JPH04128289A - Production of fr900506 substance - Google Patents

Abstract

PURPOSE:To produce the subject substance useful as an immunosuppressant, antibacterial agent, etc., on an industrial scale by culturing an FR900506 substance-producing strain in a medium added with pipecolic acid. CONSTITUTION:The objective substance {chemical name: 17-allyl-1,14- dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25- dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[2,2,3,1,0< 4.9>]octacos-18- ene-2,3,10,16-tetraone} can be produced by culturing an FR900506 substance- producing strain [e.g. Streptomyces tsukubaensis No.9993 (FERM BP-927)] in a medium added with preferably 0.5-2.0wt.% of pipecolic acid of formula at 25-35 deg.C for 50-150hr by submerged aerobic culture technique.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial application field The present invention provides F.
This invention relates to a method for producing R900506 substance. In particular, this invention relates to a method for increasing the production of FR900506 substance by fermentation.

(B) Conventional technology -dimethoxy-11,19,21,
27-tetramethyl-11,28-dioxa-4-azatricyclo[22,3,1,O'1o9tacox-18-
xC2.3,10.16-titraone, which is known to be produced by fermentation (for example, JP-A-61-1999)
(See Publication No. 148181). The substance exhibits various pharmacological activities, and its remarkable immunosuppressive activity has attracted particular attention, so there is a need for mass production. To this end, it has been desired to develop strains with higher productivity and methods that can increase production. Therefore, the inventors of the present invention particularly studied additives during culture, focused on the chemical structure of the PR900506 substance, tried adding pipecolic acid, and finally completed the present invention.

(C) Problems to be Solved by the Invention According to the present invention, there is provided a method for producing PR900506 substance, which comprises culturing PR900506 substance-producing bacteria in a medium to which pipecolic acid is added.

The PR900506 substance-producing bacteria used in this invention are:
PR means a bacterium that can produce the substance PR900506. As a specific example, Streptomyces tsukubaensis No. 9993 (Feikoken Article No. 927, deposited with the Research Institute of Microbiology, National Institute of Technology), its natural mutant bacteria, X-ray irradiation, ultraviolet rays, etc. irradiation, N-methyl-N
Examples include artificial mutant bacteria obtained by conventional treatment with N-nitrosoguanidine, 2-aminopurine, and the like.

Pipecolic acid is a compound having a chemical structure of -cooH, and the carbon atom to which the carboxyl group is bonded is an asymmetric carbon. , in this invention, its DL-form is usually used. However, it may be an optically active form thereof. Optionally, a precursor of pipecolic acid such as lysine may be used.

The amount of pipecolic acid added is not particularly limited, but is preferably about 0.02 to 3.0% by weight, more preferably about 0.5 to 2.0% by weight, based on the production medium.

The F R900506 substance can be produced by culturing the P R900506 substance-producing strain in an aqueous medium containing assimilable carbon and nitrogen sources, preferably under aerobic conditions, such as shaking culture or submerged culture. .

Preferred carbon sources for the medium include carbohydrates such as glucose, xylose, galactose, glycerin, starch, dextrin, and the like. Other carbon sources include maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like.

Preferred nitrogen sources include yeast extract,
Hepton, gluten meal, cottonseed flour, soybean flour, corn
Steep liquor, dry yeast, wheat germ, feather flour, fallen flour, and the like, as well as inorganic or organic nitrogen compounds, such as ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium phosphate, urea, amino acids, and the like.

Carbon and nitrogen sources are preferably used in combination, but less pure substances can be used as long as they contain trace amounts of growth factors and significant amounts of inorganic nutrients and do not necessarily need to be used in pure form. There isn't. If desired, inorganic salts such as sodium carbonate, sodium phosphate, or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, etc. may be added to the medium. If necessary, antifoaming agents such as liquid paraffin, fatty oils, vegetable oils, mineral oils or silicones may be added, especially if the culture medium foams significantly.

Deep aerobic culture is preferred as a condition for mass production of FR90 (1506 substance. For small scale production, shaking culture or surface culture is carried out in flasks or bottles.Furthermore, growth is carried out in large tanks. In this case, P
In order to avoid growth delays during the production process of the R900506 substance, it is preferred to use a pre-culture of the microorganism to inoculate the production tank.

That is, a relatively small amount of culture medium is inoculated with microbial spores or hyphae, the inoculated medium is cultured to first produce a microbial preculture inoculum, and then the cultured preculture inoculum is aseptically transferred to a large tank. It is desirable to move it. The medium in which this preculture inoculum is produced may be substantially the same as the medium used to produce the FR900506 material, or it may be different.

Agitation and aeration of the culture mixture can be accomplished in a variety of ways. For stirring, use a propeller or similar stirring device, rotate the fermenter, or shake it.
This can also be done using various pump devices or by passing sterile air through the medium. Aeration may be accomplished by passing sterile air through the fermentation mixture.

Fermentation is usually carried out at a temperature range of about 20°C to 40°C, preferably 25 to 35°C, for about 50 to 150 hours, but the temperature may be changed as appropriate depending on the fermentation conditions and fermentation scale.

The FR900506 substance produced in this way is
It can be recovered from the culture medium by conventional methods commonly used for recovery of other known biologically active substances. The PR900506 substance produced is found in the culture mycelium and in the filtrate, and therefore the PR90050B substance can be extracted from the mycelium and filtrate obtained by filtration or centrifugation of the culture broth, by vacuum concentration, lyophilization, or by conventional solvents. Extraction, pH! treatment with conventional resins such as anion exchange resins or cation exchange resins, nonionic adsorption resins, treatment with conventional adsorbents such as activated carbon, silicic acid, silica gel, cellulose, alumina, crystallization,
It can be separated and purified by conventional methods such as recrystallization.

In addition, when a substance FR900520, which is a similar compound of substance FR900506, is produced by the method of the present invention, the production of this substance is also included in the present invention.

(d) Examples The invention will now be illustrated by examples.

One ship! Put 40 ml of seed medium with the following composition into a 250 g + 1 capacity Erlenmeyer flask, and add 1 strain of Streptomyces tsukubaensis No. 9993 (Feikoken Joyori No. 927) to this.
A platinum loop was inoculated and seed culture was performed at 30°C for 3 days.

Seed medium cornstarch! (wt%) glycerin
1 Glucose 0.5 Cottonseed flour 1 Constable liquor 0.5 Dried yeast 0.5 Calcium carbonate 0.2 pH 6.5 1.
ml, add 1% of the above seed culture to it, and
Aerated agitation culture was performed at 5° C. for 7 days using a rotary shaker (250 revolutions per minute with a width of 2 inches).

Production medium Soluble starch 2 (wt%) Fatty wheat germ 0.8 Dry yeast 0.4 Constable liquor 0.6 Calcium carbonate 0.1 pH 6,8 In the same manner as above, each concentration of DL-pipecolic acid was added. Culture was carried out.

Centrifuge 10ml of culture solution (2000pra, 1
0 minutes) to obtain bacterial cells, which were stirred with acetone loml for 1 hour. Regarding the acetone extract, high performance liquid chromatography (column: 4φx250xL Recrocart RP-18 (manufactured by Merck), mobile phase 0% acetonitrile, 0.1% phosphoric acid (50°C), detection: UV
220 nm, flow rate: 1 ml/min, retention time = 7.9 min)
The production titer of the PR900506 substance was measured.

DL-pipecolic acid PR Added amount (wt%) of substance PR900506 Potency (ug/a+) 0
135.4 0.02 154.4 0.1 176.9 0.5 194.9 1.0 205.8 2.0 209.5 From the above results, DL-pipecolic acid has a Showing a significant production addition effect at a concentration of .02%,
At a concentration of 0.5%, an increase in production of about 44% was observed, and at a concentration of 2.0, an increase of about 65% was observed.

(E) Effects of the Invention According to the present invention, a significant increase in the production amount can be achieved by adding a small amount of pipecolic acid in the fermentation production of F K506 substance, and a method for producing F R900506 substance that has high industrial utility value. brings about.

Claims (1)

[Claims] 1. FR90050, which is characterized by culturing FR900506 substance-producing bacteria in a medium added with pipecolic acid.
Manufacturing method of 6 substances.