JPS6030689A - Physiologically active substance spf-1 and its preparation - Google Patents

Abstract

PURPOSE:To produce a novel physiologically active substance SPF-1 extremely hopeful as an antitumor substance, by culturing a microbial strain belonging to Streptomyces genus. CONSTITUTION:A microbial strain belonging to Streptomyces genus and capable of producing physiologically active substance SPF-1, e.g. Streptomyces pyogenes ATCC21060, is cultured in a medium added with penicillin or its relating substance such as penicillin G at a proper stage during the cultivation, and the objective physiologically active substance SPF-1 is separated from the culture liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel physiologically active substance 5PF-1 and a method for producing the same.

More specifically, the present invention relates to 5PF-1, a physiologically active substance that is extremely promising as an antitumor component, and a method for producing the same.

Conventionally, preparations made by attenuating viable streptococcus cells,
It is already used as an anticancer drug.

A method of crushing the bacterial cells of Streptococcus pyogenes, extracting the active ingredients with drained water or saline solution, adding an organic solvent, and collecting the antitumor ingredients as a precipitate (Special Publication No. 3B-117). A method of lysing bacteria and fractionating the active fraction as a water-soluble fraction using a lytic enzyme, lysozyme, cellulose or proteolytic enzyme (British Patent No. 1163)
No. 865), etc. are known.

As described above, it is widely known that Streptococcus bacteria themselves or their bacterial cell components have antitumor activity. It was nothing more than a cellular constituent. In order to isolate the active ingredient from the bacterial cells or inside the bacterial cells, the entire bacterial cell had to be fractionated by lysis or mechanical crushing. According to such treatment, purification was complicated and isolation of the active ingredient was extremely difficult. It is known that a protein with a molecular weight of 150,000 has been actually isolated and measured as an active ingredient. (0 Publication No. 48-43841) The present inventors have conducted extensive research on superior antitumor active ingredients against streptococcus, and as a result, 1. A completely new physiologically active substance, 5PF-1, was discovered in the culture medium.

The physiologically active substance 8PF-1 of the present invention is excreted from the cultured living bacteria and exists in the culture solution, so it can be removed by filtering the bacteria and purifying the culture P solution. Therefore, isolation is quite easy.

The physiologically active substance 5PF-1 of the present invention is excreted into the culture medium and is characterized by its extremely small molecular weight of about 500 to 7,000 (!:ζ).

Hitherto, no physiologically active substance related to streptococcus has accumulated in the culture solution, and no known substance with a molecular weight of less than tens of thousands of thousands of people has been found.1The physiologically active substance 5PF-1 of the present invention is a completely new substance. It is recognized that

In addition, the physiologically active substance SPF'-1 of the present invention is recognized as a peptide substance based on elemental analysis, color reaction, etc., but it has a unique absorption in the ultraviolet absorption spectrum and is a well-known antitumor active substance. It is a substance that is clearly different from the above, and is recognized as a novel substance.

The present invention provides a method for producing the physiologically active substance 5pF-1, which is characterized by culturing a physiologically active substance S P F-1 producing bacterium belonging to the genus Streptococcus and collecting the physiologically active substance 5PF-1 from the culture. It is inclusive.

In the present invention, bacteria that produce the physiologically active substance 5PF-1 belonging to the genus Streptococcus are widely used;
Streptococcus pyogenes as a column (5 trep
tococcus pyogenes) AT CC
21060 is mentioned.

Culture media include meat extract medium, yeast extract medium, plain,
Natural media such as Heart Infusion Medium (B1 Technique Medium) are often used, but general media containing carbon sources, nitrogen sources, etc. can also be used as long as the medium allows Streptococcus bacteria to grow effectively. I can do this.

The culture is carried out at pH 6.0 to 8.0, preferably 6.8 to Z2, at 60 to 40°C, or preferably at 35 to 57°C, under anaerobic conditions.
This static culture is generally performed, but other modified methods such as agitation culture may also be employed.

In the present invention ζ, adding penicillin or its related substances at an appropriate time during the culture is a physiologically active substance 5P.
It will play an important role in the acquisition of F-1.

The timing of addition of penicillin or related substances is 5% to 15% for 5 to 15 hours after the logarithmic growth phase of the culture at 67°C.
10 hours is preferred. Thereafter, by continuing the culture for 1 to 20 hours or 5 to 15 hours, a large amount of the physiologically active substance 8PF-1 can be accumulated in the culture solution.

Penicillin or its related substances may be any related substance that has a similar action to the well-known penicillin, but penicillin G is commonly used. The amount of penicillin G to be added is 100 to 3000 units/d, preferably about 1000 units/s+/culture solution.

The obtained culture solution is centrifuged to remove the bacterial cells, ammonium sulfate is added to the obtained solution, and both portions with a saturation level of 50 to 80% are separated.The resulting precipitate is added to a buffer solution containing a stabilizer. dissolve in

A solution containing the biologically active substance JSPF-1 can be stored in a frozen state or by lyophilization. This material can be further purified by contacting it with an ion exchanger or gel filtration agent, if necessary. As the ion exchanger, ion exchange resin, ion exchange cellulose, ion exchange Sephadex (manufactured by Pharmacia), etc. are used, and as the gel filtration agent, Toyobar HW50Ft or HW-50SFC is used.
Toyo 1st (Ei 11), Sephadex (manufactured by Pharmacia), etc. are used. Although the aged calcium phosphate gel can be used as is, it is convenient to use it in the form of hytroxylapatite. The aqueous solution of the substance obtained as described above is placed in a column packed with these ion exchangers or gel filtration agents.

The aqueous solution is brought into contact with these treatment agents by passing it through at a suitable rate or by adding the aqueous solution all at once into a container containing an ion exchanger or F7 superagent. Elution is performed using a buffer solution of appropriate salt concentration and pH. Two or more types of ion exchangers, gel filtration agents, or calcium phosphate gels can also be used in combination. For example, DF
The purification effect can be further improved by contacting with 3AD Sephadex and further contacting the eluted solution with Toyovar HW50F or 508F for elution.

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

Next, the physicochemical properties of the physiologically active substance 8PF-1 will be shown.

1. Elemental analysis 2. Molecular weight As measured by gel filtration method, the molecular weight is approximately 500 to 7. (I
It is QO.

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

4. Specific rotation [α], = +65.5 to 64.3° (c = 1.04)5
, ultraviolet absorption spectrum of this substance 3. The ultraviolet absorption spectrum of a 5% aqueous solution is shown in Figure 1F9H, and the ultraviolet absorption spectrum of a 3.1% aqueous solution is shown in Figure 2.

All are 257 nm, 265 nm, and 280 nm.

Absorption is observed at 287 nm, which is characteristic.

6. Infrared absorption spectrum It is shown in FIG.

Soluble in water, but methanol, ethanol, n-butanol, imbutanol, n-propanol, n-hexane, chloroform.

It is insoluble in solvents such as 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 10. Puret reaction 10. Molisch reaction - Decier reaction - Anthrone reaction - Cystine sulfuric acid reaction - 11. Stabilized This substance is L-cysteine, dithiothreitol (DTT)
), glycerol, albumin, globulin, (NH*
)t80 It is stabilized by adding salt, etc.

Next, examples of the present invention will be shown.

In the Examples, the activity unit of the physiologically active substance 5PF-1 was measured using the Udaka method (Tournal of Antih
ioticsvol 55 No, 101319-1
3250CT 19B2). The measurement was carried out using polymer-permeable E. coli mutant strain MP-2 (FERM-P 543).
2) (A+zric, Biol-Chem.

43371 (1979) to perform a bioassay using antibacterial activity against MP-2t as an indicator.

i.e. Bacto Antibiotic Mediamuro (
Difco product) A medium (M, medium) consisting of 1.75% agar and 1.6% agar was heat sterilized at 120°C for 15 minutes to reduce the concentration to 2%.
Dispense 0-10ml into Petri dishes and let it stand to prepare a plate medium.

On the other hand, a medium consisting of 70.5% Pebut, 0.5% meat extract, 0.3% NaC, and 0.8% agar was heated at 120°C.

Sterilize by heating for 15 minutes. After that, keep it in a constant temperature bath at 42℃.
When the temperature of the culture medium reaches 42℃, incubate it at 37℃ for 17 minutes.
MP-2 bacteria cultured for an hour are added to the medium so that 10' cells are present in each cell.

Collect 2- with 2 pipettes, add it to the surface of the medium prepared in advance, and spread it quickly and uniformly to solidify. The test solution containing the physiologically active substance 5PF-1 is diluted appropriately, and 0.05 ml of the solution is applied to a paper disk (diameter 81i1++) (Toyo Roshi).

Place this paper disk on the fabrication plate,
Cultured at 37°C for 17 hours.

The size of the inhibition circle created by the physiologically active substance 5PF-1 is measured. The concentration of 5PF-1 that provides an inhibition circle diameter of 10 mm is defined as 1 unit (1 u).

Example 1゜ To medium A2 with the following composition.

Meat extract 1% Polypeptone 1% NaC! 0.5% pH=ZI Streptococcus pyogencs AT
Seed CC21060 (BHI medium 100 #3)
Inoculate 100@/ of the pre-cultured culture solution by static culture at 7°C for 8 hours, and culture anaerobically with stirring for 15 hours at 67°C, then add 1000 units of penicillin G/#It and continue culturing. Continues for 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contains 120 units of the physiologically active substance 5PF-1.
It contained vrl.

Example 2 Medium Belt with the following composition: Yeast extract 3.0% BHI 1.0% Polypeptone 1.0% Maltose 0.3% KH, Po, 0.1% Mg80.・7 H, OC1.02% State = 7.2 Streptococcus pyogenes AT
Culture solution 1 in which CC21060 was precultured in the same manner as in Example 1
Inoculate with 00*/.

After culturing anaerobically at 37°C for 15 hours with stirring, 1000 units/- of penicillin G was added, and the culture was further continued for 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

Culture P solution contains 152 units/d of physiologically active substance 5PF-1.
It was contained.

Example 3゜ in medium C2J with the following composition.

Meat extract 1% Polypeptone 1% NaC 0.5% Maltose 0.6% KH, Po, 0.1% Mg5O, 7H, OO, 02% pl (=7.1 Streptococcus pyogenes AT
Culture solution 1 in which CC21060 was precultured in the same manner as in Example 1
After anaerobic cultivation at 37°C for 15 hours with stirring, 1000 units/- of penicillin G was added.
Culture is continued for an additional 5 hours. The resulting culture solution was centrifuged to remove bacterial cells.

Culture P solution contains 240 units/w of physiologically active substance 8PF-1.
It contained 11.

Example 4 Medium D2) with the following composition: Meat extract 1.5% Polypeptone 1.0% NaC 05% Casamino acids 05% Yeast extract 02% pH = ZI Streptococcus pyngenes AT
Culture solution 1 in which CC21060 was precultured in the same manner as in Example 1
00 mJ of the sample stored at 20°C was inoculated and kept at 37°C for 15 h.
After culturing anaerobically with stirring, add 1000 units/ml of penicillin G and continue culturing for an additional 5 hr. The obtained culture solution was centrifuged to remove bacterial cells.

The culture furnace solution contains 100 units of the physiologically active substance 5PF-1.
-Contained.

Example 5゜ to medium E2 with the following composition.

Meat extract 1.5% Polypeptone 1.0% NaC 0.5% Casamino acids 0.25% Yeast extract 0.25%...= 7.1 Streptococcus pyogeneg AT
CC21060 was precultured in the same manner as in Example 1, and 100 m/d of the seed culture was added and inoculated, and cultured anaerobically at 67° C. for 15 hr with stirring. 1000 units/d of penicillin G was added, and the culture was further continued for 5 hr. The obtained culture solution was centrifuged to remove bacterial cells.

The culture P solution contains 180 units/- of the physiologically active substance 5PF-1.
It was contained.

Example 6 5 treptococcus pyo in the medium E2
Genes ATCC 21060 was precultured in the same manner as in Example 1, and 100 ml of the culture solution stored at 20°C was inoculated.
57℃.

After culturing anaerobically with stirring for 15 hours, penicillin G1
000 units/d was added and the culture was continued for an additional 5 hr.

The obtained culture solution was centrifuged to remove bacterial cells.

The culture P solution contains 100 units/100 units of the physiologically active substance 5PF-1.
It contained ml.

Example Z In the following medium F2, meat extract 6.0% polypeptone 1.0% NaC 0.5% casamino acid 0.5%...=Z1 Streptococcus pyogenes AT
CC21060 was precultured in the same manner as in Example 1, inoculated with 1 oov of the culture solution stored at 20°C, and kept at 37°C for 5.5
After anaerobic incubation with stirring for hr, penicillin G100
Add 0 units/- and continue culturing for an additional 15 hr. The obtained culture solution was centrifuged to remove bacterial cells.

The culture P solution contains 120 units/- of the physiologically active substance 5PF-1.
It was contained.

Example 8: B HI 5.7% Polypeptone 0.5% Maltose 0.6% KH2P0. 0.1% Mg5O, 7H200, 02% pH = 7.1 Streptococcus pyogenes AT
Inoculate 100 m of a culture solution pre-cultured with CC21060 in the same manner as in Example 1, and culture it anaerobically at 37°C for 15 hours with stirring. Add 100 units of penicillin G and continue the further culture for 5 hours. . The obtained culture solution was centrifuged to remove bacterial cells.

Drain the culture p solution and collect 300 units of physiologically active substance 5PF-1/
It contained gold.

Example 9 In the medium E3), 5 treptococcus py
Add and inoculate 300 ml of the seed culture obtained by statically culturing Ogenes ATCC 21060 at 67°C for 8 hours.
After culturing anaerobically at 7°C for 15 hours with stirring, 1000 units/- of penicillin G is added, and the culture is continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

56×10 of the physiologically active substance 5PF-1 was added to the culture solution 3.
'It contained units.

Ammonium sulfate was added to the culture P solution, and a fraction having a saturation level of 50 to 80% was collected to obtain a precipitate. This precipitate is a physiologically active substance S
It contained 50 x 1 inch units of Pli'-1.

The entire axillary precipitate was dissolved in 5 GO ml of stabilizer-containing buffer, and the solution was poured into a DEAE-cellulose column (5) <73
cm), and the physiologically active substance SPF was adsorbed. This was eluted stepwise using a 0.3M NaC solution.
Separate the active part. The yield or activity was 30 x 10' units.

The active moieties are dialyzed against the buffer and then applied to a DEAE-Sephadex A-25 column (2,6 x 50σ) to adsorb the active moieties, which are then linearly adjusted to the saline concentration in the phosphate buffer. Elution is performed while increasing the temperature, and the active portion is collected. The activity obtained was 106 x 10' units.

Furthermore, this eluate was concentrated and filtered using gel filtration material TOYOPEARL HW-
Add 50F column (2x100cmH).

Separate the active fraction. The activity obtained was 2.2 x 10' units.

The obtained eluate was freeze-dried and the physiologically active substance 5P
5 Dvg of white powder of F-1 was obtained.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of a 3% aqueous solution of the physiologically active substance 5PF-13, FIG. 2 similarly shows the ultraviolet absorption spectrum of a 3.1% aqueous solution, and FIG. 3 similarly shows the infrared absorption spectrum. Agent Patent attorney Door 1) Parent Man No. 1 250 275 300 m Diagram 2 m

Claims (1)

[Claims] tit 8P physiologically active substances having the following physicochemical properties
F-1゜1, Elemental Analysis 2, Molecular Weight As measured by gel filtration method, the molecular weight is approximately 500 to 7,000. 3. Decomposition point This substance turns brown at 170°C, turns black and decomposes at 200°C. 4. Specific rotation [α), = +63. ! 1~64.3@(c=1.
04) 5. Ultraviolet Absorption Spectrum The ultraviolet absorption spectrum of a 3.3% aqueous solution of this substance is shown in FIG. 1, and the ultraviolet absorption spectrum of a 3.1% aqueous solution is shown in FIG. Both are 257 nm and 265 nm.
Absorption is observed at nm, 280 nm, and 287 nm, which is characteristic. 6. Infrared absorption spectrum shown in Figure 3. Z Solubility in solvents Soluble in water, but insoluble in solvents such as methanol, ethanol, n-butanol, imbutanol, n-propanol, n-hexane, chloroform, acetone, methyl isobutyl ketone, and ethyl ether. The pH of a 0.85% aqueous solution of this substance is 6.5. 9 The substance is in the form of a white powder. 10. Color reaction Ninhydrin reaction → Biuret reaction 10. Molisch reaction - Decier reaction - Anthrone reaction - Cystine sulfuric acid reaction - 11. Stabilized This substance contains L-cysteine, dithiothreitol (DTT), glycerol, albumin, globulin, ( NH4)2 Soy It is stabilized by the addition of salt, etc. (2) 5P physiologically active substance belonging to the genus Streptococcus
Cultivate F-1 producing bacteria and extract the physiologically active substance 5PF from the culture.
-Physiologically active substance 5PF-
1 manufacturing method. (3) 8P physiologically active substances belonging to the genus Streptococcus
When culturing the F-1 producing bacteria, penicillin or a related substance is added to the culture at an appropriate time during the culturing. Manufacturing method.