JPS58170495A - Preparation of apramycin and oxyapramycin - Google Patents

Abstract

PURPOSE:To prepare apramycin and oxyapramycin, by cultivating a microorganism belonging to the genus Saccharopolyspora. CONSTITUTION:A microorganism such as Saccharopolyspora sp. Ac 2580(FERM- P 6239) belonging to the genus Saccharopolyspora, capable of producing apramycin and/or oxyapramycin is inoculated into a nutritive medium, cultivated at pH slightly acidic or neutral at 30-45 deg.C under aerobic conditions, apramycin and/or oxyapramycin is collected from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Apramycin and Oxyapramycin.
Concerning the manufacturing method.

Nebramycin 7:y (Nebramycin) complex is produced by Streptomyces teneprarius (Strept).
omyceslcnchrarius) AT C
8 amino sugar antibiotic components produced by C17920,
i.e. nebramycin f'l, I, Il, I
II, fV, V, Vl and
obial Agents and Chemo
therapy, 1967°314-348. U.S. Pat.
6. Streptomyces soutene prarius ATCC17
It has been published in US Pat. No. 3,853,709 that neplumine factors ■ and ■ are produced by a mutant strain of 920.

Nebramycin factor ■ is currently named Abramycin, but its chemical structure is AnnualLcpnr.
t in Medicinal Chemistry
, 9.99 (1974), J, Org, Chcm.
, 41 (12), 2087-2092 (197
6) and is described as being useful as an antibacterial agent for the treatment of various animal and plant diseases (US Pat. No. 3).
No. 691,279, US Pat. No. 3,853,709, US Pat. No. 3,876,767). In addition, Abramain/ is Streptarotyx e Hindustanus (Slr-epto
alloteicus hindustanus) A
TCC:d 1217.

31218 and 312.19° was also reported in JP-A-58-20491 and J, Ant.
1biotics.

31.497-510 (1978).

Nebulomyces nebula/factor ■ is named oxyapraminone, but its chemical structure is J.

Org, Chem, 43 (7), 1430
~1434 (1978), etc., and in addition to the aforementioned U.S. Patent No. 3,853,709, U.S. Patent No. 89
No. 62427 discloses its manufacturing method.

The present inventors isolated pure actinomycetes classified into the family Rare Actinomycetes, and as a result of continuing to search for amine-sugar antibiotics for the isolated strains, isolated i4 from field soil in Shimooshima-cho, Tokyo. It has been found that the actinomycete strain AC2580 produces abramycin and oxyapramysone. As a result of identification to clarify the taxonomic position of this strain, this strain was identified as a microorganism belonging to the genus Tusocalopolyspora.The present invention was completed based on the above findings. .

The present invention relates to horseflies belonging to the genus Satucharopolyspora and/or
Abramycin and/or abramycin are collected from the culture.
or Abramycin using a microorganism belonging to Yanocalopolisbora maple, which is not known to produce Vl, -aplamain and oxyapramain and by providing a new method for producing Ogi/γbramynon.

“f zolamayinji and/or oxyapramai7/
Producing bacteria (referred to as antibiotic producing bacteria) &LX'J
Although it belongs to the genus Nocaloborispora, for example, strain AC2580, which belongs to the genus Yaccaloborispora isolated by the present inventors, is an example of a strain most effectively used in the present invention, and the mycological properties of this strain have been investigated. It is shown as follows.

a Morphological characteristics AC2580 strain was grown on Sudachi inorganic salt agar medium (ISP
Medium 4) (Inter, J, System,
Bacleriol, , 16° 313-340 (19
After culturing for ~14 days at 37°C and 1O2 on 66)), the observed findings were as follows: The substratum hyphae were curved and elongated with branching] 2. It is fragmented and has a diameter of 04 to 06 μm, and no spores adhere to it.

Aerial hyphae generated from substratum hyphae extend in a curved or straight shape with simple branching, and have a diameter of 05 to 07 μm, and the tip usually forms a loop or a loose spiral with 2 to 8 turns. Some are curved or straight. Aerial hyphae segment to form multiple spores, usually 10 or more linked like biz chains, and the spores are often separated by empty hyphal parts.

The follicle r is oval or short cylindrical, and the size is 05-0,7
x 0.7 to 13μ, and its surface is lined with a shell with many tufts of straight or curved long hair-like substances.

It does not form sporangia, sclerotia, or flagellated spores in the ground hyphae or aerial hyphae.

[) Growth status on each of the following media After culturing at 37° C. for 14 days on each of the following media, the findings observed are as shown in Table 1. Color display is Co1or11 old mon
y Manual 4th edition 1958 (Contain
r (1 organization of America)
According to the color classification by.

(Except for each physiological property (1) in
``It grows at 1, but the optimum temperature is 80 to 45 degrees Celsius.

■) Liquefaction of gelatin (glucose/peptone gelatin f/
Positive a) Hydrolysis of starch (ISP medium 4 positive (0 coagulation of skimmed milk, peptonization; coagulation: negative, peptonization 8 positive 6) Production of melanin-like pigments (ISP medium kA6 and 7 H); Negative (0511T hydrogen hydride formation (ISP medium);
Tested with p-paper containing lead acetate); positive 0゛] Reduction of nitrate; positive (J, Bacteri
ol,,78.

15-27 (1957)] (Mark Oxygen requirement; Aerobic (51) Resistance to lysozyme; Sensitive IJn
ter.

J. System, Bacteriol, 27
, 176-178 (1977)] ω Tolerance to sodium chloride # (basal medium: 1sp
Medium 42): Grows at 0 to 14 degrees, grows at 15 degrees at 1 degree (7 no).

■ Resistance to antibiotics J, Antibiotics, 3'2, 180-1
According to the method of 86 (1979), the minimum growth concentration (MIC) for each antibiotic is determined as shown in Table 2.

Table 2 Antibiotic name MIC (try/m
l) Kanamain 〉100 gentamain/n 〉100 paromomain/n
50 streptomycin
25 neomynon 100 tobramycin) 100 rifanpin
<1z50 icomycin A,,1
>to.

■ Resolution of various substances Edited by T, R, G, Gray et al.; Ecology of
5oilhatlt1ri 11. 293~
'+21. Liverpool University
si ty'Press, Liverpool,
1967, J. Gen.

Microbiol, , 69.88-80 (197
1) and J, (ien, Microbiol, 8
8, 75-85 (1975), the resolution of various substances was measured and the results are shown in Table 3.

Table 3 (10: positive, -: negative) Chiro7; + Keratin; - Casei/
; 10 Urea ; +Kya/Zan;
10 Elastin; .

±; doubtful, −; negative] l, −°γ rabinose; trehalose; +l)
-Sorbitol;+ Sucrose;+1) Noj
Lactose;→ L-Sorbose;+1) Glucose;10 D-Sorbitol;+Glycerin;+
Zuruntol; -1-inositol; + Quinolose; ID-mannose; + Salicin
;+D-Mannitol;+Cellobiose;1Melizitose;-Starch;+Melibiose;-Adonitol;
;) Maltose ;+ α-D-Methylgraphinose ;+ Rico/Do ;←L Rhamnose
;+ Cellulose ;-D-ribose ;+ (11) Organic acid (J, Bacteriol, 78
, 15-27 (1957). +; positive, -
; Negative 1 Sodium acetate ; + Sodium propionate ; Sodium decabutyrate ; + Sodium citrate ; + Sodium fumarate ; 4 Sodium malate ; + Sodium succinate;
+ Sodium diruby/acid; Sodium tobenzoate; - Sodium diruby/acid; Sodium tobenzoate; + (・, Staining grano, staining is positive, acid-fast staining is negative.

f bacterial cell composition (DH, Bckcr et al. method (Appl, Mi
crobiol.

12.421-423 (1964)), meso-type diaminopimelic acid was detected.

In addition, Lcchevalier's method (J, Lab,
CI in.

Mcd., 71.934-944 (1968)) (1. The method of Mnrdarska et al. (J, G) detected arabinose and galactose.
en, Microbiol.

'11. In the analysis of lipids according to TT~86 (1972), the lipid LCN-A was not detected.

The method of E, Minnikin et al. (J, Gen, Mic
robiol,.

8, 200-204 (1975)), no nocardomicolic acid or mycolic acid was detected.

From the above mycological properties, the characteristic properties of the AC2580 strain are summarized as follows: 1) In terms of morphology, it has a spiral, linear or curved spore chain loosely wound around aerial hyphae generated from fragmented substratum hyphae. 2) In cell analysis, meso-diaminopimelic acid, arabinose, and galactose were detected, and the spores were trapped in a shell with long hair-like substances.
N-A, nocaldomycolic acid and mycolic acid were not detected; 3) Durham staining was positive and acid-fast staining was negative; 4) it was aerobic.

Based on these characteristic properties, we searched various literature to identify the taxonomic position of strain AC2580.
Saccaropolisbora (Sa cc ha ro -p)
olyspora Lacey & Goodfell
ow) (J, 0(In.

Microbiol, 88, 75-85 (19
751) for the first meeting, so AC'23g
0 plants were identified as belonging to the genus Polisbora, and 1 plant was identified as belonging to the Sacca sp.
ropolysporasp, )AC23gO, this bacterial strain was donated to the Research Institute of Microbial Industry, Institute of Microbiology, and was transferred to
RM P-6239).

Up to C, Sulbutomyces tenebrarius is known as an abramycin-producing bacterium, and Streptomyces tenebralius is known as an oxyabramycin-producing bacterium. It is being

Therefore, an attempt was made to compare the AC2SgO strain with Streptomyces varius and Streptomycetes efiliaus, which are known as abramycin-producing strains.

Mar1 A note on the mycological characteristics of Streptomyces tenebralix ulc Antlrnicrob
ial Agents and Chemothera
py, /967.3217-33/] and Streptomyces autotenebrarius ATCC/97:
20 (hereinafter simply referred to as ATCC/[7co0)] and a description of the mycological properties of Streptarotyx φhindustanus (J, Antibi
otics, LL, II 97-510 (/7g), JP-A-33-2017/issue] and Streptarotychus hindustanus ATCC 3/2/
7 (hereinafter referred to as single fi, TCC3/2/7) f
A comparison of the observation results using ThXI and the mycological properties of the ThXI strain and the mycological properties of the ThXI carbon source strain is shown in Table 5 and Table 5 below. ±; Usability is questionable; Streptomyces tenebrarius and AC23gO strain are similar in that they form an aerial mycelium-like spore chain. However, (1) Streptomyces tenebrarius Aerial hyphae form characteristic structures such as sclerotia and clusters, whereas the AC23gO strain does not have such structures in m% on all the media shown in the growth conditions on each medium in the previous section. (2) The cyst surface of Streptomyces tenebrarius is smooth, whereas the AC23gO strain has a long hair-like structure. The color of aerial mycelia is yellow or tan, whereas Ac,
:zsgo strain should be white, (4) The town bath f1 pigment of Streptomyces tenebrarius is red, and it is yellower than 1 liter of 0.0' 3N hydrochloric acid. -1AC23
gO stock), yellow (Yu (kai is color, 0.03N
Hydrochloric acid or 0.0! (5) The formation of aerial hyphae of Strephtomyces teneprarius is inhibited by fluorescence, whereas the AC; 15g0 strain No inhibition of aerial mycelium formation was observed in
, -(6) previous get t tko note 4dt
Method 4 4) In the analysis of bacterial cell composition according to ATCC/97 approval, meso-diaminopimelic acid, galactose and mannose were added to Ml, and 7) was added with 23 g of IL% AC.
Mesodiaminobimeli acid, galactose, and arabinose have been detected in the O strain, and differences are recognized in the presence or absence of mannose, arabino, and -s.
, ATCC/9720 was investigated for its resistance to 1 lysozyme, and found that it was ll1lj resistant, whereas k'(#
3KO strain is susceptible, (8) Basal jLjtj (ISP medium A2) Ill-
When I investigated the resistance to 1 g of sodium chloride, it was found that it grew in 7% 1 of sodium chloride (it did not grow in 1 g or more). , the AC23gO strain grows at /g% 1 and does not grow at 75% or higher, (9) Streptomyces ° tenebrarius grows at I
IRl1mCoagulate milk σ)? Against this 2, AC2! ;
(10) There is a clear difference between raffinose and L-rhamnose in terms of carbon source utilization, and the above-mentioned product properties clearly differ. 1),
AC23gOa and Streptomyces tefubularius should be taxonomically distinguished.
The 23gO strain forms a spiral in the spore chain on the aerial hyphae,
It is similar in that the soil hyphae are yellowish, do not produce melanin-like pigments, and are heat resistant to growth temperatures.

However, (Listreptarotychus hindustanus forms a single nucleus or cluster [1], and it also forms a sporangium, inside which is a flagellum-bearing sporangiospore (
- On mother extract/malt extract agar medium and glycerin/asparagine agar medium, cultured at 2 g °C for 3 to 4 weeks and on all the media shown in the previous section. Observation of the ridge of culture at 37°C for 2 to q weeks - Even in this, no mononuclei, clusters, sporangia, or IIMs were detected. (2) The spore surface of Streptarotychus hindustanus was smooth. In contrast, the AC23IO strain has a long hair-like structure; (5) Streptarotychus hindustanus has abundant protuberant thread formation, whereas Ac, 2j, rO
The color of the KC231rO strain is white, whereas that of the KC231rO strain is white.

(S) X-Treptarotyx Hindustanus forms spherules in the substratum, whereas AC23Z0
(6) Streptarotychus hindustanus does not have a dividing property in its substratum hyphae, whereas the AC, 2350 strain has a dividing property; (7) The method described in f above. Su, ATCC3/
In the body composition analysis of 2/7, meso-diaminopimelic acid, galactose, and mannose were detected, whereas in the %AC2310 strain, meso-diaminopimelic acid, galactose, and arabinose were detected, indicating a difference in the presence or absence of mannose and arabinose. (8) The method described in C■ above, ATCCJ/
When we investigated the degree of resistance to lysozyme in Co/7, we found that it was resistant, whereas the AC23IO strain was susceptible. (9) AT in the basal medium (ISP medium, %2)
The resistance of CC3/2/7 to sodium chloride and thorium is 14-9, while it grows up to sodium chloride SIs and does not grow above 6-9.
The AC2580 strain does not coagulate, grows to 15Is or more, and does not coagulate.
Munose, 1)-sorbitol, and D-xylose are clearly different from each other, and the properties listed above are clearly different.
Strain C2580 and Streptarotychus hindustanus should be taxonomically distinguished.

As described above, this antibiotic-producing bacterium is explained, but the general properties of radiation [1] according to Dutch science are extremely variable and not constant. It is a well-known fact that mutations can occur by artificial mutation means using mutagenic agents, and such artificial mutant strains as well as natural mutant strains belong to the genus Satucharopolyspora and are capable of producing abramycin and/or Alternatively, any strain capable of producing oxyapramycin can be used in the present invention.

In the present invention, first, the present antibiotic-producing bacterium belonging to Satucalopolysbora maple is cultured in an appropriate medium. In culturing this bacterium, a culture method for actinomycetes is generally used.

The medium used is a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and, if necessary, inorganic salts. , fructose, galactose, funnose, glycerin, molasses, starch, dextrin, co/kiss, dried yeast, soy flour, corn steep liquor,
Organic nitrogen sources such as cottonseed meal, casein, soybean protein decomposition products, amino acids, and urea, and inorganic nitrogen compounds such as nitrates and ammonium salts are used alone or in combination. In addition, as necessary, sodium salt, potassium salt, calcium salt,
Inorganic salts such as magnesium salts and phosphates are added. Furthermore, the culture medium may contain micronutrients that promote the production of the antibiotic-producing bacteria and/or 1'geramain and/or oki7apramain; -t, growth promoting substances, and precursor substances may be appropriately added.

Cultivation is usually carried out under aerobic conditions F, such as shaking or aerated agitation culture, and industrially, deep aeration agitation culture is preferred. It is preferable to culture the medium at a neutral pH, rather than acidic. The culture temperature is usually kept at around 30 to 45°C. When culturing, 1111 is usually 2 in the case of static culture.
When the culture is carried out for ~5 days, the present antibiotic is produced and accumulated. Preferably, +7<1 should be terminated when the amount of the present antibiotic accumulated in the culture reaches the maximum. These culture conditions such as medium composition, liquid properties of the medium, culture temperature, stirring speed, and aeration amount, 1) are adjusted and selected as appropriate to obtain favorable results, depending on the type of strain used and external conditions. Sat
It goes without saying that I do. When -C foaming occurs in liquid culture, an antifoaming agent such as silicone oil, vegetable oil, or surfactant is appropriately used.

Since the antibiotic accumulated in the culture obtained in this way is mainly contained in the culture F solution, the culture is treated with a lachrymal agent such as Celite, Perlite, Hi, Flow-Subacel, etc. In addition, it is advantageous to separate the culture F solution and the bacterial cells by filtration or centrifugation, and then collect the present antibiotic from the culture F solution.

In order to separate and purify the present antibiotic from the culture solution F, various methods known in the art of separating amino sugar antibiotics can be used. It can be separated from the culture medium by methods such as chromatography using positive A/exchange resins or other solid adsorbents. Take care of the culture F solution.
Adjust to H7, Amberlight I RC-50, CG-
A chromatographic method using a weakly acidic cation exchange resin such as 50, the most preferable ammonium type, or an ion exchange cellulose/10-acetate such as CM-cellulose, an ion exchange Sephadex such as CM-Sephadex, etc. is used. This is a method using chromatography.

Next, the adsorbed antibiotic may be eluted with a weak base eluent, such as dilute ammonium hydroxide, changing the concentration order as necessary. The eluate thus obtained can be combined with fractions containing the same components and concentrated or lyophilized to obtain apramine and oxyf-solafine, respectively.

Furthermore, if purification is required, separation and purification can be achieved by repeatedly performing the above chromatography.

Next, the method of the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 5 In a 110 m/' Erlenmeyer flask, 1 t glucose, 1% dextrif, 15% casein decomposition product, 0 yeast extract.
Liquid medium (+3 I) containing 5% Cal/Rum 01
f? O) 100m/ml was dispensed and sterilized at 120°C for 20 minutes, then a platinum loop of Socalopolyspora subinose AC2180 from a slanted agar medium was inoculated into 10 of each medium, and cultured with stirring at 30°C for 72 hours. .

The obtained seed culture was transferred to a 301-volume jar fermentor containing medium 201 having the same composition as above, and stirred at 30° C. for 47 hours at a stirring speed of 25 Or, p, m. .

Aerated agitation culture was carried out under aeration conditions at an aeration rate of 151/min.

In a 2501 volume fermentation tank, a liquid medium containing 2% glucose α, 4% glycerin, 02% soluble starch, 05% peptone, 05% soybean flour, 05% Ebios, 05% salt, and 02% calcium carbonate/rum 1 pH? o) 200m/! After preparing and heat sterilizing, the above culture 101 was transplanted, and 30
Culture 1901 was obtained by culturing with aeration at a temperature of 120 hours at a stirring speed of 110 r, pom, and aeration rate of 10,017 minutes. To this, 5 kg of Barley) was added and filtered, and the resulting culture F solution was added to Amberlite JRC-50.
(manufactured by Rohm and Haas) (ammonium type) 1
The mixture was charged with 201 ml of 2N ammonia water after being charged with water and washed with water. The entire eluate was concentrated under reduced pressure to 100 m/ml. Then, this concentrate was diluted with 6N sulfuric acid I)
I-1? 0 to Ah”b L, CM-Sephadex C-25 (manufactured by Pharma/-γ Fine Chemicals)
(Aqueous ammonia, type) Charged to a column at 500 mC. After washing with water, it was eluted with ammonia water using a linear concentration gradient from 0 to 008 N, and the eluate was fractionated into 20 ml portions. Each fraction was divided into chloroform-methanol-1
The product was traced by thin-layer gel chromatography using $4000 water (1:2:1) as a developing solvent, and the target product was confirmed by color development. The samples from 194 to 206 contained only oxyabramyone, and the samples from 219 to 242 contained only acramainone. Each of these first and other products was collected, concentrated under reduced pressure, and then lyophilized to obtain oxoapramycin 1,82 and apramycin a7f.

fNoramain properties; white powder melting point 245°C (decomposition) lα 1162° (C=1.water) 11v; end MW (F T) mass ); 539 molecular formula:
C21H4+ Ns 0n TLC; nfA=(132, Rf, =Q22 carrier; developing solvent; A; chloroform-methanol-28thia/monia water (
2'l:2) B: Chloroform-methanol-14%-f/monia water (1:2:1) Oxygen/agglamy7 properties; white powder melting point;) 265°C (decomposition) [α+170'(( '=l, water) U■; End MW (P Dmass); 555 Molecular formula: C
21H41H5012 TLC'; RfA = (1:31, RfB = Qxt
; Abramine described in literature [J, Org, Chem, 41
(12), 2087-2092 (1976)) and Okinabramin/N (J, Org, Chem.,
48(7).

1430-1434 (1978) 1.

Patent Applicant Toyo Jozo Co., Ltd. Representative Ito Fujima Commissioner of the Patent Office Mr. Kazuo Wakasugi/Indication of the Case 1981 Patent Application No. 416 Name of the Invention Process for Producing Abramycin and Oquinapramycin 3 Relationship with the case of the person making the amendment Patent applicant address: 632 Mifuku, Ohito-cho, Tagata-gun, Shizuoka Prefecture /≠ Date of amendment order Voluntary Claims column of the specification to be amended and (2) Invention of the invention ``Antibiotic equipment'' in line g of page 2 of the specification in the detailed explanation column is corrected to ``antibiotics''.

[Rare Actinomycetes] on page 3, line 1S of the specification is corrected to "Rare Actinomycetes."

Page 3 of the specification, lines 1g to 79, page 5, line 2, line O, page 11, line 3, / line O, page 75, line 1, + line, line /4Z, 1st page O, line 7, 17th page table, 1st page
Page table, page 20, line 1, // line, line 73, / line, page 27, line 2, line 7, line 72, line 1g, line 22
Page 3, line 7, line 72, /O line, page 23, line 72, /S line, /g line, page 211, line 1, line 1, line 75, 20 line, page 2S, line 70, line 2, same page
On page 9, line 1S, "Acxsgo stock" is corrected to "Ae 23za0 shares."

In the specification, page 3, lines 1 to 2, "In the first observation, sclerotia" is corrected to read "Ac, 25go strain" in the first observation.

"Cotton seed meal," in the first row of page 26 of the specification, is replaced with "cotton seed flour."
I am corrected.

! Claims (1) Abramycin and/or oxyapramycin-producing bacteria belonging to the genus Satucalopolysvora are cultured in a medium to accumulate abramycin and/or oxyapramycin in the culture, and the culture A method for producing abramycin and/or oxyapramycin, which comprises collecting abramycin and/or oxyapramycin from.

(2) Abramyzone and/or oxyapfumycin-producing bacteria belonging to the genus Satucalopolyspora are Satucalopolyspora sp. Ac23gQ (FEBM).
P 6239)"'Q The manufacturing method according to claim 1.

Claims (2)

[Claims] (1) Cultivate apramain and/or oxyapramycin-producing bacteria belonging to the genus Satucharopolisvora in a medium, accumulate abramycin and/or oxyapramycin in the culture, and collect abramain and/or oxyapramycin from the culture. abramycin and/or oxyabramycin/
Apramine manufacturing method. (2) Abramai belonging to the genus Satucharopolyspora/
and/or oxyabramain-producing bacteria is Satucalopolysvora sp. AC2580 (FETtM).
P-6289), the manufacturing method according to claim 1.