JPS5995897A - Preparation of antibiotic substance - Google Patents

Abstract

PURPOSE:To obtain antibiotic substance Y-02077Hgamma and delta substance, by a fermentation method, by culturing a microbial strain belonging to Micromonospora genus and capable of producing said substances, and separating the objective substances from the cultured product. CONSTITUTION:A microbial strain belonging to Micromonospora genus and capable of producing Y-02077Hgamma and delta substance [e.g. Micromonospora sp. Y-2077H strain (FERM-P No.6557)] is cultured, and the objective Y-02077gamma substance of formula (R1 is methyl) and Y-02077Hdelta substance (R1 is H) accumulated in the cultured product are separated therefrom.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for the microbial production of antibiotics. More specifically, two microorganisms belonging to the genus Micromonospora isolated by the present inventors were cultured, and from the culture Y-02077Hγ (wherein R1 means a hydrogen atom or a methyl group) was obtained. (R1=methyl group) and Y-02077Hδ (R, -hydrogen atom) substance is collected.

Y-02077Hr and δ substances are disclosed in JP-A-51-864.
It is known as a substance exhibiting antibacterial activity as described in Japanese Patent No. 44.

According to the same publication, the Y-02077Hγ and δ substances can also be produced by a chemical synthesis method using an oxidative demethylation reaction of dientamicin (Gentamicin C1).

The present invention provides a method for producing Y-02077Hγ and δ substances using a 9-fermentation method.

The Y-020778γ and δ substance-producing bacteria belonging to the genus Micromonospora used in the present invention include, for example, Micromonospora echinospora subsp.
Comoroenosis can be mentioned.

This microorganism is an actinomycete isolated by the present inventors from the soil of Komoro City, Nagano Prefecture, and is a new strain with the strain number Y-02077H. The mycological properties of the Y-02077H strain are as follows.

■ Morphological properties Strain Y-02077H does not form aerial hyphae on various types of agar medium that are commonly used. 0.
5 μm), no septa are observed in the basal hyphae, and spores are attached one by one to the apex of the sporophore branched from the hyphae. Under an electron microscope, the spores are approximately spherical with a diameter of about 1 μm, and many slightly rounded projections are observed on the spore surface.

2. Growth status on various media Table 2 shows the growth status and colony shape when cultured on various media at 28 tr for 7 to 21 days.

Table 2 3 Physiological properties Table 3 shows the physiological properties of the Y-02077H strain.

Regarding things other than the growth temperature range, effects on milk and cellulose, the observation results after 2 weeks at 28 tr are shown. Furthermore, the growth temperature shows the observation results from 7 to 30 days, and the effects on milk and cellulose show the observation results from 7 to 21 days.

To summarize the properties shown in Table 3 and above, strain Y-02077H does not form true aerial hyphae on various agar media, but forms a single spore on the sporophore produced from basal hyphae.

Analysis of the cell wall revealed that it contained meso-diaminopimelic acid, so it was recognized as an actinomycete belonging to the genus Micromonospora. Bergey's known strains similar to this strain
Manual of Determinattve
Bacterio-1ogy 8th Edition
(1974) and various literature, it was found that Micromonospora echinospora (Micro-monospora echinospora) is a similar strain to the Y-02077H strain.
spora echinospora), Micromonospora dionenosis (Micromonospo
ra jionen3is, JP-A-49-55895
) Micromonospora sagaminenosis (Micromo
nospora sagaminensis, JP-A-4
9-47590). However, Y-020
77H strain has a different color of growth on Bennett agar, oatmeal agar, etc. when compared to the descriptions of Micromonospora dionenosis and Micromonospora sagaminensis F) 2) Also, the former has extremely good utilization of rhamnose, whereas the latter 2) The two strains do not take advantage of this (・.On the other hand, the Y-02077H strain has the same color as Micromonospora echinospora when growing on various media (dark brown to black) and has rounded protrusions on the spore surface. The Y-02077H strain is in good agreement with the microorganisms in terms of sugar utilization, growth temperature range, optimal growth pH, cellulin decomposition action, melanin-like pigment production (negative), etc. It was determined that the strain belonged to Monospora echinospora.It was determined that the strain belonged to Micromonospora echinospora.
CC15838).

Three strains have been reported: Micromonospora-Echinospora subspaces Echinospora (ATCC 1,5837) and Micromonospora Echinospora subspaces Feruginia (ATCC 15836).
Table 4 shows the differences compared with the Y-02077H strain.

Table 4 Differences between strain Y-02077H and three strains belonging to Micromonospora echinospora Based on these results, strain Y-02077H has the morphology, color tone of bacterial colony on agar medium, spore shape, growth temperature range,
Although it has characteristics of Micromonospora and Echinospora in terms of optimum growth pH, melanin-like pigment production (negative), cellulin decomposition action, and sugar utilization, it belongs to Micromonospora and Echinospora. It exhibits different properties from the three known strains in terms of gelatin liquefying action and milk coagulating action. In addition, a medium containing 1% glucose (e.g. glucose
The Y-02077H strain grows poorly on asparagine agar, Benenoto agar, etc. (on the other hand, the three known Micromonospora strains show normal to slightly good growth.
There are also slight differences in sugar utilization. Based on the above, strain Y-02077H is recognized as a new subspecies belonging to Micromonospora echinospora, and Micromonospora echinospora zabuspacis comoroensis (M
icromonospora echinospora
5bsp.

Komroensis). Y-02077
H strain is Micromonospora niss B Y-02077H
It has been deposited as a strain at FIKEN, and its deposit number is FIKEN Bacteria No. 6557.

The 5y-02077H strain used in the present invention is a naturally occurring actinomycete, which is susceptible to artificial and natural mutations, as seen in other actinomycetes. Alternatively, it also includes strains that have been artificially mutated using ultraviolet rays, X-rays, chemical agents, etc., as well as natural mutant strains thereof.

Y-020 belonging to the genus Micromonospora in the present invention
In culturing the 77Hγ and δ substance-producing bacteria, the usual culture method for actinomycetes can be used. As a nutrient source for culturing, ℃ and strainer are used. As the carbon source, any assimilable carbon compound may be used.2For example, glucose, starch, glycerin, fructose, arabinose, rhamno-4° sucrose, dextrin, coconut oil, oilseed oil, etc. can be used alone or in combination. It leaks. Furthermore, alcohols, organic acids, etc. may also be used. Examples of inorganic and organic nitrogen sources include monochloride, sodium nitrate, monosulfate, mononitrate, urea, etc., and organic nitrogen sources include peb)-', i? Mother's Kiss, meat extract, corn stew liquor, soybean flour, fish meal, cottonseed meal, casamino acids, etc. are used alone or in combination.The culture method is a liquid culture method, especially a deep aeration agitation culture method. It is advantageous to culture at a temperature of 27 to 30C.The pH is preferably near neutral.

The culture time varies somewhat depending on the conditions, but is usually about 3 to 5 days, and the culture is terminated at an appropriate time, taking into account the time when the 't-020778 γ and δ substances reach their maximum titer.

Isolation and purification of Y-02077Hr and δ substances from the culture solution was performed by a method commonly used for separating water-soluble basic substances.
·sell. That is, it can be carried out by appropriately combining methods such as adsorption/desorption using a cation exchange resin, activated carbon, cellulose, and silica gel column chromatography.

The physicochemical properties of the antibiotic Y-02077H γ and δ substances thus obtained are as follows.

1) Physical and chemical properties of Y-02077Hγ substance ■ Basic white powder ■ Melting point = 137-143C ■ Specific optical rotation: [α]', ' = +115.3 (C = 0
．． 68. , O) ■ Ultraviolet absorption spectrum: (R
20) Only shows terminal absorption.

■ Infrared absorption spectrum (KBr) (crn')
: The infrared absorption spectrum of this substance is shown in Figure 1.

■Nuclear magnetic resonance spectrum (R20): Shown in Figure 2 ■Mass spectrum: From the mass spectrum of this substance shown in Figure 3, its molecular weight is 4637, and its molecular formula is Cy, U4.
It was determined to be tN507.

■ Rf of silica gel thin layer chromatography of this substance
Value: Y-02077H-γ 049 Die/Tamaishino C,0,57 // C20,53 〃 CIa 047 Developer: Chloroform: Methanol: Aqueous ammonia (20:15:8) ■ Color reaction: Ninhydrin reaction ■ Sakaguchi reaction 0 2) Physical and chemical properties of Y-02077Hδ substance ■ Basic white powder ■ Melting point: ]26-145C ■ Specific optical rotation: C(112N)1=+93.9 (
C=0.6 R20)■ Ultraviolet absorption spectrum (R
20): Shows only terminal absorption.

■ Infrared absorption spectrum (KBr) (cm-')
: The infrared absorption spectrum of this substance is shown in Figure 4.

■Nuclear magnetic resonance spectrum (R20) Shown in Figure 5 ■Mass spectrum: From the mass spectrum of this substance shown in Figure 6, its molecular weight is 4491, and its molecular formula is C, R3.
, N507.

■ Rf of silica gel thin layer chromatography of this substance
Value: Antibiotic name Rf value Y-02077Hδ 0.48 Die/Tamaishino C+ 0.57/IC20,53 // C,, 0,47 ■ Color reaction: Ninhydrin reaction ■ Sakaguchi reaction O Based on the above physical and chemical properties 2 The product obtained by the fermentation method of the present invention is:

Y-02077Hγ substance represented by the formula (R,=C)]3)
and Y-02077Hδ substance < R1=H).

Example extract l-IJ /2.0%, Curcose 0.5%, Corn steep liquor 05%, Polypeptone 05%,
Yeast extract (manufactured by Oriental Yeast Co., Ltd.) 05%, meat extract (manufactured by Kyokuto Pharmaceutical Industries KK) 0.3%, Play/Heart Inquiry Nobuillon "Eiken" 05%, Ca COs
0.2%.

Dispense 50 ml of a medium with a pH of 8.0 into a 500 ml Erlenmeyer flask, sterilize it as usual, and use Y-0.
Inoculate the 2077H strain and culture at 28C for 72 hours to use as a seed culture.

In addition, 3% potato starch and 1.5% defatted soybean flour.

Corn stable liquor 0.5%, yeast extract (manufactured by Oriental Yeast Co., Ltd.) 0.2%, MgSO4.7H200
,05%.

NaC10.3%, COCl20.02g/l,
A medium with a pH of 7.3 was added to a 20 ton jar fermenter, and Adekanol (manufactured by Mudenka Co., Ltd.) was added as an antifoaming agent.
Added 0.03%.

After sterilization as usual, the above seed culture solution is inoculated at a rate of 3% and cultured at 29°C for 120 hours (250-27 rpm). Obtain 15Z as a culture solution. Repeat this operation 14 times to obtain about 205 cells of culture solution.

Add 205tVc of culture solution to adjust the pH to 2, and leave at room temperature at 30°C.
Stir for a minute. Radiorai) 600 (Showa Chemical Industry K
K) Add 10 kg and press and filter. After adding sodium hydroxide solution to the slurry and adjusting the pH to 7, JRC-
50 (manufactured by Rohm and Haas) [NH4+]2
01 was adsorbed onto a column packed with water, and after washing with water, it was eluted with 50 tons of IN-ammonia water. The eluate was concentrated under reduced pressure, and concentrated using iwocG-50 (manufactured by Rohm At)・-S Co., Ltd.)C.
The active fraction was adsorbed onto a column packed with 2.8 t of NH4"] and eluted using a water-washed active water-ammonia water concentration gradient method. After concentrating this under reduced pressure, it was packed with silica gel (Wako Tsumugi Pharmaceutical Co., Ltd.). Using a column (3.3 x 50 cm), chloroform: methanol: aqueous ammonia (20
: 15 : 8). The active fractions were collected and concentrated, and the concentrated liquid was collected using CG-50 Type Il (ROHM &
)・-S Co., Ltd. H) [NH4+) Adsorbed in a column packed with 30 ml.

Washed with water, active fractions were eluted by active water-ammonia water concentration gradient method and concentrated, Y-02077Hγ, Y-02077
77111 g of a mixture of both Hδ substances was obtained.

This mixture was subjected to preparative thin layer chromatography (silica gel), and Y-020778γ substance and Y-
02077Hδ material was isolated. The city is (・Zeremo Do)
wex I
g.

Also, the Y-02077Hδ substance was used as a white powder llrr1
I got g.

Confirmation of the active fraction of this substance is done by Bacillus spuptilus ATC.
A bioassay using C6633 and silica gel thin layer chromatography were performed at 0°C.

[Brief explanation of the drawing]

(11 Figures 1 and 4 are Y-02077Hγ
The infrared absorption spectra of the substance and Y-02077Hδ substance are shown. (2) Figures 2 and 5 are respectively Y-020771 (
Nuclear magnetic resonance spectra of γ material and Y-02077Hδ material are shown. (3) Figures 3 and 6 are respectively Y-020771 (
The mass spectra of the γ substance and the Y-02077Hδ substance are shown. 58 Continued from page 1 0 Inventor Masaru Iwanami 8-1 Nishikigaoka, Kohoku-ku, Yokohama 589-

Claims (1)

[Claims] 1. Y-02077Hγ belonging to the genus Micromonospora
and δ substance-producing bacteria were cultured, and Y-0207 was added to the culture.
7Hγ and δ substances are accumulated and Y-020 is removed from the culture.
Y- characterized by collecting 77Hγ and δ substances
02077H-Method for producing γ and δ substances. 2. Y-02077Hγ belonging to the genus Micromonospora
and the δ substance-producing bacterium is Micromonospora varnis b. B. strain Y-02077H.