes
Add and inoculate 300 ml of the seed culture obtained by statically culturing ATCC21060 at 37°C for 8 hours. After culturing anaerobically at 37°C for 15 hours with stirring, add 1000 units/ml of penicillin G and continue culturing for an additional 5 hours. . The obtained culture solution was centrifuged to remove bacterial cells.

Culture solution 3 contains 56x the physiologically active substance SPF-1.
It contained 10 ⁴ units.

Ammonium sulfate was added to the culture solution, and a fraction with a saturation level of 50 to 80% was collected to obtain a precipitate. This precipitate contained 50×10 ⁴ units of the physiologically active substance SPF-1.

The entire amount of this precipitate was dissolved in 300 ml of stabilizer-containing buffer solution, and the solution was transferred to a DEAE-cellulose column (5×
70cm), and the physiologically active substance SPF was adsorbed.
This is eluted stepwise using 0.3M NaCl solution, and the active portion is separated. The activity obtained is 30×
It was 10 ⁴ units.

The active moiety is dialyzed against buffer and then DEAE
-Sephadex A-25 column (2.6 x 50cm)
In addition, the active moiety is adsorbed, and elution is performed while linearly increasing the salt concentration in the phosphate buffer.
Separate the active portion. The activity obtained is 10.6×10 ⁴
It was a unit.

Furthermore, this eluate is concentrated and applied to a gel filtration material Toyopearl HW-50F column (2 x 100 cm) to separate the active fraction. The activity obtained was 2.2×10 ⁴ units.

The eluate thus obtained was freeze-dried to obtain 350 mg of a white powder of the physiologically active substance SPF-1.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of a 3.3% aqueous solution of the physiologically active substance SPF-1, and Figure 2 shows the same.
The ultraviolet absorption spectrum of a 3.1% aqueous solution is shown, and FIG. 3 also shows the infrared absorption spectrum.

Claims (1)

[Claims] 1. A physiologically active substance having the following physicochemical properties:
SPF-1. 1 Elemental analysis C: 41.26%, H: 4.91%, N: 6.52% 〓 〓 〓 36.64% 4.10% 5.40% 2 Molecular weight When measured by gel permeation method, the molecular weight is about 500~
7000. 3. Decomposition point This substance turns brown at 170℃ and turns black at 200℃ and decomposes. 4 Specific rotation [α] ²⁰ _D = +63.3 to 64.3° (c = 1.04) 5 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 3.3% aqueous solution of this substance is shown in Figure 1, and the ultraviolet absorption spectrum of a 3.1% aqueous solution The absorption spectrum is shown in FIG. All have characteristic absorption at 257nm, 265nm, 280nm, and 287nm. 6 Infrared absorption spectrum Shown in Figure 3. 7 Solubility in solvents Soluble in water, but insoluble in solvents such as methanol, ethanol, n-butanol, isobutanol, n-propanol, n-hexane, chloroform, acetone, methyl isobutyl ketone, and ethyl ether. . 8. Distinction between basic, acidic, and neutral The pH of a 0.85% aqueous solution of this substance is 6.5. 9. Color of substance: White powder. 10 Color reaction Ninhydrin reaction + Biuretz reaction + Mauritsch reaction - Decier reaction - Anthrone reaction - Cysteine sulfate reaction - 11 Stabilization This substance is L-cysteine, dithiothreitol (DTT), glycerol, albumin, globulin, (NH ₄ ) ₂ Stabilized by adding SO ₄ , salt, etc. 2 Physiologically active substances belonging to the genus Streptococcus
A method for producing the physiologically active substance SPF-1, which is characterized by culturing SPF-1 producing bacteria and collecting the physiologically active substance SPF-1 from the culture. 3 Physiologically active substances belonging to the genus Streptococcus
When culturing the SPF-1 producing bacteria, penicillin or a related substance is added to the culture at an appropriate time during the culture. Manufacturing method.