JPH0541996A - Production enhancer or mycinamins and its production - Google Patents

Abstract

(57) [Summary] [Purpose] To supply mycinamicins in large quantities and at low cost. [Structure] At least one or more kinds of dotriacolides selected from the group consisting of MG299A substance, MG299B substance and salts thereof are added, and mycinamicin-producing bacteria are cultured in a medium to produce mycinamicins.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an agent for increasing the production of macrolide antibiotic mycinamicins and a method for producing the same.

[0002]

2. Description of the Related Art Hitherto, mycinamicins I to XVIII have been known as mycinamicin antibiotics [J. A
ntibiotics, 33, 4, 364 to 376 (1
980): ibid. 3,3,276-281 (1981):
36, 2, 175-178 (1983): JP-A-61-61
-18557 Publication: JP-A-61-21480 Publication:
JP-A-63-5399: JP-A-63-31479
Publication: JP-A-64-40495 Publication: JP-A-2-1
7197 and JP-A-3-11096].

Among these mycinamicins I to XVIII, mycinamicins I, II, IV and V show extremely excellent antibacterial activity especially against staphylococcus and mycoplasma and are useful as therapeutic agents for human and animal infectious diseases. ..
Conventionally, mycinamicins have been produced by aerobically culturing microorganisms belonging to the genus Micromonospora in a normal medium.

[0004]

The productivity of mycinamicins is not sufficient by the conventional method, and further higher productivity is required. Therefore, a method for supplying a large amount of mycinamicins at low cost is required. It was

[0005]

Means for Solving the Problems The inventors of the present invention have been studying a method for increasing the production of mycinamicin antibiotics, and have surprisingly been known as enzyme inhibitors such as β-lactamase, DNase and topoisomerase I. Compound Dotriacolides (J. Antibiotics, 34 ,
12, 1628-1630 (1981) and Japanese Patent Laid-Open No.
7-200380) was added to the culture of a mycinamicin-producing bacterium to improve the productivity of mycinamicins, and the present invention has been completed.

That is, the present invention relates to MG299A substance, M
It is a mycinamicin production-increasing agent containing as an active ingredient at least one or more dotricholides selected from the group consisting of G299B substances and salts thereof. The present invention also provides a MG299A substance (hereinafter referred to as MG299A substance in the method for producing a mycinamicin which comprises culturing a mycinamicin-producing bacterium in a medium.
299A. ), MG299B substance (hereinafter referred to as MG2
It is called 99B. ) And at least one or more dotricholides selected from the group consisting of salts thereof, which is suitable as a method for producing mycinamicins, that is, a method for increasing production.

In the present invention, the mycinamicins are represented by the following formula (1)

[Chemical 1] [In the formula, A represents an oxygen atom or a single bond, and R ₁ and R ₂
And R ₆ are each a hydrogen atom or a methyl group, R ₃
Is an oxygen atom or (a hydrogen atom and a hydroxyl group), R ₄ is a hydrogen atom or a formula (2)

[Chemical 2] R ₅ is a hydrogen atom or a hydroxyl group, ─ is a single bond or a double bond, and Q is a methyl group or a hydroxymethyl group or the formula (3)

[Chemical 3] (Wherein n represents 0 or 1, and Y and Z each represent a hydrogen atom or a methyl group), and is not particularly limited as long as it has a basic structure represented by Specific examples thereof include mycinamicin I to XVIII shown in Table 1 below.

[0008]

[Table 1]

The mycinamicin-producing bacterium is not particularly limited as long as it is a microorganism capable of producing the above-mentioned mycinamicins, but a microorganism belonging to the genus Micromonospora is preferable,
Further, Micromonospora griseorubida is more preferable, and Micromonospora griseorubida A11725 (Micromonosporagr) is more preferable.
iseorubida A11725, FERM BP
-705) strains. This strain has the ability to produce known mycinamicins, and its mycological properties are described in J. Antibiotics, 3
3 , 4, 364-376 (1980), Japanese Patent Publication 51-1
It is described in Japanese Patent No. 8557. In addition, it is a well-known fact that the general properties of actinomycetes are such that the mycological properties are extremely variable and are not constant, and that they are mutated naturally or by UV irradiation or a mutagenesis agent that is usually performed. Therefore, not only such artificial mutants, but also natural mutants, strains having the ability to produce mycinamicins can be used in the present invention. In addition, a mycinamicin-producing bacterium transformed by genetic engineering can also be used.

The dotricholides in the present invention refer to MG299A and MG299B described in JP-A-57-200380, or a mixture thereof, or salts thereof. Heretofore, dotricholides have been known as substances that inhibit the enzymatic action of β-lactamase, which mainly hydrolyzes and inactivates penicillins or cephalosporin antibiotics (J. Antibiotics).
s, 34 , 12, 1628-1630 (1981) and JP-A-57-200380). Furthermore, it is also known to inhibit the enzymatic activities of various DNA degrading enzymes and topoisomerase I (J. Antibiotic).
s, 35 , 2, 248-250 (1982)).

Dotriacolides are micromonospora Echinospora MG299-fF35 (Microm
onospora echinospora MG29
9-fF35) is known to be produced in culture (Japanese Patent Application Laid-Open No. 57-200380), but a mycinamicin-producing bacterium, Micromonospora griseorubida A11725 (Micromonospora g).
Riseorubida A11725, FERM B
The present inventor has confirmed that P-705) also produces dotricholides, and this strain can be advantageously used. The physicochemical properties of dotricholides are as follows.
That is, when using n-butanol: methanol: water (4: 1: 2 by volume) as a developing solvent in white powder and silica gel thin layer chromatography, Rf was 0.2.
It is 9 to 0.30. The red external absorption spectrum is as shown in FIG. MG299A has an absorption maximum at 210 nm in the ultraviolet absorption curve measured in an aqueous solution.

The dotriacolides of the present invention include M
It may be any ratio of G299A and MG299B, and it is usually preferable to use it without changing the ratio of the product of the microorganism producing dotriacolides,
Usually, MG299A: MG299B is about 7: 3. The salt is not particularly limited as long as it does not adversely affect the growth of microorganisms, and examples thereof include sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt and the like.

The addition amount of dotricholides is usually 0.1 to 20 g, preferably 1 to 10 g per liter of medium. The addition time of dotriacolides can be added at all times from the time of medium preparation or after transplantation of the seed culture solution to the end of the culture, but addition of dotriacolides from the beginning of the culture is not always preferable for the growth of mycinamicin-producing bacteria. Particularly preferably 3 to 3 after the start of culture.
It's the sixth day. As a method of addition, the medium may be added to the medium, the medium may be added to dotricholides, or the mixture may be added after mixing with the microorganism. Also, shot (single time) addition and continuous addition are possible. In adding the dotriacolides, in consideration of the above-mentioned use amount, in powder form or in an appropriate concentration of, for example, 1 to 20%, an aqueous organic solvent as long as it is not disadvantageous to the growth of water, a buffer solution or the like or microorganisms. It may be dissolved and used in an appropriate form such as a solution state.

To carry out the method of the present invention, first, a mycinamicin-producing bacterium may be cultured in the same manner as a known method, that is, in a medium containing an anabolic carbon source, an anabolic nitrogen source, and an anabolic inorganic salt source. Cultivation may be carried out with reference to the conditions for producing mycinamicins, for example, the culture method described in Japanese Patent Publication No. 61-18557.

Solid or liquid media can be used,
Liquid medium is suitable for mass production. As the culture method, any known culture method can be used, but the aeration and agitation culture method in a fermentation tank is particularly suitable for mass production. The medium components include carbon sources such as glucose, starch, glycerin, sucrose, molasses, dextrin: peptone, meat extract,
Soybean powder, casein hydrolyzate, cottonseed flour, corn steep liquor, nitrates, nitrogen salts such as ammonium salts: substances containing cations such as sodium, potassium, magnesium, calcium, cobalt, manganese, iron, chlorine, sulfuric acid, Inorganic salts such as substances containing anions such as phosphoric acid and acetic acid: Growth factors of bacteria such as dry yeast and yeast extract.

The culture temperature is usually 20 to 37 ° C., preferably 26 to 32 ° C. The culture time may be the time at which the productivity of mycinamicins is maximized,
Usually, about 4 to 10 days are exemplified. Then, the mycinamicins can be collected from the culture obtained by culturing in this manner by a known method.For example, the culture is filtered and extracted by adding an organic solvent such as ethyl acetate to the filtrate. The method of concentrating the organic layer, subjecting it to various chromatographies, and separating and purifying is exemplified. The mycinamicins may be purified as a mixture or as a single product depending on the purpose, and can be appropriately used in a known method.

By adding dotriacolides and culturing as described above, mycinamicins are remarkably increased. In particular, mycinamicin I, II, IV and V markedly increased. According to the present invention, it is possible to increase the production of various compounds belonging to the mycinamicin class.
It is also possible to selectively increase the production of only one or several compounds. In that case, appropriate culture conditions may be selected with reference to the examples of the present application.

[0018]

The present invention will be described below with reference to examples and reference examples, but the present invention is not limited thereto.

Reference Example Preparation of a mixture of MG299A and MG299B A) Culture 20 g of dextrin, 25 g of cottonseed flour, 5 g of polypeptone, 2.5 g of yeast extract, 40 mg of ferrous sulfate heptahydrate, 40 mg of cobalt chloride and 6 water per liter of culture medium. The pH of the liquid medium consisting of 2 mg of the hydrate and 1 g of calcium carbonate was adjusted to pH 7.5 by dropping a 5N sodium hydroxide aqueous solution. 900 ml of this medium was dispensed into a 5-liter round bottom flask, and steam sterilized at 120 ° C. for 30 minutes. Micromonospora on this medium
Grise Orbida A11725 (FERM BP-70
5 ml of the frozen bacterium of 5) was inoculated and cultured at 30 ° C. for 50 hours on a reciprocal shaker to obtain a seed culture solution. On the other hand, 90 g of dextrin, 25 g of cottonseed powder, 5 g of defatted wheat germ, 1 g of dry yeast, 5 g of calcium carbonate, 1.5 g of dibasic potassium phosphate, and 4 g of magnesium sulfate heptahydrate per liter of medium.
g, 40 mg of ferrous sulfate heptahydrate, 2 mg of cobalt chloride hexahydrate, and 0.5 g of a silicon-based antifoaming agent, the pH of the liquid medium was adjusted to pH 7.2 by dropping a 5N caustic soda aqueous solution. A 30 l jar fermenter containing 20 l of this medium was steam sterilized at 120 ° C for 30 minutes. The above 900 ml of the seed culture solution was inoculated into this jar fermenter, and aeration and stirring culture was carried out for 8 days under the conditions of 30 ° C., 300 rpm, and 20 l / min of sterile air supply to obtain 17 l of culture solution. The mixture of MG299A and MG299B in the culture was quantified by high performance liquid chromatography (HPLC) to yield 3.6 mg / ml. HP
The LC measurement conditions are as follows. Column: Inner diameter 6 mm x 300 mm Packing agent: AQUASIL SS-562N (manufactured by Labo-Cotech) Solvent system: Acetonitrile: Methanol: 10% Phosphoric acid water (9: 2: 1) Flow rate: 1.2 ml / min

B) Purification and collection 17 l of the above-mentioned culture was filtered to separate bacterial cells and other solids, and 15 l of filtrate was obtained. The filtrate was extracted with 5 l of butyl acetate and partitioned into a butyl acetate layer containing mycinamicins and an aqueous layer containing a mixture of MG299A and MG299B. MG299A and MG29
15 l of an aqueous layer containing a mixture of 9B with concentrated sulfuric acid to pH 1.
The mixture was adjusted to 5 and extracted with 5 l of n-butanol to obtain 3.5 l of an n-butanol solution containing the target substance. 2 l of water was added to this and the pH was adjusted to 5.5 with a 10% aqueous sodium hydroxide solution, and the target substance was redissolved in the aqueous layer. 2.3 L of this aqueous layer was concentrated under reduced pressure to 0.5 L to obtain a crudely purified solution. This crudely purified liquid was adsorbed on a column packed with Diaion HP-20 (2 l). First, the column was washed with 4 liters of 1% aqueous sodium hydroxide solution and 4 liters of 30% methanol, and then 100%.
The target substance was eluted with 6 l of methanol. The eluate was concentrated under reduced pressure, adjusted to pH 7.0 with a 10% aqueous sodium hydroxide solution, and freeze-dried to obtain 22 g of a white powder. The physicochemical properties of the target product obtained by the above operation are as follows, which are in agreement with the physicochemical properties of the dotriacolides described in JP-A-57-200380. .. The developing solvent for thin layer chromatography (TLC) is n-butanol: methanol: water (4: 1: 2), and the measurement conditions for HPLC are as described above. Melting point: 154-155 ° C. (decomposition) Specific rotation: [α] ₂₆ ^D = + 8.2 ^o UV λ _max (H ₂ O): 210 nm IR: Spectrum shown in FIG. 1 Elemental analysis: C45.08%, H6 .89%, S11.3
0% TLC Rf: 0.29 HPLC Rt: 3.06 min Note that the above target substance is about 70% MG299A and about 30% calculated from the nuclear magnetic resonance spectrum of its decomposed product.
% MG299B.

Example 1 20 g of dextrin, 25 g of cottonseed flour, per 1 l of medium,
A 5N caustic soda aqueous solution was added dropwise to a liquid medium consisting of 5 g of polypeptone, 2.5 g of yeast extract, 40 mg of ferrous sulfate heptahydrate, 2 mg of cobalt chloride hexahydrate, and 1 g of calcium carbonate to adjust the pH to 7.5. This medium 50
ml was dispensed into a 500 ml Erlenmeyer flask and steam sterilized at 120 ° C. for 20 minutes. Micromonospora on this medium
Frozen bacterium of Griseorubida A11725 was planted in 1 ml and cultured for 42 hours on a rotary shaker at 220 rpm to obtain a seed culture solution. 90 g of dextrin, 25 g of cottonseed flour, 5 g of defatted wheat germ, 1 g of dried yeast, 5 g of calcium carbonate, 1.5 g of dibasic potassium phosphate, 1 g of magnesium sulfate heptahydrate, 40 mg of ferrous sulfate heptahydrate per liter of medium. A 5N aqueous solution of sodium hydroxide was added dropwise to a liquid medium containing 2 mg of cobalt chloride hexahydrate to adjust the pH to 7.2. Next, each 15 ml of this medium was dispensed into a 150 ml Erlenmeyer flask and steam sterilized at 120 ° C. for 20 minutes. 1.5 ml of a seed culture solution of Micromonospora glyceorvida A11725 was transplanted to this medium, and cultured at 30 ° C. for 4 days on a rotary shaker at 220 rpm. Then, a mixture of MG299A and MG299B prepared in Reference Example (MG299
A about 70%, MG299B about 30%) aqueous solution or M
An aqueous solution of G299B was added so as to have a concentration shown in Table 2, and the culture was further continued for 4 days. MG299B is MG299A and MG29 prepared as described in Reference Example.
It was synthesized from the mixture of 9B according to the method of JP-B-57-200380. This MG299B and MG29
A mixture of 9A and MG299B was dissolved in water to a concentration of 100 mg / ml, 5N aqueous sodium hydroxide solution was added dropwise to adjust the pH to 7.3, and steam sterilization was performed at 120 ° C for 20 minutes. Each component of mycinamicin in the culture solution is HPL
It was quantified by C. The HPLC measurement conditions are as follows. Column: Inner diameter 4 mm × 150 mm Filler: YMC-GEL ODS S-5 AM type (manufactured by YMC) Solvent system: 0.1 M phosphate buffer: Methanol: Acetonitrile (50:31:14) Flow rate: 0.8 ml / min The results are shown in Table 2.

[0022]

[Table 2] According to the results in Table 2, MG299A and / or M
The addition of G299B markedly increased mycinamicins.

Example 2 90 g of dextrin, 25 g of cottonseed flour, per 1 liter of medium,
Defatted wheat germ 5g, dry yeast 1g, calcium carbonate 5
g, dipotassium phosphate 1.5 g, magnesium sulfate heptahydrate 1 g, ferrous sulfate heptahydrate 40 mg, and cobalt chloride hexahydrate 2 mg, a 5N aqueous caustic soda solution was added dropwise to a pH of 7. Prepared to 2. Next, this medium 1
Dispense 5 ml each into a 150 ml Erlenmeyer flask.
Steam sterilized at 0 ° C. for 20 minutes. 1.5 ml of the seed culture solution of Example 1 was transplanted, and the seed culture solution was placed on a rotary shaker at 220 rpm for 30
Cultivation was carried out at 4 ° C for 4 days. And MG prepared in the reference example
An aqueous solution of a mixture of 299A and MG299B was added so as to have a concentration shown in Table 3, and the culture was further continued for 4 days. The mixed aqueous solution of MG2994 and MG299B was adjusted to a concentration of 100 mg / ml, a 5N aqueous sodium hydroxide solution was added dropwise to adjust the pH to 7.3, and steam sterilization was performed at 120 ° C. for 20 minutes. Each component of mycinamicin in the culture solution was quantified by HPLC as in Example 1. The results are shown in Table 3.

[0024]

[Table 3]

Example 3 90 g of dextrin, 25 g of cottonseed flour, per liter of medium,
Defatted wheat germ 5g, dry yeast 1g, calcium carbonate 5
g, dipotassium phosphate 1.5 g, magnesium sulfate heptahydrate 4 g, ferrous sulfate heptahydrate 40 mg, and cobalt chloride hexahydrate 2 mg, a 5N aqueous caustic soda solution was added dropwise to a pH of 7. Prepared to 2. Next, this medium 1
Dispense 5 ml each into a 150 ml Erlenmeyer flask.
Steam sterilized at 0 ° C. for 20 minutes. The micromonospora glyceorbida A1172 obtained in Example 1 was added to this medium.
1.5 ml of the seed culture solution of No. 5 was transplanted and cultured at 30 ° C. for 4 days on a rotary shaker of 220 rpm. Then, an aqueous solution of a mixture of MG299A and MG299B prepared in Reference Example was added so as to have a concentration shown in Table 4, and further 4
Culture was continued for a day. Each component of mycinamicin in the culture solution was quantified by HPLC. The results are shown in Table 4.

[0026]

[Table 4]

[0027] Mycinamicins can be isolated and purified from the culture broth of each of the above-mentioned examples by a known method, and the yield of each mycinamicin is approximately proportional to the quantitative value of the culture broth.
I was able to get it.

[0028]

EFFECTS OF THE INVENTION By adding and culturing the dotricholides of the present invention, the productivity of mycinamicins, especially mycinamicins I, II, IV and V, was improved.

[0029]

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of dotricholides of the present invention.

Claims (4)

[Claims] 1. A mycinamicin production-increasing agent containing, as an active ingredient, at least one or more dotricholides selected from the group consisting of MG299A substances, MG299B substances and salts thereof. 2. A method for producing mycinamicins, which comprises culturing a mycinamicin-producing bacterium in a medium, comprising:
A method for producing mycinamicins, which comprises adding at least one or more dotricholides selected from the group consisting of substance A, substance MG299B and salts thereof. 3. The method according to claim 2, wherein the mycinamicin-producing bacterium is a microorganism belonging to the genus Micromonospora. 4. The addition amount of dotricholides in the medium 1
The production method according to claim 2, wherein the amount is 0.1 to 20 g per liter.