JPS6148920B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing the physiologically active substance mutastein. Mutastein is a known substance that was previously isolated by the present inventor from a culture of a bacterium belonging to the genus Aspergillus, and has the following physicochemical properties (Japanese Patent Application Laid-Open No. 146587-1987). (1) The molecular weight is 200,000 or more, and Cefdex G
-100 and Sepharose 6B, it is eluted in the void volume. (2) It has protein properties and contains about 10% sugar. (3) Soluble in water and salt solutions, but saturated with ammonium sulfate.
Salted out at 30%. Also insoluble in acetone, ethanol, ethyl acetate, and benzene. (4) Soluble in buffers with pH 7 or higher, but pH 3-3.5
A precipitate is formed. (5) Stable to heat treatment at PH9, 100℃ for 10 minutes. (6) Elemental analysis values C: approx. 44%, H: approx. 7%, N: approx. 12% (7) Color reaction Phenol sulfuric acid color reaction (orange), phorin color reaction (blue). This substance has the effect of inhibiting dextran synthesis by caries bacteria, and is useful for the prevention and treatment of dental caries. The present inventor conducted a wide search to find microorganisms with better mutastein production than the above-mentioned Aspergillus genus, and discovered that the following 16 genera produced a significant amount of mutastein, and completed the present invention. did. Cercospora genus, especially Cercospora cruenta (C.cruenta) IFO 6164
and Cercospora lapisicola (C.
rhapisicola) IFO 8433 Cladosporium genus, especially Cladosporium paeoniae (C.paeoniae)
IFO 9770 and Cladosporium resinae f.avellaneum (C.resinae f.avellaneum) IFO
6367 Colletotrichum genus, especially Colletotrichum dematium, C.dematium f.truncata IFO 6704 Cylindrosporium
Genus Dactylaria, especially C.dioscoreae IFO 9622 Genus Dactylaria, especially D.purpurella IFO
9336 The genus Dematium, especially D. nigrum IFO 6131 The genus Diplogelasinospora, especially D.inaequalis IFO
9824 The genus Drechslera, especially D.erythrospila
IFO 7378 The genus Endophragmia, especially Endophragmia alternata (E. alte-
rnata) IFO 30204 Genus Fannellia, especially F. flavipes IFO 9655 Genus Leptographium,
Especially Leptographium kitajimana (L.
Kitajmana) IFO 6908 Monascus genus, especially M. ruber IFO 9203 Mucor genus, especially M. oblongisporus IFO 7058 Doratomyces genus, especially Doratomyces stemoniteis (D.stemonitis) IFO
8314 Sporormiella genus, especially Sporormiella intermesia (S.inte-
rmedia) IFO 8392 Stachybotrys genus, especially Stachybotrys microspora (S.micros)
pora) IFO 30018 and S. nephrospora (S. nephrospora) IFO 7067. All of the above-mentioned strains used in the present invention were obtained from the Fermentation Research Institute (2-17-85 Jusanhonmachi, Yodogawa-ku, Osaka City).
more available. Therefore, the present invention is a method for producing the physiologically active substance mutastein, which comprises aerobically cultivating mutastein-producing bacteria belonging to the above-mentioned 16 genera and collecting mutastein from the culture. Mutastein is produced in a culture by aerobically cultivating a mutastein-producing bacterial strain by a culture method known as a mold culture method. For example, a mutastein-producing strain is subcultured on a potato dextrose agar medium, and for the production of mutastein, the cells grown on the agar medium can be directly inoculated into the production medium and cultured. Furthermore, the bacterial cells grown on the production medium can be cultured on a new production medium to produce mutastein there. Mutastein-producing bacteria grow at temperatures between 7 and 35 degrees Celsius, but
A temperature of 20 to 30°C is usually preferred for mutastein production. In order to culture Aspergillus bacteria that produce mutastein, nutrient sources known for culturing molds and other microorganisms can be used. For example, glucose, maltose, dextrin, starch, lactose, sutucarose, glycerin, etc. can be used as carbon sources. Among these carbon sources, glucose and glycerin are the preferred carbon sources for mutastein production. To produce mutastein, all known nitrogen sources for mold and other microbial growth can be used. For example, peptone, meat extract, yeast, yeast extract, soybean flour, peanut flour, cornstarch liquor, rice bran, inorganic nitrogen sources, etc. can be used. When producing mutastein by culturing mutastein-producing bacteria, inorganic salts and metal salts are added if necessary. Also, trace amounts of heavy metals can be added if necessary. Mutastein can be obtained by culturing its producing bacteria aerobically, and commonly used aerobic culture methods, such as solid state culture, shaking culture, and aerated agitation culture, are used. When antifoaming is required during culture or medium sterilization, antifoaming agents such as silicone oil and surfactants can be used. The culture temperature is preferably 20 to 30°C. The physiological activity of mutastein can be assayed by the following method that examines Braak formation. That is, take 10 ml of culture medium in a test tube, add nichrome wire (420 cages), and collect cariogenic bacteria (dental caries bacteria) Streptococcus.
mutans and grow at 37°C. After a certain period of time (for example, 48 hours), the amount of plaque attached to the nichrome wire is measured. The physiological activity of Mutastein can be determined by comparing the amount of plaque between a control without Mutastein and a control with a certain amount of Mutastein added [RMMcCabe, PHKeyes,
A. Howell, Jr., Arch. Oral. Biol., vol. 12, 1653~
1656 pages, 1967]. The culture is continued until mutastein is substantially accumulated, and the substance can be extracted and separated from the culture using various methods based on the properties of the substance revealed by the inventors, as shown in the Examples below. This can be done by a suitable combination of methods. Namely, salting out method using ammonium sulfate, etc., PH precipitation fractionation method by adjusting PH, various ion conversion chromatography methods, gel filtration methods using various carriers, various electrophoresis methods, ultrafiltration methods, and freeze drying. method, dialysis method, extraction treatment method using various organic solvents, etc. Mutastein can be isolated from the culture by appropriately combining and repeatedly using these methods. Mutastein obtained according to the present invention has a property of being easily polymerized due to molecular aggregation, and this is the main factor that may cause changes in some of its physical and chemical properties. That is, (1) Molecular weights ranged from around 200,000 to highly associated
5 million or so, and some with even more advanced meetings can be seen. (2) Mutastein usually contains about 10% sugar, but can sometimes contain up to 20% sugar. (3) Those with a molecular weight of 5 million or less are soluble in water and salt solutions, but may become insoluble if the association progresses further. Also, depending on the degree of molecular association, salting out may not be possible with 30% saturated ammonium sulfate, and some may require 80% saturation for precipitation. (4) Highly associated and insolubilized substances are difficult to dissolve in buffer solutions with a pH of 7 or higher. Mutastein, which is highly associated and insolubilized, is
By treating in a 0.1N NaOH solution for 20 to 60 minutes (20 to 50°C), most of the molecules can be reduced to low molecular weight and solubilized. Mutastein association may also be induced by separation operations from the culture, such as precipitation with pH 3 to 3.5, ammonium sulfate salting out, concentration, and freeze-drying. Furthermore, the degree of association may vary depending on the salt concentration in the aqueous solution, the pH, and the culture conditions during mutastein production, such as medium composition and culture time. Example 1 A 0.2 mg/ml solution of mutastein obtained by the method of Example 1 of JP-A-57-146587 (dissolved in a solvent system of 25 mM phosphate buffer containing 0.02% NaN ₃ and pH 8.0) was prepared in the same solvent. Toyopearl HW prepared using the system
65 (Toyo Soda Kogyo Co., Ltd.) column, the activity is recovered at a molecular weight of around 2 million. However, when the same solution is concentrated by ultrafiltration to 5 mg/ml, a portion of the solution becomes subsolubilized and the molecular weight becomes 5 million or more. Most of the remaining molecules have a molecular weight of 200 when developed on a Toyopearl HW-65 column under the same conditions.
Collected over ~500. Example 2 Aspergillus terreus M3328 with 1.5% sucrose, 1.5% dextrin, and 2 polypeptone
%, cornstarch liquor 2%, KH ₂ PO ₄ 0.2
%, _MgSO4・_7H2O0.1 %, _FeSO4・_7H2O0.01
% and Celite 1% (PH6.5) and cultured aerobically at 25° C. for 7 days to obtain a culture solution. Example 1: Molecular weight of mutastein in the main culture solution
When measured using the same method as above, it was 200,000 to 300,000.
On the other hand, the same strain was mixed with 1% glucose and 2% starch.
Soy bean meal 2%, KH ₂ PO ₄ 0.1%,
Mutastein was inoculated into a medium containing 0.05% MgSO ₄ 7H ₂ O (without pH adjustment) and cultured aerobically at 25°C for 7 days. Those associated with 200,000 to 300,000 (molecular weight
5 million or more). Next, examples of the present invention will be shown. Now that the above-mentioned properties have been clarified by the present invention, various modification methods can be used to collect mutastein from cultures or related materials based on these findings. It is. The present invention is not limited to the examples,
It includes all methods that can be easily extrapolated from the findings already described. Example 1 Cercospora spp. was grown in a medium containing 3.5% glucose, 1% starch, 2% soy bean meal, 0.5% meat extract, 0.5% peptone, 0.2% sodium chloride, and magnesium sulfate (MgSO ₄ 7H ₂ O).
Cruenta IFO 6164 was inoculated and cultured aerobically at 25°C for 10 days. The resulting culture solution (100 ml) was saturated with ammonium sulfate to 80%, and the resulting precipitate was thoroughly dialyzed against 25 mM phosphate buffer (PH7), which was collected by centrifugation. Then, the dialysis fluid was PHed with dilute hydrochloric acid.
Collect the precipitate produced as 3.5 by centrifugation,
25mM phosphate buffer (PH) containing 0.02% _NaN3
7) was sufficiently dialyzed. This internal fluid is 0.02%
Toyopearls prepared with the same buffer containing _NaN3
It was loaded onto a HW-65 column and developed to obtain an active fraction. After thoroughly dialyzing this active fraction against distilled water, 65 mg of purified mutastein was obtained by freeze-drying.
I got it. Example 2 Cladosporium was grown on a ground with the same composition as in Example 1.
inoculated with Lescine f. avelanem IFO 6367 at 25°C.
Culture solution obtained by culturing aerobically for 10 days (100
ml) was saturated with ammonium sulfate to 50%, and the resulting precipitate was collected by centrifugation. Following the same purification procedure as in Example 1, 42 mg of pale white purified mutastein was obtained. Example 3 Colletotrichum dematium f. Truncata IFO
A culture solution (100 ml) obtained by culturing 6704 in the same medium and culture conditions as in Example 1 was saturated with ammonium sulfate to 70%, and the resulting precipitate was collected by centrifugation. Following the same purification procedure as in Example 1, brown mutastein 68 was obtained.
I got mg. Example 4 Dactylaria purpurella IFO 9336 was mixed with 1% glucose, 2% starch, and 2% soy bean meal.
%, phosphoric acid-potassium 0.1%, magnesium sulfate (MgSO ₄ 7H ₂ O) 0.05%,
The cells were cultured aerobically at 25°C for 7 days. The resulting culture solution (100 ml) was saturated with ammonium sulfate to 50%, and the resulting precipitate was collected by centrifugation and diluted with 25 mM containing 0.02% _NaN3 .
Thorough dialysis was performed against a phosphate buffer (PH7) mixture. This internal solution was loaded onto a Sephacryl S-300 (Pharmacia, Sweden) column prepared with the same mixture and developed to obtain an active fraction. This active fraction was thoroughly dialyzed against distilled water and then freeze-dried to obtain 23 mg of purified mutastein. Example 5 Drechslera erythrospira IFO 7378 was cultured using the same medium and culture method as in Example 1. The resulting culture solution (100 ml) was saturated with ammonium sulfate to 50%, and the resulting precipitate was collected by centrifugation. Example 4 below
105 mg of purified mutastein was obtained by the same procedure as above. Example 6 Fueneria Frapipes IFO 9655 Example 1
The culture solution (100 ml) obtained by culturing using the same medium and culture method as above was saturated with ammonium sulfate to 50%, and the resulting precipitate was collected by centrifugation. Following the same procedure as in Example 1, 28 mg of purified mutastein was obtained. Example 7 A culture solution (100 ml) obtained by culturing Leptographium kitajimana IFO 6908 using the same medium and culture method as in Example 4 was saturated with ammonium sulfate to 70%, and the resulting precipitate was collected by centrifugation. Following the same procedure as in Example 4, 42 mg of purified mutastein was obtained. Example 8 Monacus rubel IFO 9203 was cultured using the same medium and culture conditions as in Example 1. The resulting culture solution (100 ml) was saturated with ammonium sulfate to 50%, and the resulting precipitate was collected by centrifugation. Thereafter, 15 mg of purified mutastein was obtained by the same operation as in Example 1. Example 9 The culture medium of Example 1 was inoculated with various bacterial strains and incubated at 25°C.
Table 1 shows the yield of the mutastein active fraction when cultured aerobically for 10 days. The purification method was the same as in Example 1 using a Toyopearl HW-65 column. The mutastein active fractions obtained by freeze-drying were all light yellow to brown in color.

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Claims (1)

[Scope of Claims] 1. Cercospora, Cladosporium, Colletotrichum, Cylindrosporium, Dactylaria, Dematium, Diplogeracinospora, Drechslera, Endophragmia, Fuennelia, Leptographi The method is characterized by culturing aerobically a mutastein-producing bacterium belonging to a genus selected from the group consisting of the genus Mucor, genus Monascus, genus Mucor, genus Polatomyces, genus Sporormiella, and genus Stachybotrys, and collecting mutastein from the culture. A method for producing the physiologically active substance mutastein. 2 Mutastein-producing bacteria are Cercospora cruenta, Cercospora lapisicola, Cladosporium peonia, and Cladosporium lecinea.
f.Averaneum, Colletotrichum dematium
f. Truncata, Cylindrosporium dioscorea, Dactylaria pulbrella, Dematium
nigrum, Diprogeracinospora inecallis,
Drechslera erythrospira, Endophragmia alternata, Fuennelia flavipes,
Leptographium kitajimana, Monascus
The method for producing the physiologically active substance mutastein according to claim 1, which is a strain of Mucor, Mucor oblongisporus, Dratomyces stemmoniteis, Sporormiella intermedia, Stachybotrys microspora or Stachybotrys nephrospora, or a variant or mutant strain thereof.