JPS5995253A - Peptide having acyl group, its preparation by microorganism and inhibitor against cyclic adenosine 3',5'-monophosphate phosphodiesterase consisting of the same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a peptide having an acyl group represented by the general formula, a method for producing the same using a microorganism, and a cyclic adenosine 3',5'-
The present invention relates to a monophosphate phosphodiesterase (hereinafter abbreviated as PDE) inhibitor.

Cyclic adenosine 3',5'-monophosphate (cAMP)
It is abbreviated as. ) has now been revealed to be the second transmitter of almost all other hormones except steroid hormones, and plays an important role in metabolic control in living organisms. Therefore, inhibiting this cAMP-degrading enzyme, PDE, can reduce intracellular cAMP.
The goal is to control the levels, and various physiological effects are expected. For example, it has the potential to improve sugar metabolism, cardiac inotropy, smooth muscle relaxation, bronchodilation, coronary artery dilation, lipid metabolism, mental stabilization, body fluid secretion promoting, and hormone secretion promoting effects.

In addition, dibutyryladenosine 3, a derivative of cAMP,
It is known that ',5'-cyclic phosphoric acid suppresses the proliferation and canceration of cancer cells in cultured cells. (Proteins, Nucleic Acids, Enzymes, Vol. 18, p. 1195, 1973).

This suggests that PDE inhibitors may have anticancer effects.

Furthermore, it has been revealed that cAMP content is low in the blood vessels of hypertensive rats (Science, Vol. 179, p. 807, 1973).
It is expected that inhibitors of cAMP is also involved in the expression of allergies, and is thought to be effective in anti-allergy and prevention of asthma. As described above, PDE inhibitors are fully expected to be used as medicines in a wide range of fields.

Conventionally, known PDE inhibitors include organic synthetic products such as methylxanthines, papaverine, quinazoline derivatives, dehydrocaffeic acid dilactone produced by microorganisms, phenazine derivatives, PDE-I, PDE-II, etc. PD caused by peptidic substances such as the inhibitor of the present invention
There are no reports on the inhibitory effect of E, and it is expected to be used as a medicine or research reagent. The method for producing the substance of the present invention is carried out by the inventors of the present invention cultivating new Bacillus bacteria from soil in a conventional aerobic manner to produce them in the culture filtrate, which is then recovered.

The substance of the present invention produced in the culture filtrate is a peptide having an acyl group represented by the above general formula, and its physicochemical properties are as follows.

The sequence order of the constituent amino acids in the fragment of the molecular ion appearing in the mass spectrometry spectrum of the permethylated compound is Glu, Leu, Leu, Val, A from the N-terminus.
sp, Leu, and Leu. In addition, the presence of lactones expressed in the infrared absorption spectrum is β-
It was confirmed by chemical analysis that the hydroxylated fatty acid and the C-terminal amino acid Leu were bonded, and the entire structure of the substance of the present invention was confirmed to be as described above.

Next, as a microorganism that produces the substance of the present invention, for example, a Bacillus bacterium newly isolated by the present inventors can be exemplified, and the mycological properties of this strain are as follows.

Mycological properties Gram-positive aerobic bacillus with periflagellated motility.
Forms spores.

The growth state on the broth agar medium is medium, butter-like pale yellow-brown colonies are formed, and the colony surface is dull and shiny on the wrinkles, and no change in the medium is observed.

Growth test 7% sodium chloride medium
+ Sabouraud dextrose medium
+Azide medium
-Anaerobic culture medium
−Growth temperature
52-15℃ Production/decomposition test Pigment production (glucose, tyrosine)
- Production of acetoin
+Acid production (glucose, arabinose xylose, mannitol) +Starch degradation +Citrate assimilation
+ Synthesis of propionate
-Lysozyme resistance
+ Generation of indole – Reduction of nitrate
+ Decomposition of casein
+ Milk hydrolysis
+ Liquefaction of gelatin
+ Tyrosine decomposition
-degradation of hippurate
+Egg yolk reaction
- Catalase test
As a result of the above morphological and physiological properties, this strain is Bergey'
s Manual of Determinative
Bacteriology (8th edition) and The G
As a result of searching for enus Bacillus, there is no match in that it decomposes hippurate, but Bacillus s
It seems most appropriate to identify it as P. ubtilis.

In addition, this strain is Bacillus subtilis
C-756, which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 6785.

Example 1 An example of the production method and separation and purification of the inhibitor of the present invention will be described.

Bacillus subtilis C- in a medium consisting of 1% glucose, 1% peptone, 0.3% yeast extract, 0.3% sodium chloride, 0.1% magnesium sulfate, and 0.1% dipotassium phosphate (pH 6.8). 756 (Feikoken Bibori No. 6785) was cultured at 30°C for 2 to 3 days, bacteria were removed, and an inhibitor was obtained from the resulting culture filtrate. When adding concentrated hydrochloric acid to 40 liters of culture filtrate to a pH of 2, or adding copper sulfate to a final concentration of 0.6%, the inhibitor will precipitate, which will be collected and extracted several times with ethyl acetate. . The ethyl acetate extracted fraction was washed with aqueous sodium bicarbonate, dehydrated over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain about 14 g of crude extract. Next, add this to a Sephadex G-50 column (
Effluent with 80mM Tris-HCl buffer (pH 7.5))
, silica gel column (chloroform-methanol, 8:
1) to obtain 8 g of active fraction. Further purification was performed sequentially using a Sephadex LH-20 column (elution with acetone) and a silica gel column (elution with chloroform-methanol, 5:1) to obtain about 3 g of colorless amorphous powder. This showed a single spot on a thin layer using various solvents, but it was further separated and purified by high performance liquid chromatography equipped with an octadecylated silica gel column, and the inhibitor of the present invention was separated and recovered.

Example 2 The following experiment was conducted to measure the PDE inhibitory activity of the substance of the present invention.

Add 40mM Tris-HCl buffer (p
H7.5), 2mM magnesium sulfate, 0.5mM c
AMP, PDE (140 μg-protein amount, manufactured by Boehringer Mannheim), alkaline phosphatase (70 μg-
Protein amount, manufactured by Boehringer Mannheim) and an inhibitor were added and allowed to react for 0 minutes at 38° C. After stopping the reaction with trichloroacetic acid, the amount of phosphorus liberated from cAMP by the enzymatic reaction was measured. It was made active. A plurality of such experiments were conducted, and the inhibition rate was calculated using the following formula.

Inhibition rate = (A-B)/A x 100 (%) A: Amount of phosphorus when no inhibitor is included B: Amount of phosphorus when an inhibitor is added and inhibitor concentration Ic50 when inhibition rate is 50% The result was 8.1×105M.

Example 3 The following chemical analysis experiment was conducted to determine the β-hydroxylated fatty acid and amino acid forming the lactone ring of the present invention.

After reducing the inhibitor with lithium borohydride by three methods, hydrochloric acid hydrolysis was performed, and the burnout of amino acids was examined.

Lithium borohydride can reduce esters and lactones, but cannot reduce carboxylic acids, so (1) Direct reduction without pretreatment.

(2) After the inhibitor is methylated, it is reduced.

(3) After opening the lactone ring of the inhibitor with an alkali, it is reduced.

After the above three types of reduction, the amino acid composition is (1) Glu
:Asp:Val:Leu=1:1:1:3(2)Gl
u:Asp:Val:Leu=0:0:1:3(3)G
lu:Asp:Val:Leu=1:1:1:4, and this result proved that it was the C-terminal Leu that was bonded to the β-hydroxylated fatty acid.

Example 4 The structure of the constituent fatty acids of the substance of the present invention was determined according to the following rules.

In analysis by gas chromatography, when the horizontal axis is the carbon number of a substance and the logarithm of the retention time of that substance is plotted on the horizontal axis, a linear relationship is established between substances within the same series, and a linear relationship is established between substances in the same series, and there is also a linear relationship between substances in the same series. Now, these straight lines are parallel to each other (``
Methods of Biochemical Analysis, Volume 8”
1, 1960). Based on this rule, methyl esters of known β-hydroxylated fatty acids and fatty acids derived from the substance of the present invention were analyzed by gas chromatography under the same conditions, and the constituent fatty acids were determined to be β-hydroxylated antiisopentadecanoic acid. admitted.

4. Detailed explanation of the drawing The diagram shows an infrared absorption spectrum of the present inhibitor measured in a potassium bromide tablet, where the vertical axis shows the absorbance and the horizontal axis shows the wavelength.

Patent applicant: Director of the Agency of Industrial Science and Technology Makoto Ishizaka Designated representative: Yoshi Takahara, Director of Microbial Technology Research Institute, Agency of Industrial Science and Technology■

Claims (3)

[Claims] (1) General formula A peptide having an acyl group represented by (2) A strain belonging to the Bacillus family and capable of producing a peptide having an acyl group represented by the general formula is cultured in a general medium consisting of a carbon source, a nitrogen source, and an inorganic salt, and the peptide having the above acyl group is extracted from the culture. A method for producing a peptide having an acyl group, characterized by separating and collecting (3) Cyclic adenosine 3',5'-monophosphate phosphodiesterase inhibitor represented by the general formula