JPH024734A - Novel antibiotic and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (R is formula II, formula III or formula IV) or salt thereof. EXAMPLE:5-Hydroxy-4-methylhepto-2-ene acid. USE:An antimicrobial agent and culture medium additive for improving productivity in fermentation of antibiotic mycinamicin. PREPARATION:A microorganism [i.e. Streptomyces griseorubida A 11725 (FERM P-705)] belonging to the genus Micromonospora and having ability capable of producing a compound expressed by formula I is cultured in a culture medium and the compound expressed by formula I is collected from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel substance useful as an antibacterial agent or as a medium additive for improving productivity in fermentation of the antibiotic mycinamicin, and a method for producing the same.

[Conventional technology]

Conventionally, Micromonospora griseorvida (Micro
-monospora griseorubida)
A 11725 (Feikoken Joyori Maishina Sewing Machine I~■
is represented by the following substituents.

[In the formula, A is an oxygen atom or a single bond, 几 is a hydrogen atom or a hydroxyl group, Q is a hydrogen atom or a group X represented by the formula % (wherein Y and Z each represent a hydrogen atom or a methyl group) It is known that macrolide thread antibiotics mycinamicin 1 to 1 are produced (J.

Antibiotics, 33 (4), 364
~376 (1980); Dokou (3), 276
~281 (1981) and 36 (2).

175-178 (1983):). The above formula CI! '
), each RQYZ mycinamicin I OHX CH3CH.

u 1 0 0HX CH, CH.

// l-HX CH, H tt l'/ HX CHcHs1/
V -OHX CH, CH3//W-H
XHH 〃 ■ -HH Among these mycinamicins, mycinamicins 1 and B exhibit particularly excellent antibacterial activity against staphylococci and mycoplasma, and are useful as therapeutic agents for infectious diseases in humans and animals.

[Problem to be solved by the invention]

The objective is to find active ingredients other than the above-mentioned mycinamicins.

[Means to solve the problem]

Under these circumstances, the present inventor continued various studies on cultures of mycinamicin-producing bacteria, and found that there were four new components in this culture that were different from the above-mentioned mycinamicins, and that these new components had antibacterial activity. The present invention has been completed based on the discovery that the carvone has the same properties and is also an intermediate in the biosynthesis of mycinamicins.

That is, the present invention is a compound represented by the formula, a salt thereof, or a method for producing them.

The object compound [I] of the present invention is produced by, for example, culturing a microorganism belonging to the genus Micromonospora and having the ability to produce compound [I] in a medium, and collecting the microorganism from the culture.

Microorganisms that have the ability to produce compound (1) include:
For example, Micromonospora griseorvida A 1172
5 (Feikoken Article No. 705') fJ1 is mentioned. This strain has the ability to produce the known mycinamicins 1 to 1, and its mycological properties are described in J.
Antibiotics, 33 (4) + 364
~376 (1980), and is described in Japanese Patent Publication No. 31905/1983.

To carry out the method of the present invention, first, a compound C1"l-producing bacterium is cultured. The f@culture is carried out by a method known per se, that is, a medium containing an assimilable carbon source, an assimilable nitrogen source, and an assimilable inorganic salt source. Among them, the reaction is carried out under conditions that produce compound [I].

Medium components and I- include glucose, starch, glycerin,
Carbon sources such as sucrose, molasses, dextrin: peptone,
Nitrogen sources such as meat extract, soybean flour, casein melt, cottonseed flour, hoon steep liquor, nitrate salt, ammonia salt; Contains cations such as sodium, potassium, magnesium, calcium, cobalt, manganese, iron, etc. substance, chlorine,
Examples include inorganic salts such as substances containing anions such as sulfuric acid, phosphoric acid, and acetic acid; bacterial growth factors such as dried yeast and yeast extract.

20-30℃ in a medium, preferably 4126-30
Micromonospora griseolavida A 11 at a temperature of °C
When 725 is cultured, the target compound [1] is produced in the medium. The amount usually reaches its maximum in 6-8 days.

To collect the target compound CI) from the culture obtained in this way, for example, the culture is filtered, the filtrate is adjusted to an acidic pH of about 3 with an acid such as hydrochloric acid, and then chloroform, ethyl acetate, butyl acetate, etc. , extracted with a non-hydrophilic organic solvent such as diethyl ether, concentrated the organic solvent layer, and subjected to column chromatography for separation and purification.

The objective compound [1] thus obtained, S2.4
．． 10-trimethyl-5-oxo-trideca-6,8-
The physicochemical properties of dienoic acid (hereinafter referred to as IPML-fi) are shown in Table 1.

Methylhept-2-enoic acid (hereinafter referred to as IPML-3) - Methylnoner-2,4-dienoic acid (hereinafter referred to as IPML-4) and *; Support: Merck & Co. silica gel Art 5715 Developing solvent: Chloroform-methanol (10:1 ) **; Column: Internal diameter 41111
:5) Flow rate: 0.8-/min Detection: UV (215 nm, 260 nm,
280 nm) Retention time: expressed in minutes The object compound (1) of the present invention can form various salts using known methods. For example, alkali metal salts such as potassium salts and sodium salts, alkaline earth metal salts such as magnesium salts and calcium salts, salts with basic amino acids such as lysine and arginine, ammonium salts, and salts with known organic amines. can be mentioned.

〔Effect of the invention〕

The antibacterial spectrum [minimum inhibitory concentration (MIC)] of the object compound CD of the present invention measured by the agar dilution method is shown in Table 2.

Table 2 MIC (meC) As mentioned above, the target compound of the present invention [■] not only has antibacterial properties itself, but also is useful as an intermediate for the biosynthesis of the antibiotic mycinamicin. If the target compound [1] is added to the culture medium and cultured, the productivity of mycinamicin can be improved.

〔Example〕

Examples of the present invention will be shown below, but the present invention is not limited thereto.

A) Dextrin 1%, glucose 1%, casein thawed product 0.5%, yeast extract 0.5%, calcium carbonate 0.1
A liquid medium (pH 7,0) 201.7e containing %
The mixture was dispensed into 150-mL Erlenmeyer flasks and steam sterilized at 120 degrees for 20 minutes. This medium was inoculated with a loopful of Micromonospora, Nis, P. A 11725 from a slant culture medium.
A primary seed culture was obtained by culturing on a rotary shaker at 30 degrees for 48 hours.

B) 5! Containing liquid medium 11 similar to A)! The mixture was dispensed into round flasks and steam sterilized at 120 degrees for 20 minutes. This medium was inoculated with 10 volumes of the primary seed culture obtained in A), and cultured on a reciprocating shaker at 30 degrees for 48 hours to obtain a secondary seed culture.

C) On the other hand, 7% dextrin, 0.5% glucose, 5% cottonseed flour, 0.5% defatted soybean flour, 0.5% calcium carbonate.
%, K2HPO40,1%, MgSO4・7H200,
30 containing a liquid medium 201 consisting of 4% CoC, 200,0002% CoC12.6H, and 0.02% silicone antifoaming agent.
Transfer the 11 secondary seed cultures obtained in B) to a 1-volume jar fermenter, and heat at 30 degrees at 300 r, p, m, 201 m/min.
Culture with aeration was carried out for 7 days under conditions of sterile air supply, and 17 culture solutions were obtained.

D) The above culture solution 171! was filtered to separate the bacterial cells and other solids to obtain 6ζ liquid 141. This culture filtrate was extracted with the same amount of chloroform to obtain an aqueous layer containing the target product. The pH of the aqueous layer was then adjusted to 3K with 6N hydrochloric acid, and extracted with 141 ethyl acetate to obtain an organic layer containing the desired product. This organic layer was concentrated to dryness to obtain 8.5 g of residue. This balance 8,
59 dissolved in a small amount of chloroform and filled with silica gel (Merck & Co., grade 60), inner diameter 25 mm x 600
+ Inject into the glass column of the kite. Elution was performed using chloroform:methanol (20:1) as an eluent, and both fractions containing the target product were concentrated to dryness, dissolved in 1 volume of methanol, and Y.
MC-GELODS 5-51'li manufactured by Yamamura Kagaku Kenkyusho, filled with silica gel treated with dimethyloctadecylsilyl chloride, inner diameter 20 fJX 30Q 1m
into a stainless steel column. For elution, use 0.1M phosphoric acid solution: methanol (8:2) 500+ at 10 min/min.
Elute with a linear concentration gradient of 500+t + (4:6) and the eluate is monitored by an ultraviolet spectrophotometer detector at 220 nm and 280 nm and fractionated in 20- increments. Fractions A, B, C, and 38 containing the target product were each concentrated, extracted with ethyl acetate, and concentrated to dryness to obtain the A fraction (f
r8~11) to IPML-3(5'?) +
I PML-4 (15 m
7), IPML-6 (1
21,'+7) was obtained.

[Brief explanation of the drawing]

Figures 1 to 3 show 5-hydroxy-4-methylhept-2-enoic acid, 7-hydroxy-6-methylnoner-2,4-dienoic acid and 11-hydroxy-2,4-dienoic acid, respectively.
,10-trimethyl-5-oxo-trideca-6,8-
FIG. 4 is a diagram showing the infrared absorption spectrum of dienoic acid.
Figures 6 to 6 show 5-hydroxy-4-methylhept-2-enoic acid, 7-bitroxy-6-methylnona-
2゜4-dienoic acid and 11-hydroxy-2,4゜1
It is a drawing showing the nuclear magnetic resonance spectrum of 0-trimethyl-5-oxo-trideca-6°8-dienoic acid. Agent Masao Miyake (and 1 other person) Procedural leader Masao (voluntarily) 1989

Claims (1)

[Claims] 1) Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R is ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ H or ▲ Mathematical formulas , chemical formula, table, etc. ▼ indicates a group) or its salt. 2) Belongs to the genus Micromonospora and has formulas ▲Mathematical formulas, chemical formulas, tables, etc.▼〔I〕 (In the formula, R is ▲Mathematical formulas, chemical formulas, tables, etc.▼, ▲Mathematical formulas, chemical formulas, tables, etc.) or ▲There are mathematical formulas, chemical formulas, tables, etc. ▼Indicates groups) A new compound characterized by culturing in a medium a microorganism that has the ability to produce a compound expressed by ▲ and collecting the compound [I] from the culture. [I] or the method for producing its salt. 3) The microorganism is Streptomyces griseorvida A1
1725 (Feikoken Joyori No. 705).