JPS60176595A - Preparation of physiologically active substance ml-236a and monacolin j - Google Patents

Abstract

PURPOSE:To prepare a physiologically active substance ML-236A from ML-236B or monacolin J from monacolin K, by using a microorganism belonging to the genus Mortierella, etc. CONSTITUTION:Live or dead microbial cells of a microorganism belonging to the genus Mortierella, Emericella, Diheterospora, Himicola, etc., e.g. Mortierella isabellina IFO7884 or Emericella unguis IFO8087, or an enzyme extracted from the above-mentioned microorganism is brought into contact with a substance ML-236B or monacolin K.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides physiologically active substances ML-236 and Nanacolin J.
Concerning a new manufacturing method.

ML-236A and Monacolin J are known substances previously discovered by the present inventors in the culture of molds belonging to the genus Penicillium and Monascus, and have the following structure <1)
has.

(1) ML-236A (R=H) Monacolin J (R=CH,) ML-236B and Monacolin are also known substances discovered by the present inventors from the genus Penicillium and the genus Monascus, respectively, and have the following structures. (Unexamined Japanese Patent Publication No. 50-155
No. 690 and JP-A-55-111790).

@ ML-236B (R=H) Monacolin K (R=CH,) ML-236A, ML-236B, Sevennacolin J
Monacolin strongly inhibits the biosynthesis of cholesterol μm and has the effect of lowering blood cholesterol in animals.

ML-236B and monacolin can be converted to ML-236A and monacolin J, respectively, by alkali treatment, but the yield is usually 50- or less.

ML-236A and Monafrin J themselves have a blood level μm-lowering effect, but they are also valuable raw materials for synthesizing various derivatives with even better activity.

The present inventor has developed a kite-236B that can be manufactured in large quantities using microorganisms.
We conducted extensive research on converting 7 nacolin K to ML-236A and monacolin JK, respectively.
ML-2 with almost 100% conversion rate by specific microorganisms
The present invention was completed by discovering that 36B and monacolin K can be converted into ML-236A and monacolin J, respectively.

Therefore, the present invention provides ML-236A with excellent characteristics.
and a novel method for producing Monacolin J.

ML-236B and Hepnacolin K respectively in ML-236
Microorganisms that convert into A and Monacolin J include Mortillera, Emericella, and Diheterospora.

Humicola spp., Dichotomomyces spp., Neocosmosvora spp.
Examples include molds belonging to the genus Xylbone, genus Torpices, and genus Cherabia. Among these molds, Mortillera fistula Isabelina rF07884 (Mort.
lerella l5aballna) r z Mericela Languis IFO8087 (Eh+erice
lla ungulg), Diheterospora chlamydosporia IFO9249 (Dlheterospora
achlamydosporla) + Humicola・
7 score tiger IFO9530 (, Humlcola
fuacoatra) + Di3 Tomomyces 0 Sejupi IFO9929
Jp) I Neo 7 Smospora africana IFO759
0 (Neocoa + nospora afrlean &
) +Xilbon Sufzp Spora IFO9516 (X
ylogone gphaeroapora)',
) Rumises laguna IFO30008, Chelavia himetei IFO30419 (Th1elavla f
lmetl ) is preferred. All of these microorganisms are available from public microorganism repositories (IFO or NRRL).

ML-236B and Monacolin K were added to ML-23, respectively.
To convert to 6A and Monacolin J, not only live microbial cells, but also killed microbial cells (e.g., acetone-dried cells), enzymes extracted from microorganisms (e.g., esterase), and immobilized cells and enzymes are converted into ML-236B and monacolin J. This is achieved by contacting with Monacolin. In this case, M
It is preferable to use L-236B and monacolin as an alkali metal salt with a cleaved lactone ring.

Depending on the conditions, ML-236A and Monacolin J are mostly produced as alkali metal salts or free acids with cleaved lactone rings. The method of contact is not limited to 20%, but
Preferably it is carried out at -40°C.

From the culture solution or reaction solution obtained in this way, the target product ML is obtained by solvent extraction, various μmography, crystallization operations, etc.
-236A and monacolin J can be isolated.

Next, examples will be shown.

Example 1 1 glutoose, 0.2% peptone, meat extract ol
H. Sakaro flasks (2 bottles) containing 100-ml medium consisting of 0.1% yeast extract and 0.3% IC8L were inoculated with Emelysella languis IF08087 and incubated at 25°C for 2 hours.
Cultured with shaking for 1 day. After that, the Na salt of ML-236B (
(lactone ring cleavage) was added to each flask.
In addition, shaking was continued at 25° C. for 20 hours. This is K
More than 90% of ML-236B is ML-236A.
Converted to . Next, the culture filtrate was obtained, adjusted to pH 3 with trifluoroacetic acid, and then diluted with ethyl acetate of about 300-236 mm.
A was extracted. After dehydrating the extract to dryness, the dried product was IO
The solution was dissolved in +d of ethyl acetate and washed with 11 um solution of 5≠l carbonate. The ethyl acetate layer was dried and then crystallized in aqueous ethanol to obtain ML-236A 6ft. Various physical properties of the obtained standard (UV absorption + IRE?
+NMR spectrum, -q soot vector, etc.) were consistent with the known physical properties of ML-2361.

Example 2 Emericera languis IF08 under the same conditions as Example 1
087 was grown, and 50 μ of Na salt from monacolin was added to each of two Sakaro flasks. Thereafter, the same operation as in Example 1 was carried out to obtain 62 mg of Monacolin J crystals. The pickle properties of the obtained sample matched those of the known Monacolin J.

Example 3 In a Sakalo flask containing the same medium as in Example 1, Mortillera.
Isabelina IFO 7884, Jihete μspora thalamidospoli 7 IFQ 9249, Humicola fusuf 7
) 5 IFO9530, Shichotomomyces sedupi I
FO9929, Neocosmos Bora africana IFO7
590゜Xyvon Spheluspora IFO9516,
) Rumyces laguna rFo 30008 and Chelavia himetei IF030419 were each inoculated and cultured at 25° C. for 3 days with shaking (2 Sakaro flasks for each strain).

Next, 50 μ of ML-236B (lactone type) was added to 10,000 of the two Sakaro flasks in which each strain was cultured.
In addition, 50 monacolin K (lactone type) was added to the other tube, and the culture was continued at 25° C. for 2 days with shaking. After completion of the culture, the contents of each flask were collected to obtain a filtrate, adjusted to pH 3 with trifluoroacetic acid, and extracted with 100 m/ml of ethyl acetate. The extract was dehydrated to dryness, the resulting dry matter was dissolved in methanol, and subjected to a rapid liquid chromatography method (manufactured by JASCO Corporation).
Silica ODS column) K shadow, generated ML-236
A or monacolin K was fractionated. The respective yields (ki) from one Sakaro flask were as shown in Table 1.

Example 4 Into a Sakalo flask containing the same medium as in Example 1, Emericella
Languis IF08087 was inoculated and cultured at 25°C for 2 days with shaking. The cells were cooled by filtration, thoroughly dehydrated, and then added to acetone that had been previously cooled to -30°C. After stirring gently, collect the bacterial cells by filtration, and leave for another 70 minutes.
After washing with setone, the cells were vacuum dried to obtain 7 setone-dried cells.

50mM phosphate potassium 7- (pH 8,0) jO
7 setsone dried bacterial cells 1° in each of 2 flasks containing yd.
In addition, one of the two bottles contains ML-236BNa.
5~ of salt, and Monacolin K Na iX for the other one.
519 was added to the mixture, and the mixture was gently shaken at 30° C. for 20 hours.

Hereinafter, the same operation as in Example 3 was performed to obtain ML-236A4.
2 kg and 3.6 N of Monafrin J were obtained.

Example 5 The 7setone dried bacterial cells 19 obtained in Example 4 were added to 0.2M phosphate potassium buff 7- (pH 8,0) 40ml 1lfC
The mixture was stirred and subjected to ultrasonic ripening for 5 minutes, and then extracted at 4°C for 20 hours with occasional stirring. Prepare two test tubes containing the transparent extract obtained by filtration and 5 ml of 50 mM potassium phosphate (pH 8,0).
ML-236B Nm salt was added to one side, and monacolin K Na salt was added to the other side for 5 hours, and the mixture was allowed to stand at 30°C for 5 hours.

After that, trifluoroacetic acid was added to the reaction solution to adjust the pH to 3,
The extraction operation was then carried out in the same manner as in Example 3.

High-speed liquid printer ML-236A4
, 5 ~ and Monacolin J 3.8■ were obtained, respectively.

Applicant: Akira Endo

Claims (1)

[Scope of Claims] 1. ``Mortinula sp., Emericella j1. ML-236B and Monacolin K were obtained using molds belonging to the genus Dihetepspora, 7 Mycolas, Dichotomomyces, Neocosmospora, Xygo/, Torpices, and Cherabia.
A method for producing ML-236A and monacolin J, comprising producing ML-236A and monacolin J, respectively. 2. Molds Mortillera Isabelina, Emerisella fistulae, Jihete p sborarilamidospori 7. Fumicola・
The physiologically active substance ML-236 according to Claim 1 C, which is Fuscoatla, Dichotomyces senupi, Neocosmospora africana, Xylvon sfuerspora, Torcx Myces laguna, Chelavia hymetei, or a variant or mutant strain thereof -B- and a method for producing Monacolin J.