JPS5991891A - Preparation of mycophenolic acid by fermentation method - Google Patents

Abstract

PURPOSE:To prepare mycophenolic acid in high yield, by cultivating a microorganism capable of producing the mycophenolic acid in a culture medium containing a porous material. CONSTITUTION:A porous material, e.g. alumina, activated carbon or zeolite, in an amount of about 1-50g/l is added to a well-known culture medium, e.g. a culture medium containing 1% glucose, 0.2% peptone, 0.1% malt extract, 0.1% yeast extract and 1.5% agar for use. A microorganism, e.g. Penicillium brevicompactum QM-8406 (ATCC16024), belonging to the Penicillium, and having the ability to produce mycophenolic acid is inoculated into the above-mentioned culture medium and cultivated under conditions of about 20-30 deg.C and about 2-10pH for about 4-20 days by the submerged culture method. The aimed mycophenolic acid is then separated and collected from the culture fluid by the well-known method.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing mycophenolic acid by a fermentation method.

Mycophenolic acid has antiviral, antitumor, antifungal, and antibacterial effects, and is a useful compound with many uses including medicine. Mycophenolic acid is present in many strains of bacteria belonging to the genus Penicillium, e.g. Penicillium brevi-c.
ompactum), Penicillium stoloniferum (P, stoloniferum)
Scabrum (P, scabrum), Penicillium nagemi (P, nagemi), Penicillium sutuaferi (P, 5Zateri), Henicillium Φbatus-mei (P, patus-mei)
), Penicillium glycobrunneum (P, gr
iscobrun-neun) and Penicillium
It is known to be produced by P. viridicatum (P, viridicatum) and the like.

Are the inventors of the Penicillium genus? When producing mycophenolic acid using this mycophenolic acid producing strain, it is necessary to add porous substances such as various types of celite and zeolite to the medium in the form of powders of 0.1 to 5 cm. This significantly promotes growth rate and mycophenolic acid production,
Moreover, it was discovered that the amount of mycophenolic acid accumulated was significantly increased, and V was able to complete the present invention.

When culturing mycophenolic acid-producing bacteria belonging to the genus Penicillium, when a powder of a porous substance is added to the medium and cultured aerobically, spores and hyphae are grown in the porous substance. One particle attaches to the seedlings and germinates using this as a nucleus to grow mycelium. Since the number of hyphal pellets increases in proportion to the number of particles, the size of the pellets conversely decreases.

In other words, by adding porous substance powder to the culture solution, the mycelia are kept in fine pellets, which improves the stirring efficiency of the culture medium and minimizes the adhesion of mycelia to the culture vessel walls.
Furthermore, the remarkable effect of increasing the growth rate of bacteria and promoting mycophenolic acid production was observed.

The microorganism used in the present invention belongs to the genus Penicillium, and is a microorganism that grows in a normal nutrient medium and has the ability to produce mycophenolic acid inside and outside the bacterial cells.

For example, Penicillium breviconbactam QM-840
Clofibrate and dodecyltrimethyl chloride resistant strain AJ derived from 6ATCC16024 or QM-8406]l7096 FERM BP-5
5 (see Japanese Unexamined Patent Publication No. 57-50889), and further AJ
AJ's 7114FERM-P6649, which has monofluoroacetic acid resistance in addition to 117096, is used.

Fermentation medium used when carrying out the present invention 3
- a known culture medium, for example V/, 17. Muth e
tal.

Anti-m1sorob+ Ageuts che
mtherap, +8 +321~327 (+9
75) The medium described above is used. The culture conditions are deep culture. At this time, the temperature is 20-30C1, preferably 25-27tT, and the pH is 2-10, preferably 3
~8, and the time is 4 to 20 days, preferably 5 to 15 days.
It is day.

During this culture, porous substances added to promote mycophenolic acid production include alumina, activated carbon, clay,
Any substance belonging to diatomaceous earth, natural or synthetic zeolite, etc., or similar substances thereof may be used.

Examples of diatomaceous earths include commercially available Celite (A501.503.505.535.545), Filter Cell, Hyfloss-Purcel, and Standard Super Cell. Zeolites include analcime and chavite.
bazite), clinoptilolite-heulandit
e), erionite, ferrierite, 4-aumontite, mordenite (mordenite, mordenite) and kaishuusite. These polyphilic substances are used in fine granular form. As shown in the examples below, the optimum amount to add varies depending on the type of porous material, but the optimum amount for medium 1.0
ranges from 1 to 509.

Mycophenolic acid was separated from the culture solution thus obtained.
For collection, known methods can be used.

The amount of mycophenolic acid accumulated in the culture solution can be easily analyzed and quantified using high performance liquid chromatography.

Example 1 Penicillium breviconbactum QM-8406AT
CC+ 6024 and clofibrate dodecyltrimethylammonium chloride resistant strain AJ 117096 (FERM BP-55) derived from this strain
was inoculated into the natural medium slant shown in Table 1 and 27t? After culturing for 14 days, the spores were collected and added to 0.1M phosphate buffer (p
A certain volume of H6,8) was suspended in a medium containing various concentrations of Celite 545 and having the composition shown in Table 2 (1 50 ml V Erlenmeyer flask (filled with Jtrtl)). Approximately 1o6
/ml), and rotational culture was performed at 20 Orpm for 10 days using a 27 Cr trowel. Table 1 shows the amount of mycophenolic acid in the obtained culture solution.
1.0 pebton
0.2 malt extract 0
．． 1 yeast extract
0.1 agar
1.51) H7.0 6- Table 2 Medium composition (pH 6.0) Component Content Glucose 10.0 Tiglycine
1.46//Methionine
0.05 NKH2PO40,311 MgSO4・7H,OO,1// FeSO42,Oppm CuSO40,3n ZnSO40,25tt Mn S04 0
．． 16 nK2MoO40,02n Celite 545 0-4 Chi (biting) Table 3 Effect of addition of Celite on accumulated amount of mycophenolic acid t Celite Mycophenolic acid (my/de
)0 66 192
0゜4 283 3831
221417 2 181 3334
158 358 Measured by high performance liquid chromatography. The results are shown in Table 3. This result shows that the addition of Celite to the medium significantly increases mycophenolic acid accumulation.

Example 2 Penicillium fistula breviconbactum AJ+17096 and monofluoracetic acid resistant strain AJ I derived from this strain
+7114 (FERM-P6649) in Table 1
Inoculate 1 natural medium (Slan) shown below, and use a 27rt trowel 11
After culturing for one day, a spore suspension was prepared in the same manner as in Example 1, and this was inoculated into a medium containing 1% of various adsorbents and having the composition shown in Table 2 (one 300-volume Erlenmeyer flask filled with 50 ml). Rotary culture was performed at 27C at 1200 rpm for 15 days. To check the growth of bacteria, one sample was taken every 7 days, and after natural filtration with a Toyo Roshi paper A 2 t trowel, the bacterial cells were expressed as dry weight at 105C for 18 hours. The adsorbents added were Celite 535, Celite 545, and artificial zeolite ZCP.
-50 and natural zeolite CLINOPTILOLIT
It is a powder of E. The results are shown in Table 4.

= 9- Table 4 No effect of addition of various adsorbents on accumulated amount of mycophenolic acid Addition 1.6 110
2.4 110 Celite 535 3.8
412 4.0 440 Celite 545
3.6 477 4.0 4
57 Zeolite ZCP-503, 43583, 6477
Zeolite CLINO, 3,63853,8394 *The dry weight of bacterial cells per 5 ml of culture solution on the 7th day of culture is 2
Seed celite and natural and synthetic zeolites are both AJ
+17096 and AJ117114 and mycophenolic acid production were promoted.

Example 3 The spore suspensions of AJ +17096 and AJ-10-+17114 obtained in Example 2 were inoculated into a medium containing various concentrations of zeolite, and cultured under the same conditions as Example 2. The results of quantifying the amount of mycophenolic acid accumulated are shown in Table 5.

Table 5 Effect of added amount of zeolite on accumulated amount of mycophenolic acid Zeolite* Mycophenol M (m9/dl
) (a) AJI+7096 AJI+7
1140 1 10
1 100.2
4 5 8 4 3 1
0.4 3 9 4
3 7 61.0
3 8 5 3 9 4*
CLINOPTILOLITE Patent applicant Ajinomoto Co., Inc.-I 1-560-

Claims (1)

[Claims] A method for producing mycophenolic acid by culturing mycophenolic acid-producing bacteria belonging to the Penicillium genus, a fermentation method characterized in that the mycophenolic acid-producing bacteria are cultured in a medium containing a porous substance. A method for producing mycophenolic acid.