CA1151091A

CA1151091A - Oa-7653 substance

Info

Publication number: CA1151091A
Application number: CA000356397A
Authority: CA
Inventors: Tsutomu Nishida; Takashi Kamogashira; Michiharu Sugawara
Original assignee: Otsuka Pharmaceutical Co Ltd
Current assignee: Otsuka Pharmaceutical Co Ltd
Priority date: 1980-07-17
Filing date: 1980-07-17
Publication date: 1983-08-02

Abstract

ABSTRACT
The antibiotic of the invention is produced from a novel strain belonging to Streptomyces hygroscopicus, acts against various bacteria and is useful, for example, as active components of antibacterial compositions.

Description

115109~
This invention relates to a novel antibiotic (given the designation OA-7653 substance)prepared from a novel strain belonging to Streptomyces hygroscopicus and to antibacterial compositions comprising the antibiotic.

While we have found that the strain of this invention belongs to Streptomyces hygroscopicus as will be described later, the strain is clearly distinct from Streptomyces hygroscopicus (Jensen) and Streptomyces hygroscopicus ISP
5578 which are typical of the species and is also distinct from the strains belonging to any known species or sub-species. Further the substance of this invention cannot be isolated from the strains of any of known species or sub-species.
The substance of the invention is characterized by the following physicochemical properties.

(1) Appearance White particulate crystals.

(2) Solubility Sparingly solubel in 0.lN aqueous solution of hydrochloric acid. Insoluble in methanol, ethanol, butanol, acetone and ethyl acetate. Soluble in 0.lN aqueous solution of sodium hydroxide.

'~t .

115~09~

~3) Spcci~`ic ro-tatory po~er ta)V3'5 = +76.~ (C ~ 63, water) (4) ~lementary analysis C: 46.1~,';, H: 4.4770, N: 7.1~
(5) I,olecv.lar wei~ht determined by ultra-centrifugal analysis About 2000.
(6) Isoelectric point determined by electrophoresis pH 5 - 6.
The isoelectric point is determined by the following method. In 1 liter of distilled water are dissolved 6.008 g of citric acid, 3.893 g of Kh-2P04, 1.769 g of H3P04 and 5.266 g of 5,5-diethylbarbituric acid. One-liter quantities of such solution are adjusted to pH 3, 4, 5, 6, 7 and 8 with 0.2N aqueous sodium hydroxide solution to obtain bvffer solutions. Using the buff~er Q ~a~ k for solutions and Whatman filter paper No. 1 ~roduct of' W. and H. ~alston Ltd~) spotted with an a~ueous solution o~ the substance of the invention, the substance is subjected to electrophoresis at 300 V f'or 4 hours to ob-tain an analysis diagram. The result is represented by Curve 1, ~ig. 3, which shows that the isoelectric point is at pH 5 to 6.
(7) IR absorption spectrum ~'ig. 4 shows an IR absorption spectrv~n o~tained v~ith use of a KBr disk, indicating main absorption pea~s ~t 32~0 (s), ~660 (s), 16~,0 (5), 151'~ (s), 1490 (s), 1~95 (s), 1235 (s), 1150 (m), 1062 (s) and 1020 (s) cm 1.
(8) UV absorption spectra i) 'l'he substance of the invention (1 mg/10 ml) is subjected to UV analysis at a cell length of 1 cm, using O.lN aqueous solution of hydrochloric acid as a solvent.
Fi~. 5 showin~ the result reveals an absorption maximum at 278 m~ (El~Cm = 56).
ii) ~'ig. 6 sho~s the result of UV analysis similarly conducted with use of distilled water as a solvent.
The spectrum reveals an absorption rnaximum also at 278 m~ (F~l~m = 56).
iii) The substance of the invention (0.5 mg/l~ ml3 is subjected to UV analysis at a cell length of 1 cm, using O.lN aqueous solution of sodium hydroxide as a solven-t.
~ig. 7 sho~ing the result reveals an absorption maximum at 298 m~ m = 100.8).
(9) lH-I~qi~R spectr m Fig. 8 shows a H-~ R spectrum of the present substance in dimethyl sulfoxide substituted with deu~erium (d6-~S0). Table 1 below shows the shifts (ppm) of peaks.

::

Table 1 No. _ ~m I~o. ppm No. ~m 1 1.05 - 1.35 53.0 - a.l 9 6.6- 7.0 2 1.60 - 2.0 6~.2 - 4.9 10 7.0- 7.55

3 2.2 75.05 - 5.6 11 7.6- 8.0

4 2.50 86.3 - 6.5 (10) 13C-~iR spectrum ~ . 10 shows a 13C-~iR spectrum of the present substance in d6-D~iIS0. Table 2 below shows -the shifts (ppm) of peaks.
Table 2 No. pPm No. ppm No. ppm 1 173.2 14 148.7 27 105.9 2 173.0 15 146.8 28 104.6 - 3 172.4 16 138.3 29 102.3 4 172.2 17 136.5 30 96.3 170.8 18 135.5 31 73.6 6 169.7 19 134.1 32 69.3 7 1~9.0 20 131.6 33 62.5 8 167.1 21 129.7 34 60.6 9 157.0 22 128.8 35 56.7 156.8 23 127.8 36 53.9 11 155.8 24 125.3 37 31.1 12 149.7 25 121.5 38 9.6 13 149.5 2~ 117.9 ~ ~r ~

(11) Thin-layer chromatography The substance of the invention is dissolved in w~ater and subjected to thin-layer chromatography with use ~4~ p~r ~
of a thin-layer plate of "Silica Gel 60 F254" ~product of r&erck), with the following results.
i) Developing solvent: butanol-acetic acid-water (4:1:1 in volume ratio). Rf = 0.
ii) Developing solvent: propanol-2N ammonia water (7:3 in volume ratio). Rf = O.
iii) Developing solvent: chloroform-ethanol-water (4:7:2 in vol~me ratio). Rf = 0.45.
iv) Developing solvent: ethanol-water (7:3 in volume ratio). Rf - 0.8.
(12)-Paper chromatography The substance of the invention is dissolved in water and subjected to paper chromatography with use of Whatman chromatograpkic paper, Whatman No. 1.
The results are as follows.
i) Developing solvent: butanol-acetic acid-water (4:3:7 in volume ratio). Rf = O.83.
ii) Developing solvent: butanol-pyridine-water (4:3:7 in volume ratio). Rf = 0.76.
(13) Color reactions The substance of the invention is dissolved in water and su~jected to color reactions on the same TL~ plates as used for the thin-layer chromatography above. The results ære as follows.
Iodine reaction Positive Ninhydrin reaction ~Lolisch reaction "
~hrlich's reaction "
Amino acid analysis and gas chromato~raphic analysis have revealed that the substance of this invention contains 0.58 ~ mole/mg of L-glutamic acid and 0.6~ ~ mole/mg of glucose as components thereof.
The substance of the invention is further characterized by its antibacterial activity.
The activity of the substance against various -, .
bacteria is determined in terms of minimal inhibitory concentrations (~IC) according to agar-dilution tests (culture medium: Heart Infusion (product of Ei~en Kagaku Co., ~td., Japan), incubation: 37 C, 18 hours).
Table 3 below shows the results thus determined.
~able 3 Test No. Test bacteria ~IC
_ (mc~/ml) 1 Bacillus subtilis P~I-219 6.25 2 ~acillus ~nthracis 12.5 3 3ac_ lus cereus IF0 3001 12.5 4 acillus cereus I~0 3466 12.5 .

ilS~O91 Test No. Test bacteria MIC
_ (mc ~ml) Bacillus cereus ATCC 1177812.5 6 Bacillus pumilus IFO 3813 6.25 7 Bacillus circulans ATCC 82416.25 8 Staph~lococcus aureus FDA 20gP 3.13 9 Sta~h.~rlococcus aureus Newman 3.13 Staph~lococcus æureus IFO 30606. 25 (Terazima) 11 Sta~h~lococcus aureus Smith6.25 12 Sta~h~lococcus epidermidis12.5 ~o 3762 13 Streptococcus ~o~enes aureus3.13 r 14 Streptococcus p~o~enes IID s23 6.25 Stre~tococcus faecalis IFO 12580 6.25 16 Streptococcus viridans 3.13 17 Sarcina lutea ~CI-1001 3.13 18 Salmonella t~ 0-901 NCTC 8393> loo 19 Proteus vu.lgaris IID 0~-19 us> 100 Yersinia enterocolitica 0-3~ loo 21 Candida albicans KYU ~ 100 22 Saccharo~ces cerevisiae ~ 100 23 ~,ycobacterium phlei IFO 315812.5 24 rrycobacterium sme~matis ATCC 607 25 : 25 r~cobacterium ~ O 3083 1.56 ~ 26 rtl~cobacterium avium IFO 3082 25 115109~

l'hc suistance ol`-thi; invention ~:a intrav~nousl~.r administ;er~d tc~ male ~lice ~f DD strain wei~hin~ 20 to 21 g and checked for acu-te toxicity.
Conseqvently no aeath resulted at a dose of 1500 m ~kg.
Biopsy of all the survivals revealed no noticeable in~`luence on organs.
In view of the ~hysicochemical roæerties and the -~har.~.acolo~ical test results stated above, in combination ~with the origin of the present com~ound, - 10 the compound ap~ear~ most analo~ous to vanco~ycin, a known antibiotic tShionogi's Annual Report, Vol.7, . 465 (1957), Anti~iotics Annual, 1957-1958, p. ~06 (1958), Journal of ~edical Chemistry, Vol. 8, p. 18 (1~65), and Antibiotics Annual, 1955-1956, ~. 60~ (1956)~.
However, vancomycin is produced from StrePtOmvCes orientalis as ~ill be described later. As disclosed in the above literature, it proves negative when subjected to ninhydrin reaction and ~hrlich's reaction. An electrophoretic analysis of the compound in the form of vancomycin hydrochloride shows that it has an isoelectric ~oint at a ~P. of 7 to 8 as represented by Curve 2 in E'ig. 3. ~ig. ~ showing a lH-~IR spectrum of vancomycin hydrochloride reveals that the spectral chart differs i`rom that OT the ~resent substance. T~ analyses Y~ith use of the developing solvents l~entioned in para. (11), ii ) bO iV) show that va~comycin hydrochloricc exhibits the spots in~iclted at A in Pigs. 11 to 13 ( in which indicatcd at B are the spots developed ou-t with use of the present substance as an aqueous solution).
When tested for acute toxicity by intravenous administra-tion to male mice of DD strain weighing 20 to 21 g, vancomycin exhibits LD50 of 400 to 500 mg/kg. In these features, vancomycin distinctly differs from the ; substance of the invention. Additionally vancomycin contains aspartic acid as a component thereof but contains no glutar.ic acid as disclosed in J. Med. ~hem., Vol. 8, p. 18 (1955). In this res~ect also, the ~resent substance differs from vancomycin. Although ,~,....... . .
we have compared the present substance with various 15 known antibio+ics derived from microor~anisms, es~ecially from those belonging to the genus Strepto.~yces like the present substance, none other than vancomycin have been found analo~ous to the ~resent sub~-tance. Thus we have identified the substance of this invention, 20 OA-7653 substance, as a novel compound.
~n~ c A The novel nub~t~ncc of the invention, namely, OA-7653 substance is produced from a strain belonging to Stre~tom~ces h~rosco~icus.
WD have isoiated the strain from soil in 25 Hiwasa-cho, ~aifu-gun, Tokushima-ken, Japan. The strain - 115109~

has the following features and has been identified as a novel strain.
I. Morphology Fig. 1 is a photomicrograph (at a magnifica-tion of 800X) showing the strain as incubated at 28C for14 days on glycerin-asparagine agar medium, while Fig. 2 is a electronmicrograph tat a magnification of 9000X) ^ showing the strain as incubated at 28C for 14 days on oatmeal agar medium. These photographs show that spore forming mycelia of the strain are branched out in a simple fashion from a long stem formed in an aerial mycelium and have two to three turns of spiral portions at the ends of branches. A very few spore forming mycelia do not have a stem and are in the form of irregular complex branches.
An observation under a scanning electron micro-scope reveals that spores are smooth-surfaced, are cylindrical to ellipsoidal in shape and are about 0.7 to 1.0 x 0.9 to 1.6 ~ in size. Usually about 15 to 40 spores are connected together in the form of a chain.
II. Cultural characteristics 1) Yeast-malt agar medium a) Abundant growth. Markedly wrinkled.
b) Aerial mycelia are moderate, powdery and covert tan (2 ge) to white.

:

1~51091 c) Substrat~ mycelia are light wheat (2 ea) on the reverse side.
d) Soluble pigment is honey gold (2 ic).
2) Oatmeal agar medium a) Moderate growth.
b) Aerial mycelia are abundant, powdery and beige (3 ge). Many black hygroscopic patches develop later and spread entirely.
c) Substrate mycelia are light beige (3 ec) on the reverse side.
d) Soluble pigment is honey gold (2 ie).
3) Inorganic salts-starch agar medium a) Abundant growth.
b) Aerial mycelia are abundant, powdery and covert 1~ gray (2 fe). Many black hygroscopic patches develop later and spread entirely.
c) Substrate mycelia are dull gold (2 ng) on the reverse side.
d) Soluble pigment is light citron gray (1 ec).
4) Glycerin-asparagine agar medium a) Abundant growth.
- b) Aerial mycelia are abundant, powdery and beige (3 ge). Many black hygroscopic patches develop later and spread ent~rely.
c) Substrate mycelia are gold (112 lc) on the reverse side.
.

. . .

d) Soluble pigment is pale yellow (1 ca).

5) Peptone-yeast-iron agar medium a) Moderate growth. Slightly wrinkled.
b) Aerial mycelia, none.
c) Substrate mycelia are cream (112 ca) on the reverse side.
d) Production o~ soluble pigment, none.

6) Tyrosine agar medium a) Abundant growth. Slightly wrinkled.
b) Aerial mycelia are abundant, powdery and covert - gray (2 fe). Black hygroscopic patches develop later and spread entirely.
c) Substrate mycelia are gold (112 pc) on the reverse side.
d) Soluble pigment is honey gold (2 ic).
~5 7) Sucrose-nitrate agar medium a) Abundant growth, flat.
b) Aerial mycelia, none.
c) Substrate mycelia are citron yellow (1 lc) on the reverse side.
d) Soluble pigment is canary yellow (1 ea).
8) Glucose-asparagine agar medium a) Moderate growth.
b) Aerial mycelia are abundant, po~dery and covert gray (2 fe). Blackish brown and greenish patches develop from the periphery later and spread entirely.

c) Substrate mycelia are gold (112 lc) on the reverse side.
d) Soluble pigment is cream (12 ca).
9) Nutrient agar medium a) Moderate growth.
b) Aerial mycelia, none.
c) Substrate mycelia are yellow tint (1 ba) on the reverse side.
d) Production of soluble pigment, none.
10) Calcium maleate agar medium a) Moderate growth, flat.
b) Aerial mycelia, none.
c) Substrate mycelia are canary yellow (1 ea) on the reverse side.
d) Production of soluble pigment, none.
11) Bennet's agar a) Abundant growth. Wrlnkled.
b) Aerial mycelia are abundant, powdery and siliver gray (3 fe). Black hygroscopic patches develop later and spread entirely.
c) Substrate mycelia are mustard (2 le) on the reverse side.
d) Soluble pigment is light antique gold (112 ic).
The strain was incubated on the above media all at 28C for 21 days. The colors are indicated according to Color Harmony Manual (Container Corporation 1~51091 of Amcrica).
III. Phy~iological properties 1) Optimum temnerature for growth: 28-30 C
(No growth below 10 C and above 45 C.) 2) Optimum pH for growth: 6.5-8.5 (No growth below pH 4.0 and above pH 11Ø) 3) ~iquefaction of gelatin: Negative : 4) Hydrolysis of starch: Positive 5) Coagulation of skim milk: Negative 6) Peptonization of skim milk: Positive (acidic)

7) Reduction of nitrate: Negative

8) Production of melanoid plgment-, Ne~ative

9) NaCQ tolerance: Growth at 7~
No ~rowth at 10,~,

10) Utilization of carbon sources:
D-Glucose ++
I,-Arabinose ++
:~ ~ Sucrose +~
. D-Y~ylose ++
`~ 20 Inositol ++
.;~ D-~annitol ++
~-Fructose ++
Xhamnose ++
Raffinose ++
Cellulose ,'; .
' ' -1~--:,' :
.
, . ~ . . .. .

(The mark "++" means "well u~ilized" and "-" means "not utilized~") IV. Type of cell wall The type of the cell wall according to the classification of Lechevalier et al. is I type (LL-diaminopimelic acid).
Based on the above mycological characteristics, the strain was taxonomically investigated with the follow-ing results.
With reference to H. D. Tresner and E.J. Backus, Applied Microbiology, 4, 243-250 (1956), the strain has the three fundamental characteristics of Streptomyces hygroscopicus that spore forming mycelia extend as short side branches from the long stem of an aerial mycelium and have at least two turns of spiral portions at the -ends of the branches, that the mature aerial mycelia are brownish gray and that they exhibit distinctly black hygroscopic properties. Investigations with reference to the following pieces of literature also show that the present strain belongs to Streptomyces hygroscopicus.
Bergey's Manual of Determinative Bacteriology, 8th Edition (1974).
Waksman, The Actinomycetes, Vol. 2 (1961).
International Journal of Systematic Bacteriology, Vol.18, pp. 69-189 (1968), ~ol.18, pp. 279-392 .~

$151091 ~l9~ vo~. 19~ n~ 391-512 (1~9) and Vol. 22, P;~). 265--39~`! (1~372).
The res~llts listed in Table 4 below v~ere obtained from -the co~parison of the present strain with a -typical strain, ';-tre~tom~ces h,v~rosco~icus (Jensen), Wak~man and Henrici (1948), and ~ith an ISP standard strain, Stre~tom.~ces hyrrosco~icu.s ISP 5578.

r a~
~, s~
t~ ~ h ~-rl ~ ~ ~
:>, O
h h J~ G) h J~ rl h t~ ~ O ~
~ ~ h O a, U~ Z ~ :~
cq o ~
H Z O

O~

Q.~ r~ S 3 o a~ ~ ~ o h O ~ G) H
~ ~: rJ O ~ ~ ~/ + + I +
O a~ ~ ~ h O
h ~ t/~ Z
X
.
r rl ~, ~
P ~ 3 O
~I S ~( 3 J~ ~1 0 h O ~ a) a) H
rl o ~ ~ ~ I + I +
~ ~ h O a) ~n u~ ~nc~ ~ a ~>
S~ ~d O

., o .. ~
o h ~ O .
v~ h H
C~ O O O~ O ~
rl ~ ;~ o ~ o C~ 0 ~00 ~ ~ o O X ~
t~ ~ O rl bO O S:; rl ~1 0 rl ~ ~ V~ O
h t~ h ~ O O Q, J~ rl rl ~: t~ ~
,~ ~ ~I N O
c> ~I c> oc~ ~! ~ rl r~ ~ H E
t~ ~ ,o ~ ~ o C) ~ o ~ c~ ~
S~ S~ h r3 ~ td~ ~ ::5 rd h h b~ 1 h t~ O O h O ~1O H ci H ~ t~ t~ rl S~ ~ ~
S ~ ~ ~ ~ a)~ o rl ~ :~ O ~a, ~ a~ rl C~ cq cq ¢ F4 E~ ~Q ~ bD ~ u~ ~ o ~; ~

: h h + ~
]~e +l ++++l ~

. ' ~ U~
., '~ . ~,~

~ . ~ ~
~ " ~ ~

o + I ~ +~ ~:

a The above table indicates that the present strain is distinct from Streptomyces hygroscopicus (Jensen) in respect of the liquefaction of gelatin.
The present strain also differs from the Jensen strain in that the former involves no development of aerial mycelia on sucrose-nitrate agar medium and on nutrien~
agar medium, whereas the latter involves development of aérial mycelia on such media.
Furthermore the present strain ~pparently differs from the ISP standard strain in the spore surface as well as in the utilization of carbon sources.
Since the present strain differs from the r - above-mentioned known strai~s of S. hygroscopicus, the present strain was further compared with four subspecies of S. hygroscopicus described in Bergey's Manual (~th ed.), with the results given in Table 5 belo~. The physiological characteristics of these species are based on the following literature.
Subspecies 1: Streptomyces hygroscopicus subsp.
~ 20 angustmyceticus ; The Journal of Antibiotics, Ser. A, Vol. 7, pp. 116-119 (1954).
Subspecies 2: Streptomyces hygroscopicus subsp.
decoyicus Antibiotics and Chemotherapy, Vol. 9, pp. 427-431 (1957).

~1510gl Subspecies 3: Streptomyces hygroscopicus subsp.
glebosus The Journal of Antibiotics, Ser. A, Vol~ 15, pp. 21-27 (1962).
Subspecies 4: Streptomyces hygroscopicus subsp.
ossamyceticus . The Journal of Antibiotics, Ser. A, Vol. 18, . ~. pp. 82-88 (1~65).
~' :
:
, ~,. .....

~. .
' ~ - 20 -.~ .

o Q~ ~s ~r~ ~~
.~ o ~ + ~o , + I , +
Q. Q~ E~ h S~
~nU~ U~ r,~ ~ 3 O
::~ O h u~ ~z: p u~
S
c~ ~
~-1 0 ~ I + I I +
Q~ ~3 h O
~q U~

N 3:
r~ S ri 3 ~ ~ ~ ~ O
~ S~ O ~ ~
h u~ u~ u~ ~ a, n ,/
t~ O
~q ~a ~ - '~1 - rl ~ ~
r C~ h O ~ + + I +
0~ Q~ ~ h O
~Q u~ 5 Z
v~

~1 ~
a~ rl ~ ~ r-l O
h O :~ a) ~
a) ~r~ O ~ ~i I + I +
~ J~~
c~

O
.
.
O
~q c~ O
r~
J~ C~ O bDO ~ ~ ~ O X
u~ ~ ~d tJ~ rl bD O ~ r~
O O Q. ~ r~ ~r( S
~)S O ~ ~ rl ~rlC) ~ ~n c) ~ ~ o t~l C> ~ c~ OC) ~1~ ~I r-l cd r~ ~
t~G) S ~ ~ J O ~ ~ .Y O X c> ~1 ~O S~ O S~ O
SQ~ O ~ G) ~ O rl ~ ~ O ~ a~
cq ~ ~ ¢ P~ ~ O :~ O ~ O ~ O ~ O

115~091 1.

~r~
+ + + + ~- + + + +
~o a~
~ + I + I + + + , +
o~
.
, Lq ~
r~
~ ~ + ' + + + + ~ , . ~q - . . ~ ~
0 ~
.,, . ~ ~ + ' + l l + I +
,~

~ ~ ~ , a) ~ I + + + + + + + + +
a~ h .. , h ~ ~D`O
'I
C~ Q
~ C) O ~ Q~
+~
. u~ ~ ~: h ~ O O O t : ,1 ~ o ~ a) ~ ~ o a) u~
, $~ C) ~-~ O O ~ O
~1 +~ O ~ o o ~ o ,~
C~ '~I ~ ~ X H ~ 4 C;~
c~ z ~ c~

,1 .
.

I Table 5 shows that the present strain is in ¦ agreement with the subspecies 4 in utilization of carbon ¦ sources but is distinct therefrom in the production of melanoid pigment, peptonization of skim milk and reduction of nitrate and differs from the subspecies 1 to 3 in the utilization of carbon sources.
The characteristics described above indicate that the present strain, although belonging to Streptomices hy~roscopicus, is distinct from the type strain, ISP standard strain, and the strains belonging to subspecies. Furthermore the antibiotic according to this invention has ne~er been isolated from the strains of any known species or subspecies. Thus we ha~e ~k-found the present strain to be novel and named the strain Streptomyces hygroscopicus subsp. hiwasaensis FB-5.
An application for the deposition of the microorganism was filed with Fermentation Research Institute of Agency of Industrial Science and Technology, Japan as Application No.4573, and the strain has been deposited in the Institute as FE~-P No.4573. The strain has been deposited also in American Type Culture Collection (ATCC) as ATCC
No.31613 since March 6, 1980.
International Journal of Systematic Bacteriology, Vol. 18, p. 154 ~1968) and Waksman, The Actinomycetes, ~ol. 2, p. 254 (lg6l) state the characteristics of ~, .

llSlO9i Streptomyces orientalis which affords vancomycin that appears most analogous to the antibiotic of the inven-tion, oA-7653 substance. The aerial mycelium is not spiral, is whitish on yeast-malt agar and glycerin-asparagine agar media and whitish to grayish on inorganicsalts starch agar media and involves poor development on oatmeal agar medium. The characteristics are therefore entirely different from those of the microorganism . utilized in the present invention.
The antibiotic of this invention, oA-7653 substance, can be obtained by incubating Streptomyces hygroscopicus subsp. hiwasaensis FB-5 on a culture ,1 I medium and isolating the substance from the resulting culture.
The strain is incubated with use of a usual `I liquid or solid culture medium. It is preferable to use a liquid medium for shake culture or for submerged culture with aeration and agitation.
The fermentation medium may have a composition 2a generally used for the culture of common actinomycetes.
Examples of useful carbon sources are starch, glucose, glycerin, etc. Examples of useful nitrogen sources are soybean flour, peptone, meat extract, cotton seed flour, . inorganic salts such as ammonium sulfate and sodium nitrate, etc. When desired, the medium my further il51091 incorporate sodium chloride, calcium carbonate, phosphate, magnesium sulfate and like inorganic salts.

Preferably the strain is incubated at a medium pH of 6.5 to 7.5 and at a temperature of 28 to 30C. With use of a liquid medium, the accumulated yield of the present substance reaches an approximate maximum about 72 hours after the start of incubation.

The present substance produced and accumulated in the culture can be collected by usual methods utilizing the physicochemical properties of the substance, for example, by resorting to the difference in adsorption force of ion exchange resin, silica gel, Sephadex (a trade mark product of Sei Kagaku Kogyo Co., Ltd., Japan), active carbon, etc., or to the difference in partition ratio between two liquids.
Such methods are usable in combination. Stated more speci-fically the culture is filtered or centrifuged for the removal of the cells, the resulting supernatant is subjected to active carbon column chromatography , Dowex 50~ (a trade mark) x 4 column chromatography, Ecteaolacellulose column chro-matography, Dowex 50W (a trade mark) column chromatography, and silica gel column chromatography in the order mentioned for elution, and the active fractions are co~lected and distilled for the removal of solvent, whereby OA-7653 substance, the antibiotic of the invention, can be isolated for recovery.

~7 , . ~

1151V9~

l~`or use as antibacterial compositions, the substancc of the invention is formulated into ~harma-ceutical preparations in combination wi~h usual carriers.
~;xarnples of useful carriers are those usually used for preparing medicinal com~ositions in the desired form, such as diluents and excipients including filler, extender, binder, wetting agent, disintegrator, surfactant and glazing agent.
'ihe antibacterial compositions can be in any of various dosage forms in accordance ~ith the contemplated purpose of treatment. Typically they are in the form of tablets~ pills, po~ders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections (solutions, suspenslons ? etc.), ointments, etc. r'or -the pre~aralion OI tablets, extensive use ~ay be made of carriers already known in the art. Useful examples are excipien-ts sucll as lactose, white sugar, sodium chloride, glucose solution, urea, starch, calciwm carbonate, kaolin, crystalline cellulose and silicic acid;
binders such as water, ethanol, propanol, syrup, glucose, starch solution, gelatin solution, carboxy-methylcellulose, shellac, methylcellulose, potassium phosphate and polyvinylpyrrolidone; disintegrators such as dried starch, sodium alginate, agar powder, laminaria po~der, SOG' ~m hydrogencarbonate, calcium carbonate, ~151091 ;-'ru~een, soclium lauryl sulf`ate, glyceryl monostearate,starch and lactose; disintegration suppressants such as white sugar, stearin, cacao butter and hydrogenated oils; absorption promoters such as quaternary ah~monium salt and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; glazing agents such as purified talc, stearic acid salts, boric acid powder and olyethylene glycol; etc. For the preparation of pills, a wide variety of carriers are usable which are already known in the art, useful examples being excipients such as glucose, lactose, starch, cacao fat, hardened vegetable oils, kaolin and talc; binders such as gum ærabic powder, tragacanth po~vder, gelatin and ethanol; disintegrators such as laminaria and agar; etc. ~ihen desired, tablets can be provided with a usual coating. Thus useful are sugar-coated, gelatin-coated, enteric coated, fil~-coated, double-layer znd multiple-~ayer tablets. Suppositories may be formulated with use of a wide variety of known carriers, such as polyethylene glycol, cacao fat, hi~her alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. The solutions and suspensions for injection should be sterilized and are preferably isotonic with the blood. ~or the preparation of such ~olutions, e~ulsions and susensions, any diluent is usable vihich is usually used in the art. ~xarnples of useful diluents are ~iater, ethyl alcohol, propylene ~lycol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyo~yethylene sorbitol, sorbitan esters and the li~e. ll'or the pre~aration of antibacterial solutions, sodium chloride, glucose or ~lycerin may be incorporated therein in an zmount ~ufficient to render the solutions isotonic. ~uch solutions ma~ further incorporate usua- solubilizin~ agents, buffers, analgesics, ` preservatives, etc. 'ihe antibacterial compositions may contain coloring agents, preservatives, perfumes, flavoring agents, sweetening agents, etc. as well as o-ther drugs. 1lor the pre~aration of pastes, creams and gels, a '~ti de variety of diluents are usable ~Jhich are ~ kno~n in the art, useful exam~les being white petrolatum, ~araffin, glycerin, cellulose derivatives, polyethylene ~lycol, silicone, bentonite, etc.
The amount of the substance of this invention to be contained in the antibacterial compositions is not specifically limited but can be suita~ly determined over a wide range. Usually the ~ount is about l to about 70~ by wei~ht of the whole com~osition.
The antibacterial compositions are not specifically limited in the mode of administration but , ~
-2~-Call be ~-iven by a sllitable method in accordance with the particular form of the comPosition~ ~'or example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally. Injections are given intravenously, singly or as adrnixed with an au~iliary solution of glucose, amino acids, etc. When desired, injections are singly given intramuscularly, intradermally, subcutaneously or intraperitoneally.
Su~positories are given to the rectu~., while ointments are applied by coating.
l;he antibacterial compositions are given at a dose suitably deter.~ined according to the purpose, symptor,ls, etc. U~ually the compositions are administered in an amount of about 100 mg to about 2 g/body/day, calculated as the present substance, in three to four divided doses.
The su~stance or antibacterial compositions oI this invention are usable as uniforml~l admixed with feed for animals. The amount of the ac-tive co~ponent to be used i5 about 1~ to about 100 g per ton of the feed although suitably variable with the kind of the animal and the degree and kind of infection. When fed to animals as admixed with the feed, the present substance ic useful for the ~revention or treatment of infections caused by various bacteria.

115~091 The inven-tion ~vill be described below i~
~reater detail vith re~erence to the followin~ exam~le and preparation exam~les.
Exa~le 1 Streptom~ces hygroscopicus subsp. hiwasaensis FB-5 is inoculated in a liquid medium having a pH of 7 and co~osed of 1~ of starch, 0.5~ of ~lucose, 0.5~, of ~lycerin, 0.5~^ of soybean flour, 0.3~0 of meat extract ('i7ako ~ireat ~xtract, prepared fro~. fin ~.~ale meat and bonito meat by Wako Co.,Ttd., Japan), 0.3~ of Polypep-tone (prod~.ct of Dai~o Eiyo Kagaku Co.,~td., Japan), 0.5~ of sodium chloride, 0.3~ of calcium carbonate and distilled water. The strain is incubated at 28 G for 48 hours A culture medium (20 liters) havin~ the same composition as above and placed in a 30-liter stainless steel container is inoculated with the inoculum in an amo1mt of l~o based on the medium, and the inoculum is incubated at 28 C for 72 hours with aeration and a~itation. The ~edium i9 aerated at a rate of 20 liters per minute, with the a~itatin~ blade driven at 300 r.p.m.
The resultin~ culture is centrifu~ed for the removal of cells. The surernatant (18 liters) is a~plied to an active car~on colw~.n for adsorption, washed with water and therealter eluted with 1.5 liters of a mixture of 2N aqueous solution of ammonia and methanol (1:1 by volume). The resulting solution is distilled in a vacuum for the removal of ammonia and methanol. The residue is adjusted to a pH of 3 with 2N aqueous solution of hydro-chloric acid and then applied to a Dowex 50W (a trade mark)x 4 tNa type) column for adsorption. After washing the column with water, gradient elution is conducted with 1 liter of 0.1 mole phosphoric acid buffer (pH 7.0) and 1 mole of NaCl in 1 liter of 0.1 mole carbonate buffer (pH 10.0).
The resulting active fractions are collected, and the combined solution is diluted with the same quantity of distilled water, adjusted to a pH of 3 with 2N aqueous solution of hydrochloric acid and then applied to a Dowex 50W ta trade mark) x 4 (NH4 type) for adsorption. After washing the column with water, the active substance is eluted with 500 ml of O.lN
aqueous ammonia solution. The resulting active fractions are collected, and the ammonia is removed therefrom in a vacuum.
The residue is adjusted to a pH of 8.0 with lN aqueous solution of sodium hydroxide and then applied to an Ecteao-lacellulose (Cl type, product of Whatman (a trade mark))column for adsorption. After washing the column with water, gradient elution is conducted with 1 liter of distilled water and 1 liter of 1 mole aqueous sodium chloride solution.
The resulting active fractions are collected, and the combined solution is adjusted to a pH of 3 with 0.2N aqeuous solution of hydrochloric acid and then applied to a Dowex 50W (a trade mark) x 4 (NH4 type) column for adsorption. After washing the column with water, the active substance is eluted with 500 ml of O.lN aqueous ammonia solution. The resulting active fractions are collected and concentrated in a vacuum.
With addition of 3.0 g of silica gel (Wako Gel C200, product of Wako Co., Ltd., Japan), the concentrate is further con-centrated to dryness. The product is suspended in 20 ml of 90~ aqueous ethanol solution, and the suspension is applied to a column of silica gel (supplied under the trade mark Wako Gel C200). After washing the column with 100 ml of 90 , , .~

115109~

aqeuous ethanol solution, the active substance is eluted with 500 ml of 70% aqueous ethanol solution. The resulting active fractions are collected, concentrated in a vacuum and then freeze-dried, giving 1 g of OA-7653 substance of the invention 5 in the form of white particulate crystals. The product has the physicochemical properties already described.

PÆPARATION EXAMPLE 1 SuDstance of the invention 200 mg Glucose 250 mg Distilled water for injection Suitable amount , Total amount 5 ml .~ ~' ' '.
.
.-The so]ution of above composi-l,ion is placcd into a 5-ml ampule. With the air in the ampule replaced by ritro~en, the solution is sterilized at 121 C for 15 minutes to obtain an injection solution.
PreParation Example 2 Substance of the invention 100 g Avicel (trade mark of Asahi Chemical 40 Industry Co., Ltd., JAPAN) Corn starch 30 g Magnesium stearate 2 ~
TC-5 (trade mark of Shin-etsu 10 g Kagaku ~ogyo Co., ~td., Japan for hydroxypropyl-methyl cellulose) Polyethylene ~lycol-6000 3 g Castor oil 40 g ~ethanol 40 g The substance o the invention, A~lcel,;
corn starch and ~agnes~ stearate are mixed together, ~round and made into table~s with use o~ the plungers and mortars of ~lOmm. ~he ta,blets are coated with a film coatin~ composition comprising TC-5, polyethylene glycol-5000, castor oil and methanol to obtain coated tablets of the above composition.
Pre~ar~tion Exam~le 3 Substance of the invention 2 g '' Purifled hydrous lanolin 5 g ' 115109~

White beeswax 5 g White petrolatum 88 g Total amount 100 g White beeswax is heated to a liquid state, and the compound of the invention, purified lanolin and white petrolatum are added thereto. The mixture is heated to a liquid state and thereafter stirred until the mixture starts to solidify to obtain an ointment of the above composition.
Brief Description of the Drawings Fig. 1 is a photomicrograph and Fig. 2 is a electronmicrograph showing Streptomyces hygroscopicus subsp. hiwasaensis FB-5 useful for producing oA-7653 substance of the invention;
1~ Fig. 3 is a diagram showing the results of electrophoretic analysis of oA-7653 substance of the invention and vancomycin serving as a control substance;
Fig. 4 is a diagram showing an IR spectrum of the present substance;
Flgs. 5 to 7 are diagrams showing W absorption spectra of the present substance;
Figs. 8 and 9 are diagrams showing lH-NMR
spectra of the present substance and vancomycin;
~ig. 10 is a diagram showing 13C-NMR spectrum of the present substance;

~' Figs. 11 to 13 are diagrams showing the results of TLC analysis of the present substance and vancomycin hydrochloride.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of an antibiotic substance comprising L-glutamic acid and glucose as com-ponents and having the following characteristics: a. spar-ingly soluble in 0.1N aqueous solution of hydrochloric acid, insoluble in methanol, ethanol, butanol, acetone and ethyl acetate and soluble in 0.1N aqueous solution of sodium hydroxide, b. positive when subjected to iodine reaction, ninhydrin reaction, Molisch reaction and Ehrlich's reaction, c. a specific rotatory power of [.alpha.]D23.5= +76.5° (C=0.463, water), d. a composition comprising 46.10% carbon, 4.47%
hydrogen and 7.18% nitrogen, as determined by elementary analysis, e. a molecular weight of about 2000, as determin-ed by ultra-centrifugal analysis, f. An isoelectric point at a pH of 5 to 6, as determined by electrophoresis, g. an IR absorption spectrum in KBr disk with significant absorp-tion maxima at the following frequencies (cm 1): 3280 (s), 1660 (s), 1640 (s), 1515 (s), 1490 (s), 1395 (s), 1235 (s), 1150 (m), 1062 (s) and 1020 (s), h. UV absorption spectra with absorption maxima at 278 mµ in 0.1N aqueous hydrochloric acid solution, at 278 mµ in distilled water and at 298 mµ
in 0.1N aqueous sodium hydroxide solution, i. a 1H-NMR
spectrum in dimethyl sulfoxide substituted with deuterium with the following peaks:

j. a 13C-NMR spectrum in dimethyl sulfoxide substituted with deuterium with the following peaks:

k. Rf values, as determined by silica gel thin-layer chromatography, of zero with use of butanol-acetic acid-water C4:1:1 by volume) as a developing solvent, of zero with use of propanol-2N aqueous ammonia solution (7:3 by volume) as a developing solvent, of 0.45 with use of chloroform-ethanol-water (4:7:2 by volume) as a developing solvent, and of 0.8 with use of ethanol-water (7:3 by volume) as a developing solvent, and 1. Rf values, as de-termined by paper chromatography, of 0.83 with use of butanol-acetic acid-water (4:3:7 by volume) as a develop-ing solvent, and of 0.76 with use of butanol-pyridine-water (4:3:7 by volume) as a developing solvent which comprises incubating the strain Streptomyces hygroscopicus subsp. hiwasaensis FB-5 on a culture medium and isolating said substance so obtained.

2. A process according to claim 1, in which a liquid medium for shake culture or for submerged culture with aeration and agitation is used.

3. A process according to claim 1, in which the strain is incubated at a medium pH of 6. 5 to 7. 5 and at a tempera-ture form 28°C to 30°C.

4. A process according to claim 1, 2 or 3, in which the culture is filtered or centrifuged for removal of cells and the supernatant is subjected to column chromatography.

5. An antibiotic substance comprising L-glutamic acid and glucose as components and having the following charac-teristics: a. sparingly soluble in 0.1N aqueous solution of hydrochloric acid, insoluble in methanol, ethanol, butanol, acetone and ethylacetate and soluble in 0.1N aqueous solu-tion of sodium hydroxide, b. positive when subjected to iodine reaction, ninhydrin reaction, Molisch reaction and Ehrlich's reaction, c. a specific rotatory power of [.sigma.]D23.5=
+76.5° (C=0.463, water), d. a composition comprising 46.10% carbon, 4.47% hydrogen and 7.18% nitrogen, as deter-mined by elementary analysis, e. a molecular weight of about 2000, as determined by ultra-centrifugal analysis, f. an isoelectric point at a pH of 5 to 6, as determined by electrophoresis, g. an IR absorption spectrum in KBr disk with significant absorption maxima at the following frequen-cies (cm-1): 3280 (s), 1660 (s), 1640 (s), 1515 (s), 1490 (s), 1395 (s), 1235 (s), 1150 (m), 1062 (s) and 1020 (s), h. UV absorption spectra with absorption maxima at 278 mµ
in 0.1N aqueous hydrochloric acid solution, at 278 mµ in distilled water and at 298 mµ in 0.1N aqueous sodium hydrox-ide solution, i. a 1H-NMR spectrum in dimethyl sulfoxide substituted with deuterium with the following peaks:

j. a 13C-NMR spectrum in dimethyl sulfoxide substituted with deuterium with the following peaks:
k. Rf values, as determined by silica gel thin-layer chroma-tography, of zero with use of butanol-acetic acid-water (4:1:1 by volume) as a developing solvent, of zero with use of propanol-2N aqueous ammonia solution (7:3 by volume) as a developing solvent, of 0.45 with use of chloroform-ethanol-water (4:7:2 by volume) as a developing solvent, and of 0.8 with use of ethanol-water (7:3 by volume) as a de-veloping solvent, and 1. Rf values, as determined by paper chromatography, of 0.83 with use of butanol-acetic acid-water (4:3:7 by volume) as a developing solvent, and of 0.76 with use of butanol-pyridine-water (4:3:7 by volume) as a developing solvent whenever prepared or produced by the process as claimed in claim 1, 2 or 3 or an obvious chemical equivalent thereof.