JPH0155275B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel physiologically active substance SPF-2 and a method for producing the same.

More specifically, the present invention relates to SPF-2, 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 agents.

In addition, after crushing the bacterial cells of Streptococcus pyogenes, the active ingredients are extracted with water or a salt solution.
Add an organic solvent to precipitate the antitumor component,
There are known methods such as a method for recovering streptococci (Japanese Patent Publication No. 38-1647) and a method for lysing streptococci with a lytic enzyme, lysozyme, cellulase, or proteolytic enzyme and fractionating the active fraction as a water-soluble fraction (British Patent No. 1163865). It is being

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-2 in the culture solution. be.

The physiologically active substance SPF-2 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-2 of the present invention is excreted into the culture medium and is characterized by having a molecular weight of about 7,000 to 25,000.

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-2 is recognized as a completely new substance.

In addition, the physiologically active substance SPF-4 of the present invention is recognized as a peptide substance based on elemental analysis, color change, 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 both, and is recognized as a new substance.

The present invention includes a method for producing the physiologically active substance SPF-2, which is characterized by culturing a physiologically active substance SPF-2-producing bacterium belonging to the genus Streptococcus and collecting the physiologically active substance SPF-2 from the culture. It is something.

In the present invention, bacteria that produce the physiologically active substance SPF-2 belonging to the genus Streptococcus are widely used, and one example is Streptococcus pyogenes ATCC21060.
can be given.

The culture medium is meat extract medium, yeast extract medium, brain heart infusion medium (BHI).
Natural media such as Streptococcus spp.) are often used, but general media containing carbon sources, nitrogen sources, etc. can also be used as long as they allow Streptococcus bacteria to grow effectively.

Culture at PH5.0-8.0, preferably 6.1-7.2 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 stirring 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-2.

The timing of addition of penicillin or related substances is 37℃.
The period of 3 to 20 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 3 to 15 hours, the physiologically active substance SPF is added to the culture solution.
-2 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 300 to 1500 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 90% was collected. The resulting precipitate was added to a buffer containing a stabilizer. Dissolve in liquid.

A solution containing the physiologically active substance SPF-2 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 active substance SPF-2 of the present invention obtained in Example 8 is a peptide substance, and becomes a pale yellow powder when freeze-dried.

Next, the physicochemical properties of the physiologically active substance SPF-2 will be shown.

1 Elemental analysis C: 53.64%, H: 5.98%, N: 11.63% 48.57% 5.02% 10.50% 2 Molecular weight When measured by gel filtration method, the molecular weight is approximately 7000~
25000.

3. Decomposition point This substance turns brown at 160℃ and turns black at 200℃ and decomposes.

4 Specific rotation [α] ²⁰ _D = +30.7° (C = 1.00) 5 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.2% aqueous solution of this substance is shown in Figure 1. 257nm, 265nm,
It has characteristic absorption at 273nm, 280nm, 287nm and 325nm.

6 Infrared absorption spectrum It is shown in FIG.

Around 3300cm ^-1 , 3020cm ^-1 , 2970cm ^-1 , 1770cm
^-1 , 1655cm ^-1 , 1600cm ^-1 , 1515cm ^-1 , 1455cm ^-1 ,
Absorption is observed at 1400cm ^-1 , 1315cm ^-1 and 1120cm ^-1 .

7 Solubility in solvents Soluble in water, but partially soluble in methanol and ethanol, and dissolves in n-butanol, isobutanol, n-propanol, n-hexane, chloroform, acetone, methyl isobutyl ketone, ethyl ether, etc. Slightly soluble or insoluble in solvents.

8 Distinction between basic, acidic, and neutral The pH of a 1.0% aqueous solution of this substance is 6.5.

9 Color of matter It is a pale yellow powder.

10 Color reaction Ninhydrin reaction + Biuretz reaction + Mauritsch reaction - Decier reaction - Anthrone reaction - Cysteine sulfate reaction - 11 Stabilization This substance contains L-cysteine, dithiothreitol (DTT), glycerol, albumin, globulin, α- and β - Stabilized by addition of cyclodextrin, (NH ₄ ) ₂ 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-2 was measured using the Udaka method (Journal of Antibiotics
Vol 35 No. 10 1319-1325 OCT1982).
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 (product of Difco) and 1.3% agar was heat sterilized at 120°C for 15 minutes.
Dispense 20 ml into a plate and leave to cool to prepare a 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. Collect 2 ml with a pipette, add it to the surface of the _M3 medium prepared in advance, and spread it quickly and uniformly to solidify. Appropriately dilute the test solution containing the physiologically active substance SPF-2, and soak 0.05 ml of this solution into a paper disk (8 mm in diameter) (Toyo Shi). Place this paper disk on the production plate,
After culturing at 37°C for 17 hours, the size of the inhibition zone created by the physiologically active substance SPF-2 is measured. Concentration of SPF-2 that gives an inhibition circle diameter of 10 mm is 1 unit (1μ)
It is defined as

Example 1 Medium A, 9 having the following composition was used.

Meat extract 1% Polypeptone 1% Yeast extract 0.25% Casamino acids 0.25% NaCl 0.1% PH = 6.9 Streptococcus pyogenes ATCC 21060 was inoculated into 100 ml of BHI medium and the preculture obtained by static culture at 37°C for 8 hours was used as the medium. Inoculate A1,
After culturing under the same culture conditions, inoculating into medium A,
37℃ using a 10-year fermentor
After culturing anaerobically with stirring at 300 rpm for 11.5 hr,
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 contains 95 units of the physiologically active substance SPF-2.
It contained ml.

Example 2 Medium B, 9 having the following composition was used.

Meat extract 1.0% Polypeptone 1.0% Yeast extract 0.25% NaCl 0.1% PH = 7.0 Streptococcus pyogenes ATCC 21060 was precultured in the same manner as in Example 1, and culture solution 1 was inoculated into medium B, and cultured using a 10-year fermentor. °C,
After culturing anaerobically with stirring at 300 rpm for 11 hours, 800 units/ml of penicillin G was added, and the culture was further continued for 5 hours.
continue. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 105.3 units/ml of the physiologically active substance SPF-2.

Example 3 Medium C, 9 having the following composition was used.

Meat extract 1% Polypeptone 1% Yeast extract 0.25% Casamino acids 0.25% NaCl 0.5% PH = 6.2 Streptococcus pyogenes ATCC 21060 was precultured in the same manner as in Example 1. Culture solution 1 was inoculated into medium C, and cultured in 10 jars. 37℃ using
After culturing anaerobically with stirring at 300 rpm for 14 hours, add 1000 units/ml of penicillin G and continue culturing.
Continues for 5 hours. The resulting culture solution was centrifuged to remove bacterial cells.

The culture solution contains 95 units of the physiologically active substance SPF-2.
It contained ml.

Example 4 Medium D, 9 having the following composition was used.

Meat extract 0.5% Polypeptone 1.0% Yeast extract 0.25% Casamino acids 0.25% NaCl 0.5% PH = 6.5 Streptococcus pyogenes ATCC 21060 was precultured in the same manner as in Example 1, and culture solution 1 was inoculated into medium D, and cultured in 10 jars. 37℃ using
After culturing anaerobically with stirring at 300 rpm for 15.5 hours,
Add 1200 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 105.5 units/ml of the physiologically active substance SPF-2.

Example 5 Medium E, 9 having the following composition was used.

Yeast extract 3.0% PH = 6.5 Streptococcus pyogenes ATCC 21060 was precultured in the same manner as in Example 1, and seed culture solution 1 was inoculated into medium E, and cultured using a 10 jar fermentor.
After incubation anaerobically with stirring at 300 rpm for 15 hr at °C.
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 4965 units/ml of the physiologically active substance SPF-2.

Example 6 The following medium F, 9 was used.

Maltose 0.3% Meat extract 2.0% Polypeptone 1.0% Yeast extract 0.25% PH = 7.0 Streptococcus pyogenes ATCC 21060 was precultured in the same manner as in Example 1, and culture solution 1 was used as medium F.
inoculated at 37°C in a 10-year fermenter.
After culturing anaerobically with stirring at 300 rpm for 10.5 hours,
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 67.5 units/ml of the physiologically active substance SPF-2.

Example 7 The following medium G, 9 was used.

Meat extract 1.0% Polypeptone 1.0% NaCl 0.5% PH=7.0 Streptococcus pyogenes ATCC 21060 was precultured in the same manner as in Example 1, and culture solution 1 was used as medium G.
inoculate and inoculate 37 using a 10 jar
After incubation anaerobically with stirring at 300 rpm for 15 hr at °C.
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 100 units/ml of the physiologically active substance SPF-2.

Example 8 Culture solution 5 obtained in the culture of Example 3 contained 150×10 ⁴ units of physiologically active substance.

Ammonium sulfate was added to the culture solution, and a fraction with a saturation level of 50 to 90% was collected to obtain a precipitate. This precipitate contained 126×10 ⁴ units of the physiologically active substance SPF-2.

The entire amount of this precipitate was dissolved in 300 ml of stabilizer-containing buffer solution, and the solution was added to a DEAE-cellulose column (500ml).
x 70 cm), 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
It was 115×10 ⁴ units.

Add the active moiety to a DEAE A-25 column (2.6 x 70 cm) to adsorb the active moiety,
Elution is performed while linearly increasing the salt concentration in the phosphate buffer, and the active portion is fractionated. The activity obtained was 85×10 ⁴ units.

Furthermore, this eluate was concentrated and adsorbed on a column (2.6 x 100 cm) of gel filtration material Toyopearl HW50F, and then eluted with 1/100M phosphate buffer (KH ₂ PO ₄ -Na ₂ HP ₄ ). The active fraction was separated and designated as SPF-2. The SPF-2 activity obtained was 22.5×10 ⁴ units.

The eluate thus obtained was freeze-dried to obtain 750 mg of a pale yellow powder of the physiologically active substance SPF-2.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of a 0.2% aqueous solution of the physiologically active substance SPF-2, and FIG. 2 similarly shows the infrared absorption spectrum.

Claims (1)

[Claims] 1. A physiologically active substance having the following physicochemical properties:
SPF-2. 1 Elemental analysis C: 53.64% ~ 48.57% H: 5.98% ~ 5.02% N: 11.63% ~ 10.50% 2 Molecular weight Molecular weight measured by gel filtration method: approximately 7000 ~
25000. 3. Decomposition point This substance turns brown at 160℃ and turns black at 200℃ and decomposes. 4 Specific rotation [α] ²⁰ _D = +30.7° (C = 1.00) 5 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.2% aqueous solution of this substance is 257nm, 265nm, 273nm, 280nm,
Absorption is observed at 287nm and 325nm. 6 Infrared absorption spectrum around 3300cm ^-1 , 3020cm ^-1 , 2970cm ^-1 , 1770cm
^-1 , 1655cm ^-1 , 1600cm ^-1 , 1515cm ^-1 , 1455cm ^-1 ,
Absorption is observed at 1400cm ^-1 , 1315cm ^-1 and 1120cm ^-1 . 7 Solubility in solvents Soluble in water, but partially soluble in methanol and ethanol, and dissolves in n-butanol, isobutanol, n-propanol, n-hexane, chloroform, acetone, methyl isobutyl ketone, ethyl ether, etc. Slightly soluble or insoluble in solvents. 8. Distinction between basic, acidic, and neutral The pH of a 1.0% aqueous solution of this substance is 6.5. 9. Color of substance: Pale yellow powder. 10 Color reaction Ninhydrin reaction + Biuretz reaction + Mauritsch reaction - Decier reaction - Anthrone reaction - Cysteine sulfate reaction - 11 Stabilization This substance contains L-cysteine, dithiothreitol (DTT), glycerol, albumin, globulin, α- and β - Stabilized by addition of cyclodextrin, (NH ₄ ) ₂ SO ₄ , salt, etc. 2 Physiologically active substances belonging to the genus Streptococcus
A method for producing a physiologically active substance SPF-2, which comprises culturing SPF-2 producing bacteria and collecting the physiologically active substance SPF-2 from the culture. 3 Physiologically active substances belonging to the genus Streptococcus
When culturing the SPF-2 producing bacteria, penicillin or a related substance is added to the culture at an appropriate time during the culture. Manufacturing method.