JPH05236943A - Microorganism for producing new glycopeptide-based antibiotic pa-42,867-a and pa-42,867-b - Google Patents

Abstract

PURPOSE:To obtain the subject bacterium belonging to the genus Nocardia orientalis, capable of producing a new glycopeptide-based antibiotic having strongly antibacterial action on gram-positive bacteria. CONSTITUTION:The objective bacterium named PA-42,867 (FERM P-1,230), separated from a soil specimen, belonging to the genus Nocardia orientalis, capable of producing antibiotic PA-42,867-A and/or PA-42,867-B. The bacterium has the following properties. Morphological properties: An aerial mycelium adheres to yeast malt agar, tyrosine agar, etc. Spores are abundantly formed. A branch is a single branch and no circular branch is found. The tip of aerial mycelium is straight or wavy. Spores have a long cylindrical shape, 0.3-0.5mum width, 1.2-1.7mum length, a smooth surface structure and no sporangium, no flagellar spore and no sclerotium. Physiological properties. Milk is converted to peptone. Liquefying ability of gelatin is positive.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention belongs to the species Nocardia orientalis and has the formula I;

[Chemical 2] (Wherein X represents CH ₃ and Y represents NH ₂ , or X represents H and Y represents OH).
2867-A and / or PA-42867-B.

[0002]

2. Description of the Related Art With the recent widespread use of antibiotics, the emergence of multidrug-resistant bacteria, especially methicillin-resistant bacteria, has become a problem. Methicillin-resistant bacteria not only show resistance to methicillin, but also aminoglycoside antibiotics,
It is resistant to many antibiotics such as tetracycline antibiotics, cephem antibiotics, penicillin antibiotics, carbapenam antibiotics and macrolide antibiotics. It has been noted that glycopeptide antibiotics, especially vancomycin antibiotics, are active against this methicillin-resistant bacterium (antimicrovial agent and chemotherapy (ANTIMICROBIAL AGENT AN
D CHEMOTHERAPY) 28 , 660-662 (1985)). Vancomycin is a long-known antibiotic (Japanese Patent Publication No. 33-84).
50), and new vancomycin analogs have been discovered (Antimicrovial Agent and Chemotherapy (ANTIMICROBIAL AGENT AND CH
EMOTHERAPY) 28 , 660-662 (1985), THE JOURNAL OF ANTIBIOTIC
S) 37 , 446-453 (1984), 38 , 1-8 (1985), 38 , 51-57
(1985), JP-A-60-139623, 60-19939.
7, 60-231698, 60-237099, etc.). However, the antibiotic PA produced by the microorganism of the present invention
-42867-A and PA-42867-B are vancomycin antibiotics having completely new structures which are not consistent with the above compounds.

[0003]

[Problems to be Solved by the Invention] Currently commercially available vancomycin is for oral use and has a low purity, so that it is difficult to use it as an injection as it is. In addition, antibiotics having stronger activity against methicillin-resistant bacteria than conventional vancomycin antibiotics have been long-awaited.

[0004]

The present inventors have confirmed that a strain belonging to the genus Nocardia is of formula I;

[Chemical 3] (Wherein X represents CH ₃ and Y represents NH ₂ , or X represents H and Y represents OH), and a compound having strong activity against a methicillin-resistant bacterium is produced. , A compound of formula I where X is CH ₃ and Y is NH ₂
42867-A, a compound in which X is H and Y is OH is PA-
It was named 42867-B. The present invention provides PA-45867-A and / or PA-4 which produce these compounds.
2867-B producing microorganisms.

The physicochemical properties of the compounds produced by the PA-42867-A and / or PA-42867-B producing microorganisms of the present invention are shown below. Physicochemical properties PA-42867-A UV absorption spectrum λ ^{0.01N HCl} _max nm (E ^1% _1cm ): 281.8 (41.15) λ ^{0.01N NaOH ・ aq} _max nm (E ^1% _1cm ): 302.6 (43.14) Specific rotation Degree [α] ²⁵ ° _D -88.8 ± 3.1 ° (c = 0.41, water) Infrared absorption spectrum (see Fig. 1) IR (KBr): 3396, 1654, 1588 (sh), 1505, 1420, 1396, 1223,
1131, 1064, 1010 (sh) cm ^-1 mass spectrometry (see Fig. 2) MS m / z: 1557 (M + H) ⁺ elemental analysis C ₇₃ H ₈₉ O ₂₆ N ₁₀ Cl ・ 7 / 2H ₂ O theoretical value (%): C; 54.08, H; 5.97, N; 8.64, Cl; 2.19 Experimental value (%): C; 54.03, H; 6.14, N; 8.60, Cl; 2.19

Nuclear magnetic resonance spectrum ¹ H-NMR: 400 MHz, in d ₆ -DMSO + 1dropD ₂ O, internal standard TMS, 1
00 ℃ (See Fig. 3) δppm: 0.888 (d, 6.5, 3H), 0.917 (d, 6.0, 3H), 1.093 (d,
6.0, 3H), 1.156 (s, 3H), 1.167 (d, 6.2, 3H), 1.202 (s, 3
H), around 1.425 (m, 1H), around 1.525 (m, 1H), 1.630 (dd, 13.
6, 4.3, 1H), 1.640 (dd, 13.6, 4.3, 1H), 1.766 (m, 1H), 1.8
66 (d-like, 13.6, 1H), 1.915 (d-like, 13.6, 1H), 2.175 (dd, 16.
0, 7.5, 1H), 2.315 (s, 3H), 2.560 (dd, 16.0, 5.0, 1H), 2.8
92 (d, 9.5, 1H), 2.947 (d, 9.6, 1H), around 3.03 (overlapping water in solvent, 1H), around 3.31 (m, 2H), 3.428 (t-like, around 8.7,
1H), 3.540 (dd, 11.6, 4.8, 1H), 3.609 (qd, 6.2, 9.5, 1H),
3.688 (dd, 7.2, 8.7, 1H), 3.730 (dd, 11.6, 2.1, 1H), 4.141
(qd, 6.0, 9.6, 1H), 4.233 (broad s like, 1H), 4.328 (dd, 5.
0, 7.5, 1H), 4.485 (s-like, 1H), 4.516 (s, 1H), 4.674 (d-like, 4.
3 vicinity, 1H), 4.699 (d, 4.0, 1H), 5.152 (d like, 4.0 vicinity, 1H),
5.188 (broad s like, 1H), 5.190 (s like, 1H), 5.305 (d like, 4.3
Near, 1H), 5.603 (d, 7.2, 1H), 5.638 (broad s like, 1H), 5.
762 (broad s, 1H), 6.375 (d, 2.3, 1H), 6.388 (d, 2.3, 1
H), 6.712 (d, 8.3, 1H), 6.793 (dd, 8.3, 2.2, 1H), 7.087 (d
d, 8.5, 2.2, 1H), 7.120 (dd, 8.3, 2.2, 1H), 7.148 (d, 2.2, 1
H), 7.215 (d, 8.4, 1H), 7.286 (broaddd-like, around 8.3, around 2.0, 1H), 7.327 (dd, 8.4, 2.0, 1H), 7.586 (broad dd-like, 8.5
Near, 2 near, 1H), 7.863 (d, 2.0, 1H) ¹³ C-NMR: 50MHz, in D ₂ O, external standard CH ₃ CN as 1.7ppm, 6
0 ℃ (See Fig. 4) δppm: 178.9, 177.8, 174.8, 172.1, 171.9, 171.7, 1
70.3, 167.8, 158.0, 156.3, 155.9, 155.85, 155.85, 1
53.85, 151, 138.8, 137, 136.85, 136.7, 134.9, 133.
9, 131, 130, 128.8, 128.4, 127.8, 127.1, 126.2, 12
4.6, 124.2, 123.9, 122.8, 119.4, 118.6, 107.4, 107.
3, 104.9, 104, 102.3, 99.2, 94, 80.7, 77.5, 77.5,
76.9, 76.8, 75.2, 72.5, 70.4, 67.4, 67.2, 63.4, 6
3.4, 61.7, 60.5, 59.9, 56.1, 55.5, 55.5, 54.9, 52.
7, 42, 41.4, 39.85, 37.6, 34.9, 25.2, 23.3, 22.5,
20.6, 19.9, 18.4, 18.1

Thin Layer Chromatography-Merck Precoated TLC Plate Silica Gel 60F2
54 Developing solvent Chloroform: Methanol: Concentrated aqueous ammonia: Secondary butanol: Water (5: 10: 5: 5: 2) Rf = 0.28 High performance liquid chromatography (Shimadzu LC-6A) Column: Chemcopack Nucleosil 5C18 φ 4.6 x 150
mm (Chemco) Detection: UV 220 nm Flow rate: 1 ml / min. Mobile phase: 8% acetonitrile-0.05M phosphate buffer- (pH 3.
5) Retention time: 8.8 min .: 9% acetonitrile-0.05M phosphate buffer- (pH3.5) Retention time: 5.6 min. Color reaction: Ninhydrin positive Solubility: Soluble in water and dimethyl sulfoxide, alcohol Insoluble in ether, insoluble in ether, benzene, chloroform, ethyl acetate Properties: Amphoteric substance, white amorphous powder

PA-42867-B UV absorption spectrum λ ^{0.01N HCl} _max nm (E ^1% _1cm ): 281.6 (44.22) λ ^{0.01N NaOH · aq} _max nm (E ^1% _1cm ): 301 (45.94) Specific optical rotation [α] ²⁵ ° _D : -97.3 ± 3.3 ° (c = 0.41, water) Infrared absorption spectrum (see Fig. 5) IR (KBr): 3360, 1656, 1587 (sh), 1505, 1421, 1393, 1230,
1129, 1062, 1013 (sh) cm ^-1 mass spectrometry (see Figure 6) MS m / z: 1544 (M + H) ⁺

Elemental analysis C ₇₂ H ₈₆ O ₂₇ N ₉ Cl · 3 / 2H ₂ O theoretical value (%): C; 55.01, H; 5.71, N; 8.02, Cl; 2.26 experimental value (%): C; 54.83, H; 5.81, N; 8.46, Cl; 2.13 Nuclear magnetic resonance spectrum ¹ H-NMR: 400MHz, in d ₆ -DMSO + 1dropD ₂ O, internal standard TMS, 1
00 ℃ (See Fig. 7) δppm: 0.891 (d, 6.6, 3H), 0.919 (d, 6.5, 3H), 1.080 (d,
6.2, 3H), 1.159 (s, 3H), 1.173 (d, 6.0, 3H), around 1.428
(m, 1H), 1.469 (m, 1H), around 1.525 (m, 1H), 1.647 (dd, 13.
7, 4.4, 1H), 1.767 (m, 1H), 1.920 (d-like, 13.7, 1H), 2.042
(dd, around 13.0, around 5.3, 1H), 2.182 (dd, 16.0, 7.5, 1H),
2.314 (s, 3H), 2.565 (dd, 16.0, 4.8, 1H), 2.817 (t like, 9.1
Near, 1H), 2.895 (d, 9.7, 1H), around 3.03 (overlapping water in the solvent, 1H), around 3.33 (m, 2H), 3.497 (t-like, around 8.5, 1H),
3.547 (dd, 11.6, 4.9, 1H), 3.636 (qd, 6.0, 9.7, 1H), 3.66
8 (dd, 7.2, 8.5, 1H), around 3.685 (m, 1H), 3.733 (dd, 11.6,
2.3, 1H), 4.092 (qd, 6.2, 9.3, 1H), 4.234 (brd, around 1.5, 1
H), 4.330 (dd, 4.8, 7.5, 1H), 4.489 (s-like, 1H), 4.516 (s, 1
H), 4.686 (d-like, around 4.4, 1H), 4.692 (d, 3.9, 1H), 5.156
(d like, around 3.9, 1H), 5.192 (broad s like, 1H), 5.200 (s like, 1
H), 5.334 (d-like, around 3.5, 1H), 5.604 (d, 7.2, 1H), 5.641
(broad s like, 1H), 5.771 (broad s like, 1H), 6.388 (s like, 2
H), 6.719 (d, 8.5, 1H), 6.800 (dd, 8.5, 2.3, 1H), 7.107 (d
d, 8.4, 2.4, 1H), 7.147 (d, 2.3, 1H), 7.170 (dd, 8.4, 2.4, 1
H), 7.237 (d, 8.4, 1H), 7.296 (broad dd, around 8.4, around 2.0, 1H), 7.331 (dd, 8.4, 2.0, 1H), 7.590 (broad dd, 8.4,
2.2 vicinity, 1H), 7.857 (d, 2.0, 1H) ¹³ C-NMR: 50MHz, in D ₂ O, as external standard CH ₃ CN 1.7ppm,
60 ℃ (See Fig. 8) δppm: 177.9, 177.8, 174.8, 172.2, 172.1, 171.7, 1
70.3, 167.8, 157.6, 156.1, 156, 155.8, 155.5, 153.9,
151.2, 139, 137, 136.7, 136.7, 135, 134.2, 131.4,
130.9, 128.8, 128.4, 127.9, 127.1,126.4, 124.5, 1
24.3, 124.1 122.6, 119.3, 118.6, 109.3, 107.2, 104.
6, 104, 102.9, 99.3, 93.7, 79.4, 78.1, 77.9, 76.7,
76.1,75.2, 72.5, 70.2, 69.6, 68.7, 67.3, 63.1, 6
3.1, 61.7, 60.4, 59.9, 57.1, 55.5, 55.5, 53, 41.5,
40.2, 39.3, 37.7, 34.1, 25.1, 23.3, 22.5, 19.3, 1
8.3, 17.8

Thin Layer Chromatography-Merck Precoated TLC Plate Silica Gel 60F2
54 Developing solvent Chloroform: Methanol: Concentrated ammonia water: Secondary butanol: Water (5: 10: 5: 5: 2) Rf = 0.22 High performance liquid chromatography (Shimadzu LC-6A) Column: Nucleosil 5C18 φ4. 6 × 150 mm Detection: UV 220 nm Flow rate: 1 ml / min. Mobile phase: 9% acetonitrile-0.05M phosphate buffer- (pH 3.
5) Retention time: 9.4 min .: 10% acetonitrile-0.05M phosphate buffer- (pH3.5) Retention time: 6.9 min. Color reaction: Ninhydrin positive Solubility: Soluble in water and dimethylsulfoxide, alcohol Sparingly soluble in water, insoluble in ether, benzene, chloroform, ethyl acetate: amphoteric substance, white amorphous powder

From the above physicochemical properties, PA-428
67-A and PA-42867-B are represented by the following formulas;

[Chemical 4] (In the formula, X represents CH ₃ and Y represents NH ₂ , or X represents H and Y represents OH). As described above, PA-42867-A and PA-42867-B have properties different from those of known glycopeptide antibiotics and are judged to be novel glycopeptide antibiotics. PA-42867-A
Examples of the PA-42867-B-producing microorganisms include PA-42867 strain isolated from one soil sample. This strain was identified as the same species as Nocardia orientalis from the results of taxonomic examination, and Nocardia orientalis PA-42867 (No-cardia orientalis PA
No. 42867), which had been deposited as Microindustrial Research Institute No. 8601, but was transferred to an international deposit under the Budapest Treaty on December 4, 1986, and is now under Microindustry Research Article No. 1230.
Has been deposited as an issue.

The mycological properties of this strain are as follows. (1) Morphological properties Aerial mycelia are well established on east malt agar, tyrosine agar, Bennet agar, etc., and abundant in sporulation. The branch is a simple branch, and no ring branch is seen. The aerial mycelium extends relatively long and its tip is straight or wavy. As a result of observation with an electron microscope, the spores are oblong and have a width of 0.3 to 0.5 μm and a length of 1.2 to 1.7 μm.
Its surface structure is smooth type. Sporangium,
Neither flagella spores nor sclerotia are found.

[0013] The display of color names is based on "Color Standards" (Japan Color Research Institute edition).

Growth temperature (Pennett agar medium, cultivated at each temperature for 14 days) 10 ° C .: Grows fairly well but does not form aerial hyphae 28 ° C .: Good in growth, aerial hyphae formation and spore formation. 37 ° C: No growth 45 ° C: No growth

(3) Physiological properties (cultured at 28 ° C. for 14 days) Melanoid pigment-producing ability Negative tyrosinase reaction-negative milk coagulation-negative milk peptone-positive gelatin liquefying ability-positive starch hydrolyzing ability-negative

[0016]

(5) Cell Wall Composition The type of diaminopimelic acid is meso-type. Based on the above properties, this strain is judged to belong to the genus Nocardia. Watsukusman, The Actinomycetes Volume 2 (1961),
Shearing and gottrap ISP (Interna
National Streptomyces Project) [International Journal of Systematic Bact
eriology) Vol. 18 (1968), Vol. 19 (1969), Vol. 22 (1972)] and Verge's Manual of Determinating Bacteriology (Bergey's).
Manual of Determina-tive Bacteriology) 8th Edition (197
4 years) and other new species of actinomycetes published documents were searched for the near green species of this strain, Nocardia orientalis (Nocardia orientalis,
listed as treptomyces orientalis) [Inte
18th rnational Journal of Systematic Bacteriology
Volume, 154-157 (1968), The Actinomycetes, 2nd
Vol. 254-255 (1961)] is the most closely related species. Nocardia Orientalis and PA-428
As a result of comparing the properties of 67 strains, arabinose and rhamno-
Although there was a difference in the availability of water, the other major characteristics were in good agreement. Therefore, the PA-42867 strain was identified as the same species as Nocardia orientalis, and Nocardia orientalis PA-42867 (Nocardi
a orientalis PA-4286 7).

In the present invention, the above-mentioned PA-4286 is used.
Not to mention 7 strains and their natural and artificial variants,
Belongs to Nocardia orientalis species, PA-428
All strains producing 67-A and / or -B can be used and are within the scope of the invention. PA-42867-A
The production of PA-42867-A and / or -B is carried out by culturing the PA-42867-A and / or -B producing strain in a nutrient medium under aerobic conditions, and after the culture is completed, PA-42867-A and / or -B is produced from the culture. This is done by separating and collecting. Hereinafter, a general method for producing PA-42867-A and / or -B will be described. As the medium composition, medium conditions, etc., those generally used for the production of antibiotics may be used. In principle, the medium contains a carbon source, a nitrogen source, inorganic salts and the like. If necessary, vitamins, precursors, etc. may be added. As the carbon source, for example, glucose, starch, dextrin, glycerin, molasses, organic acid and the like are used alone or as a mixture. As the nitrogen source, for example,
Soybean powder, Corn plica, meat extract, yeast extract, cottonseed flour, peptone, wheat germ, ammonium sulfate,
Ammonium nitrate is used alone or as a mixture. Examples of the inorganic salt include calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, copper sulfate, manganese chloride, zinc sulfate, cobalt chloride, and various phosphates, which are added to the medium as necessary.

Cultivation may be carried out according to the method generally used for the production of antibiotics, preferably liquid culture, and deep-aeration culture in the case of mass production. PH of medium
If the pH of the medium may vary, a buffer such as calcium carbonate may be added to the medium. Cultivation may be carried out at about 20-40 ° C, preferably 28-32 ° C. The culturing time is largely dependent on the scale of fermentation, but it is about 60 to 100 hours required for mass production. When vigorous foaming occurs during culture, an antifoaming agent such as vegetable oil, lard, or polypropylene glycol may be appropriately added before or during the culture. After the completion of the culture, in order to separate and collect PA-42867-A and / or -B from the culture, an ordinary method for separating and collecting the fermentation product is appropriately used. For example, filtration, centrifugation, adsorption / desorption with various ion exchange resins and other active adsorbents, and chromatography,
Extraction with various organic solvents may be appropriately combined.

PA-42867-A and -B may be desired to be in the form of a salt for the separation operation and for the convenience of use as a medicine or veterinary medicine. PA-42867-A
And as a base capable of forming a salt with -B, an alkali metal such as potassium and sodium, an alkaline earth metal such as aluminum and magnesium, and as an acid, an inorganic acid such as hydrochloric acid, sulfuric acid and nitric acid, and acetic acid and fumaric acid. Organic acids are mentioned. PA-42867-A and -B and their pharmaceutically acceptable salts can be administered orally or parenterally to humans or animals as the active ingredient of antimicrobial agents.
Can be orally administered as tablets, capsules and powders using commonly used excipients, stabilizers, preservatives, wetting agents, surfactants, etc., and parenterally as injections, coatings and suppositories. It can also be administered locally. Its dose is for therapeutic purposes,
Although it varies depending on the patient's age, symptoms, etc., the daily dose for an adult is about 0.1 to 10 g for an adult.

[0021]

The compound PA-428 produced by the microorganism of the present invention
67-A and -B show a strong antibacterial activity against Gram-positive bacteria, especially against methicillin-resistant bacteria, and are useful as medicaments for humans or animals. Further, since it can be collected with high purity, it is suitable not only as an oral preparation but also as an injection.

[0022]

The present invention will be described in detail below with reference to Examples.
It does not limit the present invention in any way. (a) Fermentation process: soluble starch 0.5%, glucose 0.
5%, polypeptone 0.5%, beef extract 0.5%, yeast extract 0.25%, salt 0.25%, deionized water (p
Nocardia Espi-PA-4286 in a 2 L Erlenmeyer flask containing 800 ml of a medium consisting of H7.0, before sterilization).
7 (Ministry of Microbiology, Microbiology No. 8601, Microscopy Lab., No. 1230
No.) seed culture slant at 180 rpm
Shake culture is performed at 8 ° C. for 48 hours. 800 ml of this culture
30L containing 20L of medium consisting of the same components as above
Inoculated into a Jaffer mentor with an air flow of 20 L / min,
28 at an internal pressure of 0.5 kg / cm ² G and a stirring speed of 200 rpm
Incubate at 24 ° C for 24 hours. Next, 10 L of this culture broth was prepared by adding 2.4% of tomato paste, 2.4% of dextrin, 1.2% of dried yeast (beast, manufactured by Iwaki Pharmaceutical Co., Ltd.), cobalt chloride-6.
Inoculate a 250 L fermentation tank containing 140 L of a medium consisting of 0.0006% of hydrate, 0.08% of antifoaming agent P-2000 (manufactured by Dainippon Ink and Chemicals, Inc.), and tap water (pH 7.0, before sterilization),
Aeration rate 150 L / min, internal pressure 5 psi, stirring speed 325 rpm
Incubate at 28 ° C for 64 hours.

(B) Separation step: The culture broth obtained in the above step is adjusted to pH 10.5 with 10% sodium hydroxide and centrifuged to obtain 145 L of supernatant. After adjusting this supernatant to pH 4.0, 13 L of Dowex 50x
2 (Na ⁺ type) (manufactured by Dow Chemical Co., Ltd.) through a column of water 70
After washing with L, elution is carried out with 40 L of 30% acetone water containing 1% triethylamine. Collect 22 L of the active fraction by the pulp disc diffusion method using Bacillus subtilis,
Adjust to. This is concentrated under reduced pressure to remove acetone. Then, it is applied to a 2 L column of HP-20 (manufactured by Mitsubishi Kasei).
After washing with 20 L of water, elution with 50% acetone water is performed. Collect 6 L of active fraction. This is concentrated under reduced pressure and freeze-dried to obtain 35.6 g of a crude powder of PA-42867.

(C) Purification step: Using 12 g of the above crude powder, a solution of 0.01 N hydrochloric acid in 150 ml was prepared, and MIC GEL CHP-20P was prepared.
Chromatography with 100 ml (manufactured by Mitsubishi Kasei). While checking the content by HPLC, elute with 0.01N hydrochloric acid and
The fraction containing 42867-A and B was adjusted to pH 7.0, and the pH was adjusted to C again.
Chromatograph on HP-20P. After applying the PA-42867-A and B-containing solution, it was thoroughly washed with 15% methanol water and eluted with 15% methanol-0.005N hydrochloric acid.
A-42867-A containing fraction and PA-42867-
Divide into B-containing fractions. The PA-42867-B containing fraction was adjusted to pH 7.0 and concentrated and lyophilized to give a residue of 683 mg. The PA-42867-A-containing fraction was adjusted to pH 7.0, concentrated, and then chromatographed with 10 ml of CHP-20P for decolorization, eluting with diluted hydrochloric acid (pH 5.0) aqueous solution, and then PA-42867-.
Fractions containing A were collected, concentrated and lyophilized to give a residue 571.
mg (purity 70%) is obtained. After dissolving 571 mg of this in water, dilute hydrochloric acid was added to adjust the pH to 5.0, and the mixture was applied to a CHP-20P 10 ml column and eluted with water. PA-42867-A containing fractions were collected and adjusted to pH 7.0, and concentrated. CH again for desalination purpose
P-20P was applied to a column of 10 ml (stabilized with 0.05 M phosphate buffer (pH 7.0)), and 0.05 M phosphate buffer (p
H7.0), washed with water, and then eluted with 50% methanol water,
Fractions containing PA-42867-A were collected, concentrated and freeze-dried to obtain 256 mg of PA-42867-A (purity: 95%).
PA-42867-B-containing residue (683 mg) was dissolved in water, diluted hydrochloric acid was added to adjust the pH to 4.0, and CHP-20P (5 ml) was added for decolorization.
Column, and elute with a dilute hydrochloric acid aqueous solution (pH 4.0).
Fractions containing PA-42867-B were collected, adjusted to pH 7.0 and concentrated, and then applied to packed column RQ-2 (Fuji Gel Sales Co., Ltd.). While checking the purity by HPLC, 7% acetonitrile-0.05M phosphate buffer (pH 4.9),
Then 8% acetonitrile-0.05M phosphate buffer
Elute at (pH 4.9), collect fractions with a purity of 95% or higher, and pH
The concentration was adjusted to 7.0, and after concentration, 10 ml of CHP-20P (0.
Column stabilized with 05M phosphate buffer (pH 7.0). After washing with water, elute with 50% methanol-water and PA-
Fractions containing 42867-B were collected, concentrated, lyophilized and
102 mg of A-42867-B (purity 98%) are obtained.

[0025]

EFFECT OF THE INVENTION PA-42867-A and PA-42
The in vitro antibacterial activity of 867-B was measured according to the method prescribed by the Japanese Society of Chemotherapy under the following conditions. Preparation of microbial cell suspension 1 strain of test strain on a slant medium was inoculated into 1 ml of a growth medium (tryptosoy broth, Eiken Kagaku Co., Ltd.) at 37 ° C for 1 hour.
Incubate for 8-20 hours. A 100-fold dilution of the culture is used as a cell suspension for inoculation. Sample solution Weigh accurately 9-10mg of sample and add 2mg / ml to distilled water.
Dissolve so that Serially dilute the agar plate sample solution with sterile water (2000-
0.25 μg / ml). 0.5 for each diluted sample solution
Transfer ml to a sterilized plastic Petri dish (9 cm in diameter), pour 9.5 ml of agar medium (sensitivity test medium, Eiken Chemical Co., Ltd.) into it, gently stir, and then solidify. Measurement of MIC value 1 platinum loop (0.5-
1 μl) is transferred to the surface of agar plates containing samples of various concentrations prepared as above. After culturing at 37 ° C. for 18-20 hours, the growth of bacteria on the agar surface is observed. The lowest concentration at which the growth of the bacteria was observed to be completely inhibited was designated as MIC and expressed in μg / ml. Addition of horse serum in special medium 0.5% (V / V) horse serum is added to the agar medium for measuring MIC against Streptococcus before injection.
The results are shown in Table 1.

[0026]

[Table 1]

Next, in vivo PA-42867-A
The antibacterial activity of is shown in Table 2. Test method: Slc-ICR female mice (8 mice per group) were inoculated intraperitoneally with the test bacteria, and PA-4286 was performed twice after 1 hour and 5 hours.
7-A (diluted multiple) is administered subcutaneously. Results: 50% effective dose (ED ₅₀ ) from the survival rate of mice after 7 days
To calculate.

[Table 2]

[0028]

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of PA-42867-A.

FIG. 2 shows a mass spectrum of PA-42867-A.

FIG. 3 shows ¹ H-NMR of PA-42867-A.

FIG. 4 shows a ¹³ C-NMR spectrum of PA-42867-A.

FIG. 5 shows an infrared absorption spectrum of PA-42867-B.

FIG. 6 shows a mass spectrum of PA-42867-B.

FIG. 7 shows ¹ H-NMR of PA-42867-B.

FIG. 8 shows a ¹³ C-NMR spectrum of PA-42867-B.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1: 365) (C12P 19/62 C12R 1: 365) (72) Inventor Shinzo Matsuura Hyogo Prefecture 4-63 Nakanonishi, Itami City

Claims (2)

[Claims] 1. A Nocardia orientalis species of formula I; (Wherein X represents CH ₃ and Y represents NH ₂ , or X represents H and Y represents OH).
A microorganism that produces 2867-A and / or PA-42867-B. 2. Nocardia Orientalis PA-
The microorganism according to claim 1, which is 42867.