JP2689643B2 - Method for producing polyene antibiotic partlysin B - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention is concerned with the polyene antibiotic partlysin B.
More specifically, the present invention relates to a method for producing particillin B using a particillin B-producing bacterium belonging to the genus Streptomyces that is resistant to tryptophan analogues.

It is known that the partlysin B produced by the present invention and its methyl ester form are effective in the treatment of fungal diseases and the like.

[Conventional technology]

Conventionally, Streptomyces aureofa (Streptomyces aureofa) has been used as a method for producing partlysin B.
ciens) method (Pandey, RCand Rinehart, Jr.,
KL, J. Antibiotics, 35 , 997-1012 (1982), and Streptomyces arenae V
-28-3 (PERM-P 8099) (JP-A 61-189224)
It is known by. However, the method for producing partricin B using these strains is unsatisfactory due to the large amount of purification of by-products and the low accumulation amount.

[Problems to be solved by the invention]

The present invention has been made in view of the above problems, and an object of the present invention is to provide a method capable of industrially advantageously producing partricin B, which has a very small amount of by-products and a high productivity. .

[Means for solving the problem]

The present inventors have conducted extensive studies to achieve the above-mentioned object, and as a result, induce a tryptophan-related substance-resistant mutant strain from a partlysin B-producing bacterium of the genus Streptomyces to improve the partlysin B-producing ability. And completed the present invention.

That is, the method for producing partricin B of the present invention is a method in which a microorganism belonging to the genus Streptomyces having partricin B-producing ability and having resistance to tryptophan-related substances is cultured in a partricin B production medium, B or its salt is collected.

The microorganism used in the present invention is a microorganism belonging to the genus Streptomyces and having the ability to produce partricin B. For example, Streptomyces arena
e) V-28-3 (FERM P-8099), Streptomyces aureofacien
s) NRRL 3878 and so on. In addition to these bacteria, it is needless to say that it can be applied as long as it is a bacterium capable of producing partricin B.

A mutant strain can be obtained from these strains according to a usual method for mutating a microorganism. For example NTG
(N-methyl-N'-nitro-N-nitrosoguanidine), treatment with chemicals such as nitrous acid, irradiation treatment with X-rays, ultraviolet rays, and the like.

The method for selecting a tryptophan-related substance resistant strain is performed as follows. That is, a relatively high-concentration suspension of mutated cells was applied to a minimal agar medium containing a tryptophan-related substance at a concentration that inhibits the growth of the bacterium used,
By culturing the colonies that grow in culture,
A resistant strain can be obtained.

As the tryptophan analog, 5-fluorotryptophan, 6-fluorotryptophan, 5-methyltryptophan, 6-methyltryptophan, or the like can be used. At this time, two or more tryptophan analogues may be used.

Thus, the property imparted by the present invention, that is, the degree of tolerance to tryptophan analogues is increased, or
By imparting resistance to one or more tryptophan analogs, the productivity of partricin B is improved.

As the medium for producing partlysin B used in the present invention, an ordinary nutrient medium used for culturing Streptomyces bacteria can be used. For example, carbon such as glucose, maltose, sucrose, starch, molasses and glycerin can be used. A nutrient medium composed of a nitrogen source such as a source, soybean flour, corn steep liquor, peptone, yeast extract, meat extract, ammonium salt, nitrate and the like is used. In addition, calcium carbonate, sodium chloride, potassium ions, phosphates, sulfates, inorganic salts such as Mg ²⁺ , Zn ²⁺ , Fe ²⁺ and micronutrients are also supplemented as required.

The culture method may be an ordinary liquid culture method for bacteria, and the culture temperature is most preferably 20 ° C to 35 ° C, especially around 28 ° C, and the pH of the culture solution is
Is preferably 7-8. By culturing under such conditions for 4 to 6 days, preferably aerobically, partricin B is reduced to 40%.
0 to 2000 mg / accumulated.

In the method of separating particillin B from the culture solution, since particillin B is accumulated outside the bacterial cells and inside the bacterial cells, generally, culture filtration and bacterial cells separated by means such as centrifugation or filtration are used to remove antibiotics from antibiotics. It is separated and purified by the means used for isolation. For example, methanol, solvent extraction with an organic solvent such as dimethylformamide, silica gel, diatomaceous earth, Avicel, adsorption column chromatography using alumina and the like, gel filtration method using Toyopearl HW40 (tosoh gel filtration simple substance), etc., It is isolated and purified by reverse phase partition column chromatography or HPLC using octadodecylated silica gel (ODS) as a carrier, and further by sequentially combining purification means such as countercurrent partitioning method, crystallization and recrystallization.

The method for quantifying partricin B can be performed by ordinary HPLC.

〔Example〕

 Hereinafter, the present invention will be described specifically with reference to examples.

Example 1 Production of mutant strain Streptomyces arene V-28-3 (FERM P-80
Irradiate the spore suspension of 99) with ultraviolet rays so that the mortality rate is about 99% to 99.9%, and dilute the suspension appropriately to 1
It was spread on a minimal agar medium (Table 1) containing μg / ml of 5-fluorotryptophan. This is incubated at 28 ° C., grown colonies are collected, and from these, a strain having a high productivity of partricin B is selected, and Streptomyces arene V-28-3 (FERM P-11014) is selected. Got

Production of Partricin B Streptomyces arene V-28- in ISP-2 medium
3 parent strain (FERM P-8099) and mutant strain (FERM P-1101)
After culturing 4) for 5 days at 28 ° C., 1 platinum loop of the bacterial cells was inoculated into a test tube containing 5 ml of the partricin B production medium shown in Table 2 and shake-cultured for 5 days to prepare a seed culture solution. . Next, 500 ml containing 40 ml of Partricin B production medium shown in Table 2
No. Sakaguchi flask was inoculated with the seed culture solution and shake-cultured at 28 ° C. for 5 days (shaking width 5.5 cm, 120 rpm).
The results shown in Table 3 were obtained.

Example 2 One platinum loop of the cells of Streptomyces arene V-28-3 (FERM P-11014) grown in ISP-2 medium for 5 days was treated with 150 ml of the partlysin B production medium shown in Table 2. A seed culture solution was prepared by inoculating a 500 ml Sakaguchi flask containing the same and culturing with shaking for 5 days, and further inoculating 3 Sakaguchi flask containing the same medium 1 with shaking culturing for 5 days. Next, the same production medium 20 for partlycin B was used.
The mixture was placed in a 30-volume jar fermenter and sterilized at 120 ° C. for 15 minutes, allowed to cool, inoculated with the seed culture solution, and aerated and agitated at 28 ° C. for 5 days (aeration 1/4 vvm, rotation speed 350 rpm).
The obtained culture broth was centrifuged to collect the cells, 20 dimethylformamide was added, and the mixture was well stirred to extract partricin B. 20 50 mM ammonium acetate and 20 chloroform were added to the extract, and the crude partlysin B which precipitated was collected by filtration. After washing the precipitate with water, 60 g of freeze-dried crude powder of partricin B was obtained. A little tetrahydrofuran: water: triethylamine = 2: 1: 0.
Dissolve in 1 and dilute 10 times with water, then filter and load the filtrate onto the column of Capsule Pack C-18 (Shiseido product) to obtain 10 mM.
Elution was performed with a 35% aqueous acetonitrile solution containing sodium acetate, and the fractions of partricin B were collected. Next, the solution was concentrated under reduced pressure, the solvent was gradually removed until the solution became slightly cloudy, and this was left overnight at low temperature to precipitate yellow crystals. The yellow crystals were collected by centrifugation, washed with water and freeze-dried to obtain 40 g of a yellow powder. This yellow powder had a molecular weight of 1112 as measured by mass spectrum, and the pattern of UV spectrum (FIG. 1) was in good agreement with the known partricin B (for example, JP-A-61-189224) (Table 4). .

[Effects of the Invention] As described above, according to the present invention, by using a partlysin B-producing bacterium that is resistant to tryptophan analogues, partlysin B can be efficiently produced from an ordinary nutrient medium such as glucose. Therefore, the productivity is greatly improved.

[Brief description of the drawings]

FIG. 1 is a graph showing a UV spectrum absorption curve of partricin B obtained in Example 2.

Claims (1)

(57) [Claims] 1. A method of culturing a microorganism belonging to the genus Streptomyces having the ability to produce partricin B and having resistance to tryptophan analogues in a partricin B production medium, and collecting partricin B or a salt thereof from the culture solution. A method for producing partlysin B, which comprises: