JPH0488993A - Production of trans-4, 5-dehydro-l-lysine and producing bacterium thereof - Google Patents

Abstract

PURPOSE:To obtain the title compound useful as an antifungal agent industrially and advantageously by culturing a specific strain belonging to the genus Nocardiopsis. CONSTITUTION:A strain [preferably Nocardiopsis sp.KC-7,223 (FERM BP-2,993)] belonging to the genus Nocardiopsis, capable of producing trans-4,5-dehydro-L- lysine, is cultured by submerged culture under an aerobic condition at 25-30 deg.C for 2-6 days and the objective compound is obtained from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel method for producing trans-4.5-dehydro-lysine useful as an antifungal agent and a microorganism that produces the same.

[Conventional technology]

Trans-4,5-dehydro-DL-lysine is a known compound, for example J, Chem, Sac, Perk.
It can be synthesized by the method described in in r19721B25.

Furthermore, JP-A-63-183526 discloses that this trans-4,5-dehydro-DL-lysine, especially trans-4,5-dehydro-lysine, exhibits excellent antifungal activity and that DL-lysine exhibits excellent antifungal activity. A method for efficiently obtaining L-integral from an integral is described.

[Problem to be solved by the invention]

However, this trans-4,5-dehydro-L-
! Since the J-lysine production method involves removing the D-isomer from the racemic compound by optical resolution, it is theoretically impossible to increase the yield of the L-isomer in the final step to 50% or more.
It was not an industrially advantageous method.

Therefore, trans-4,5-
It has been desired to develop an industrially advantageous method for producing dehydro-lysine.

[Means to solve the problem]

Under these circumstances, the present inventors have conducted intensive studies and found that a specific bacterium belonging to the genus Nocardiopsis specifically produces trans-4,5-dehydro-L-lysine, and has devised the present invention. completed.

That is, the present invention is characterized by culturing trans-4,5-dehydro-L-lysine-producing bacteria belonging to the genus Nocardiopsis, and collecting trans-4,5-dehydro-lysine from the resulting culture solution. The present invention provides a method for producing trans-4,5-dehydro-lysine and a microorganism that produces the same.

Examples of microorganisms that produce trans-4,5-dehydro-lysine of the present invention include Nocardiopsis sp. KC-7223. This KC-722
The three strains have the following mycological properties. Unless otherwise specified, properties on various media were observed using conventional methods after culturing at 27°C for 14 days, and color expressions were based on the classification of Color Harmony Manual, 544th edition (Container Corporation America). I followed.

(2) Morphological properties Although this strain is poor on several types of media, it forms aerial mycelia. They are simply branched, and the sporophores occur in the form of branches on the aerial hyphae, and their tips are straight or partially wavy.

The spores are short cylindrical, approximately 0.3 x 1.0 μm in size, and form chains of 20 or more. The spore surface has a smooth structure. Formation of flagellated spores, sporangia, sclerotia, etc. was not observed, and fragmentation of long-living mycelium was observed.

■, Properties on various media 1, Sucrose/nitrate agar medium Growth: Good Adhesion of aerial mycelium: Not observed Color of aerial hyphae: Not observed Color of long hyphae: Slight yellow (24!c) Soluble pigment: Slight yellow (21c) 2. Glucose-asparagine agar medium Growth: Moderate Aerial hyphae settlement: Not observed Color of aerial hyphae: Not observed Color of long hyphae: Pale gray pink (3c a) Soluble pigment: Slight yellow (24!c) 3. Glycerin/asparagine agar medium Growth: Good Aerial mycelial settlement: Not observed Color of aerial hyphae: Not observed Color of long hyphae: Slight yellow (2ea) Soluble pigment: Slight yellow (2l c) ) 4. Starch/Inorganic Salt Agar Medium Growth: Good Aerial Mycelia Adhesion: Poor Aerial Mycelia Color: White (a) Long Mycelia Color: Slight yellowish white (2c a) Soluble pigment: Not observed 5. Tyrosine Agar Growth on medium: Moderate Aerial hyphae growth: Not observed Color of aerial hyphae: Not observed Color of long-growing hyphae: Pale gray brown (3ec) Soluble pigment: Not observed 6, Growth on nutrient agar medium: Moderate aerial hyphae Growth on yeast/malt agar medium: Good Growth on yeast/malt agar medium: Poor Aerial mycelium growth: Poor Color of aerial hyphae: White (a) Color of long hyphae: Slight yellowish white (2ca) Soluble pigment: Slight yellow (21c) 8. Growth on oatmeal agar medium: Moderate Adhesion of aerial hyphae: Not observed Color of aerial hyphae : Not observed Color of long-growing hyphae: White (a) Soluble pigment: Not observed ■, Physiological properties 1, Growth temperature range 15°C to 40°C Optimal growth temperature 30°C to 36°C 2, Liquefaction of gelatin Negative 3. Hydrolysis of starch Positive 4. Coagulation of skim milk
Positive Peptonization of skim milk Positive 5, Production of melanin-like pigment Negative 6, Salt tolerance
≦4% ■、Carbon source availability (Pridham Bot) As a result of examining the availability of carbon sources on IJ-bu agar medium, L-arabinose, D-xylose, D-glucose, D-fructose, and sucrose were found. and raffinose were used.

The use of D-mannitol was not allowed. The use of inositol and L-rhamnose was questionable.

■、Cell wall components 1. Analysis of diaminopimelic acid during cell wall hydrolysis Contains meso-diaminopimelic acid.

2. Analysis of sugar components during cell wall hydrolysis Contains no madulose, galactose, arabinose or xylose.

Based on the above-mentioned morphological characteristics and physiological properties, a strain similar to this strain was designated as "Bercy's Manu 7JL".

``Of Determinative Bacteriology'' 8th edition, Schirling and Gottlieb's ISP-described bacterial strains, and Waxman's ``The Perfect Chinomycetes'' Vol. 2
A search for publications on new species of actinomycetes and other actinomycete species revealed similar strains, Nocardiopsis mutabilis (Nocardiopsis mutabilis).
opsis mutabilis IFO14310)
Stocks were selected.

However, Nocardiopsis mutabilis IF01431
Strain 0 (rN, hereinafter referred to as "strain m") contains galactose as a whole cell sugar component, but strain KC-7223 does not. In addition, the two strains differed in that the N and m strains did not utilize raffinose, while the C-7223 strain did. Based on the above, the KC-7223 strain was determined to be a new strain closely related to Nocardiopsis mutabilis, and was named Nocardiopsis sp. (FE
! RM-BP-2993).

In the present invention, not only the above-mentioned strain KC-7223 but also its natural mutants and artificial mutants can be used, as long as they belong to the genus Nocardiopsis and have the ability to produce trans-4,5-dehydro-lysine. It goes without saying that you can.

In order to obtain trans-4,5-dehydro-cy-lysine using the above-mentioned microorganism of the present invention, the microorganism of the present invention may be inoculated into an appropriate medium and cultured according to the usual culture method for antibiotic production. . As the medium, one containing an appropriate combination of assimilable nitrogen sources and carbon sources is used. There are no particular restrictions on the carbon and nitrogen sources; examples of carbon sources include starch, glucose, glycerin, maltose, dextrin, sucrose, fructose, and molasses; etc. can also be used. These may be used alone or in combination of two or more. On the other hand, as nitrogen sources, soybean flour, yeast extract, dried yeast, peptone, meat extract, cornstarch liquor, casamino acids, distiller's solitaire, ammonium chloride, ammonium sulfate, urea, sodium nitrate, etc. are used alone or in combination. It will be done.

Other inorganic salts such as common salt, potassium phosphate, magnesium sulfate, calcium chloride, calcium carbonate, calcium hydroxide, cobalt chloride, zinc sulfate, iron chloride, iron sulfate, potassium phosphate, sodium phosphate, and trace amounts of Heavy metals can be added. Furthermore, organic and inorganic substances that aid the growth of bacteria and promote the production of trans-4,5-dehydro-lysine can be added as appropriate.

When using the aerated culture method, an antifoaming agent such as fatty oil, silicone oil, paraffin, etc. is further used.

As a culture method, although culture on a solid medium is also possible, it is preferable to use a liquid culture method, particularly a deep culture method, as in the general antibiotic production method.

Cultivation is carried out under aerobic conditions, with a culture temperature of 20-35
℃, especially 25 to 30℃ is preferred. When culture is carried out for 2 to 6 days, whether in shaking culture or tank culture, the active substance is produced and accumulated in the culture medium. The culture is stopped when the production amount in the culture solution reaches the maximum, and the antibiotic of interest is isolated and purified from the culture solution.

To isolate and purify this substance from the culture filtrate, the target substance is a water-soluble basic substance that is soluble in water and sparingly soluble in common organic solvents. Separation and purification methods can be used. For this purpose, adsorption/desorption methods using ion exchange resins, activated carbon, cellulose, silica gel, alumina, etc., butanol,
An appropriate combination of methods such as extraction with amyl alcohol or the like can be used.

When the culture filtrate is passed through a layer of weakly acidic cation exchange resin, the target substance is adsorbed. This is eluted with a 0.1 to 3.0 normal alkali or acid, and the active ingredient is lyophilized to obtain a coarse powder of the target substance. The weakly acidic cation exchange resin used at this time is Amberlite IRC-50.
, I RC-84 or 1tcG-50 (ROHM & Co., Ltd.)
(manufactured by Haas), Diamondion WK-10 or WK-20
(manufactured by Mitsubishi Kasei Corporation). In addition, as alkalis, ammonia water, hydroxide, IJium aqueous solution, etc.
Examples of acids include formic acid, hydrochloric acid, and sulfuric acid.

A method can also be used in which the culture filtrate is adjusted to pH 7 to 9, the target substance is adsorbed on activated carbon, and the target substance is eluted with acidic water or hydrochloric acid and methanol.

The coarse powder obtained in this way is a weakly acidic cation exchange resin,
The target product can be obtained as a free base by adsorbing it onto CM-Sephadex, CM-cellulose, etc., and eluting with aqueous ammonia, aqueous ammonium formate, etc. by a concentration gradient method or a concentration step method.

The isolated target product is further processed by adsorption chromatography using cellulose, silica gel, alumina, etc., gel filtration method using Sephadex G-10, etc., ion exchange chromatography using Dowex 1x2, etc., and high performance liquid chromatography. It can be purified by applying etc. These methods can be applied alone or in combination.

The free base thus obtained can be further treated with an inorganic acid such as hydrochloric acid,
An acid addition salt can be obtained by adding sulfuric acid, hydrobromic acid or an organic acid such as acetic acid, oxalic acid, methanesulfonic acid, etc. in a conventional manner.

〔Effect of the invention〕

According to the present invention, by using a microorganism that specifically produces trans-4,5-dehydro-lysine and culturing it, trans-4,5-dehydro-lysine, which has an excellent antifungal effect, is produced.
-Dehydro-L-lysine can be efficiently produced.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these.

Example 1 A soil sample collected in Fukaya City, Saitama Prefecture was suspended in sterilized water, and this was mixed with potato infusion solution (10% potato, 0.5% tomato paste, 1% oatmeal), 0.1% meat extract, Mycostatin 50μg/m! , penicillin GO 14μg
/ml, polymyxin B 2μg/-, nalidixic acid 8
Medium containing μg/rnl and 1.5% agar (pH 7,
Trans-4,5-dehydro-L-lysine producing bacteria (Nocardiopsis sp, KC
-7223) was obtained.

Example 2 Nocardiopsis sp, KC-7223 strain grown on a potato dextrose agar slope was treated with 1% soluble starch, 1% glucose, 1.5% soybean flour, KH, PO.

0.27%, Na, HPO40.18%, MgSO4・
7H200,05% and CoCl2 6H-00,0
005% and cultured with shaking at 28° C. for 72 hours to obtain a seed culture.

Next, glucose 2%, soybean flour 1%, meat extract 0.2
%, yeast extract 0.2%, Mg5O1.7H,00,0
A medium 151 consisting of 5% CoCf2.6H, OO, 0005% and 0.5% cottonseed oil was placed in a stainless steel tank with a capacity of 30 f.
- inoculated, aeration rate 51/min, stirring number 300 r, p,
The cells were cultured at 28°C for 72 hours.

After culturing, the culture solution was filtered and the obtained filtrate (2OA)
Amberlite I RC-50 [H”: NH, + type (
1: 4) :] adsorbed on the column (11), and after washing with water, 0
．． It was eluted with 5N ammonia water. The active fraction was collected, concentrated under reduced pressure, adsorbed on a column (100 rnl) of CM-Sephadex C-25 (NH4+ type), and mixed with water to 0.2N
Concentration gradient elution was performed with aqueous ammonia. The active fractions were collected and concentrated under reduced pressure, and the concentrated solution was passed through a cellulose column (2001
ff) and eluted and fractionated with a solvent system of methanol:water:6N-hydrochloric acid:pyridine (40:13:2:5). After concentrating the active fraction and adjusting the pH to 5, it was purified by Sephadex G-10 column chromatography (300 ml) using water as a developing solvent, and lyophilized to obtain a colorless powder (60 ml). .

The physical and chemical properties of the obtained substance are as follows.

1. Specific rotation (hydrochloride); [α] = 5-5.7° (c 0.7, H2O)2
, UV absorption spectrum; shows only terminal absorption.

3. Infrared absorption spectrum; The spectrum measured in hydrogen bromide pellets is as shown in Figure 1.

4. 'It-NMR; The 'H-NMR spectrum measured in heavy water is as shown in FIG. 2.

5. "C-NMR; Measured in heavy water" The C-NMR spectrum is as shown in FIG. 3.

Margin 6 below: Thin layer chromatography: (manufactured by Difco) is smeared with a suspension of the test bacteria and incubated at 27°C.
The cells were cultured for 2 to 4 days and measured.

Plate: Cellulose plate Art, 5718 (manufactured by Merck & Co.) 7. Color reaction; Positive for ninhydrin reaction and Lydon-Smith reaction.

From the above physical and chemical properties, this substance is trans-4,5-
It was confirmed to be dehydro-lysine.

Test Example 1 Trans-4,5-dehydro-L- obtained in Example 2
! The minimum inhibitory concentration (MIC) against J-lysine fungi was measured and was as follows. The measurement was performed using a yeast morphology agar plate.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of the material of the present invention, Figure 2C1H
-NMR spectrum, Figure 3 shows the ft13C-NMR spectrum. that's all

Claims (1)

[Claims] 1. Cultivating trans-4,5-dehydro-L-lysine producing bacteria belonging to Nocardiopsis and collecting trans-4,5-dehydro-L-lysine from the resulting culture solution. Characteristic method for producing trans-4,5-dehydro-L-lysine. 2. A trans-4,5-dehydro-L-lysine producing bacterium belonging to the genus Nocardiopsis. 3. The trans-4,5-dehydro-L- according to claim 2, which is Nocardiopsis sp. KC-7223.
Lysine producing bacteria.