JPS6339898A - Novel peptide mr-33-a substance and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula having following physicochemical properties; Elementary analysis (%): C 59.55, H 8.32, N 15.90; Molecular weight: 502.65; Molecular formula: C26H42O4N6; Melting point: 163-166 deg.C (decomposition); [alpha]<22>D = -30.5 + or - 1.5 deg. (c=1.105, CH3OH); Soluble in methanol, ethanol and dimethyl sulfoxide, and hardly soluble in water, acetone, ethyl acetate, ether, chloroform and petrolum ether; Positive to the Sakaguchi reaction, Rydon-Smith reaction and 2,3,5-triphenyltetrazolium chloride and negative to ninhydrin reaction, etc. USE:A protease activity inhibitor. PREPARATION:A bacterial strain belonging to Genus of Streptomyces such as Streptomyces.Sp-AK-33 strain (FERM P-885), etc. is cultured, preferably under an aerobic condition at 28 deg.C for 2-4 days.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel peptide and a method for producing the same. More specifically, formula (1) (wherein I5ovaltr)l in the above formula represents an invaleryl group, and Phe and Ilt represent phenylalanine and inleucine residues, respectively. ) (MR-33-A substance) and a microorganism belonging to the genus Streptomyces isolated by the present inventors are cultured, and MR-33-A substance is collected from the culture.
-35-Relating to a method for producing a substance.

The present inventors focused on the important role of proteases in biological control mechanisms, and as a result of searching for new protease inhibitors, a microorganism similar to Streptomyces tora produces a substance that strongly inhibits protease activity. After discovering the fact that this occurs and isolating the active ingredient from the culture solution, it was confirmed that this substance is a novel peptide represented by the above formula (1). An example of MR-33-A substance-producing bacteria is AK-33, which was newly isolated from Tottori soil.
There are stocks.

The mycological properties of this strain are as follows.

1. Morphology Under a microscope, aerial hyphae elongate better than well-branched substratum hyphae, and their tips become spiral-shaped. Whorl branching is not recognized.

The morphology of the spore surface observed with an electron microscope is smooth. The spores are cylindrical and the size is 0.5~G, 8x08~1,
The diameter is 6 microns, and chains of 10 or more spores are usually observed.

2. Growth status on various media The properties on various agar media are as follows.

The culture temperature was 28°C, and observations were made after 21 days of culture.

The color tones are based on Shibefu Color Chart A (Japan Color Research Institute).

(Note) G: Growth A: Aerial mycelium; Back side S: Soluble pigment 3, physiological properties (1) Growth temperature range: 15°C-37°C (2) Liquefaction of gelatin: # quality (3) Starch Hydrolysis: Positive (weak) (4) Coagulation of skim milk: Negative Peptonization: Positive (5) Melanin-like pigment tyrosine agar: Positive Peptone/yeast/iron agar: Positive 4 Carbon source availability (Pridham-Gotlieb agar medium , 25°C culture) (1) D-glucose, D-xylose, D-fructose to be used,
D-galactose, ino/tol, D-mannitol (2) Unused L-arabinose, L-rhamnose, raffinose, nucrose, SuIJshin 5 Summary of properties of diaminopininophosphate LL type and above in whole cell hydrolyzate Then, the AK-33 strain belongs to the genus Streptomyces, the tips of the aerial hyphae are spiral and form chains of 10 or more spores, and the spore surface is smooth. The color of the aerial mycelium is white to gray at the beginning, and becomes yellowish to greenish as it matures. It produces yellow to brown soluble pigments on various media, and has a strong ability to produce melanin-like pigments on tyrosine agar, peptone, yeast, and iron base.

The present inventors used Streptomyces sp. AK-33 (Slreptampces S/
IAK-33).

This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession number 8851.

[Production method of the substance of the present invention]

The substance of the present invention is produced by Streptomyces sp. AK.
-33 strain is cultured and collected from the culture. The culture method is carried out according to the culture method of general microorganisms, but deep culture method using a liquid medium is usually advantageous. Cultivation is carried out under aerobic conditions, and the temperature can be changed as appropriate as long as the Strep+-Mrs. sp. AK-33 strain grows and produces the substance of the present invention, but it is generally carried out at around 28°C. .

As the medium, ordinary raw materials used for culturing actinomycetes are used, but for the production of hiR-33A substance, L-
It is good to add an appropriate amount of yin loin.

Although the culture time varies depending on the composition of the medium and temperature conditions, in most cases, the amount of production and accumulation of the substance of the present invention is maximized after 2 to 4 days of culture under aerobic conditions.

The substance of the present invention is mainly accumulated during the night of culture. Various methods can be used to isolate and purify a target substance from a culture, depending on the chemical properties of the target substance.

For example, an appropriate combination of extraction with a solvent such as n-butanol, ion exchange chromatography using an ion exchanger such as ion exchange resin or ion exchange Sephadex, and reversed phase chromatography using silica gel bonded with an alkyl group, etc. When used, the substance of the invention is isolated in powder form.

[Physicochemical properties of the substance of the present invention]

1. Elemental analysis value: CUN Actual value 59,55 8.32 15.90 Theoretical value 5
9.97 8.52 16.14 (C26H4204N
6.L (as 20)2 Molecular weight: 502.65 (calculated value, as C26H4204N6)6 Melting point:
163-166°C (decomposition) 4, specific rotation: Ca
"JD22-30. c; + 1.5 (C = 1.1
05 in CH30H) 5, UV absorption spectrum: 250, 257 and 262M! The absorption specific to 7-enylalanine is improved.

6. Infrared absorption spectrum; KJBr-1゜ν m, 3300-3250 (NH)ax 2960 (CHand CH2) 1640 (amide/l, yuanidium)
154 Q (amide l) 1450 C1)htn&! ) 700 (phlntl) 7 Solubility in various solvents: Soluble in methanol, ethanol, and dimethyl sulfoxide, slightly soluble in water, acetone, ethyl acetate, ether, chloroform, and petroleum ether.

8 Color reaction: Sakaguchi reaction, Rpdon-Sm1th reaction, and 2, 5
．． positive for 5-triphenyltetrazolium chloride;
The ninhydrin reaction is negative.

9. Components: Acid decomposition (6N HCjl, 110°C) of this substance.
124 hours), and the decomposed product was analyzed by 7L gel thin layer chromatography, and phenylalanine and inroinine were detected. However, when the acid decomposition product was extracted with ether and the ether layer was analyzed by gas chromatography, it was found that 1 sovalerit:
Acid was detected.

Furthermore, when this substance was oxidized using K Mn 04 and the oxide was similarly analyzed for amino acids, phenylalanine, isoleucine, and arginine were detected.

This substance Oxide Phenylalanine 0.95 0.91 Isoleucine 1, DO 1,00 Arginine 1.04 Amino acid sequence: Dissolve the oxide of this substance in 0.5 yytl of Tris-HCl buffer (pH 8,0), and add it to the tarbozp lye /
ytidaseA0.5μmi' and carboscp
/ye door tidase B 5 pf added and 25°
The reaction solution was collected at fixed time intervals and analyzed by glue gel thin layer chromatography. When phenol water (3:1) was used as a developing solvent and color was developed with a ninhydrin reagent, arginine was detected first, followed by the release of isoleucine. Note that phenylalanine was not detected.

From the above results, the chemical structural formula of the substance of the present invention was determined to be as follows.

T [Physiological activity of the substance of the present invention] The substance of the present invention and the substrate solution are mixed and pre-incubated at 67°C for 5 minutes, and then the enzyme solution is mixed to start the reaction. 0.2% casein is used as a substrate, and 4 mM protein and 50% boric acid-sodium hydroxide buffer are added, and the reaction is allowed to proceed at 37°C for 30 minutes. Next, 0.17 M perchloric acid was added to the reaction solution to stop the reaction, and after centrifugation, 28071? The absorbance at ff is measured and the inhibition rate is determined from comparison with the control solution.

Results: 0.3μg of this substance inhibited the papain activity of 5μ2 by 50%, and similarly, 4.2μ2 of this substance inhibited 511? It inhibited the tributane activity of 50%.

[Example of production of the substance of the present invention]

Synthetic medium 2500ysl consisting of malt extract, yeast extract, glucose and L-inleucine (PI(7,4)
was inoculated with a pure culture of AK-33 strain and incubated at 30°C for 72 hours. The culture was filtered. The filtrate was adjusted to pH 9.0 with sodium hydroxide, and then transferred to a DOWEX 1×4 column ( CI type) (75 x 120 mm) was passed through, and impurities were removed by adsorption onto the column. The non-adsorbed fraction was dried under reduced pressure for 500 min.
Concentrate to shoulder level and add 1-butanol (50C1+tX3)
Extraction was performed using

This 1-furinol layer was concentrated and dried under reduced pressure, and then added to 0.01 M IJ phosphate buffer (pH 8,0).
40C]+t, CM-8ephadesc C
-25 (Na type) column (≠60
M) was eluted. This eluate was concentrated under reduced pressure to 100 ml, filtered using an ODS column ($10X400), and then refilled with a LiChro RP-8 column (
φ10x240m) and 100m of water? The active substance was eluted and fractionated with 80% methanol. The active fractions were combined, concentrated under reduced pressure and dried, and methanol-
By precipitation from ether, almost pure substances of the invention 8~ were obtained as white amorphous substances. Effects of the Invention The Δ1R-33-A substance of the invention strongly inhibits grotease activity. In general, proteases are closely related to various pathological phenomena such as inflammation, carcinogenesis, cancer metastasis, and muscular dystrophy, and the substances of the present invention are effective in analyzing these pathological phenomena and are expected to be therapeutic agents for diseases. It is.

Further, applications in the fermentation industry, such as use as an active group in affinity chromatography to purify protease, are also considered.

(Patent applicant Maruishi Pharmaceutical Co., Ltd.) (Agent: Patent attorney Yasu Kasuya)

Claims (1)

[Claims] 1. MR-33-A substance represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼. (However, Isovaleryl in the above formula represents an isovaleryl group, and Phe and Ile represent phenylalanine and isoleucine residues, respectively.) 2. Cultivate a strain belonging to the genus Streptomyces that has the ability to produce MR-33-A substance. , MR-33-A from the culture solution
Novel peptide MR-3 characterized by collecting substances
3-Method for producing substance A. 3. The production method according to claim 2, wherein the MR-33-A substance-producing bacterium is Streptomyces sp. AK-33 strain.