JPH0419240B2 - - Google Patents

Description

[Detailed description of the invention]

(Field of Industrial Application) The present invention relates to a novel physiologically active peptide that exhibits enzymatic inhibitory activity against cysteine proteases such as papain and calpain, and its production method and uses. (Structure of the Invention) The bioactive peptide of the present invention has the following formula: (In the formula, R represents a hydrogen atom or a hydroxyl group.) The compound of the formula of the present invention takes the aldehyde structure of the formula in anhydrous dimethyl sulfoxide solution, but
In aqueous solution, it exists as a hydrate structure of the following formula. (In the formula, Val represents a valine residue.) Any of the above structures is also included within the scope of the present invention. The compound of the formula of the present invention is derived from Staphylococcus tanavensis, which the present inventors isolated from the soil of Tanabe City, Wakayama Prefecture.
tanabensis) SAM0019 [FERM P-7891]. These compounds have the formula R
Those in which R represents a hydrogen atom were named Stacopin P-1, and those in which R represents a hydroxyl group were named Stacopin P-2 by the present inventors. Staphylococcus tanavensis SAM−
0019 has the following mycological characteristics: Morphological findings (cultured on broth agar medium, 37°C for 48 hours) Morphology: Coccus Size: Diameter 1-1.5 μm Pleomorphism: None Motility: None Flagella: None Spores Presence or absence: None Aggregation form: Often singly or in pairs, sometimes in quadruple forms. In rare cases, a sextuplet pattern is observed. Acid-fastness: Negative culture findings (cultured at 37°C for 48 hours) Broth agar plate: Colonies are flat and circular, the periphery is completely green, milky white, shiny, 1-3 mm in diameter. Juicy agar slope: milky white colonies, good growth. Meat juice liquid: Slightly cloudy, white sediment Meat juice gelatin puncture medium: Grows on the surface, does not liquefy gelatin. Physiological properties Nitrate reduction: + Denitrification salt reduction: + MR test: + VP test: + Indole formation: - Hydrogen sulfide formation: - Starch hydrolysis: - Citric acid utilization KOSER medium: + CHRISTENSEN medium :+ Nitrate usage: ± Ammonium salt usage: + Pigment production: – Urease: + Oxidase: – Catalase: + Growth range: PH5-9 Temperature 15-46°C Temperature for enzymes: Facultative anaerobic O-F test :F Generation of acid and gas from sugars

[Table] Characteristics of the new species Coagulase: - Sodium chloride tolerance: Grows on 15% sodium chloride Alkaline phosphatase: + DNA G+C content: 35.7 mol% Quinone system: MK-7 (Major component) MK-
6 & MK-8 (Minor component) Novebiocin resistance (MIC≧1.6μg/ml): - Lysostaphin (MIC≧100μg/ml): + Production of acid from sugar on Purple Agar Base medium: Sugar Acid Production D-(+)-cellobiose - D-(+)-xylose - Seuucrose - Maltose + α-lactose + D-(+)-trehalose + D-(+)-meletitose - D-manitol + xylitol - (Note) The base composition of DNA is measured by the method of Tamaoka and Komagata (Japan Agricultural Chemistry Society 1984 Abstracts, p. 68, 1984), and the measurement of quinones is by the method of Yamada and Kuraishi (Experimental method for chemical classification of microorganisms). Novobiocin and
The resistance test for lysostaphin was conducted in the Revised 5th Edition of the Bacteriology Practice Handbook [edited by the Institute of Medical Science Alumni Association, Maruzen,
Tokyo, 1976), the production of acids from sugars on a purple agar-based medium was performed using the method of Kloss & Schleifer [Journal of Clinical Microbiology (Journal of Clinical Microbiology, 1976).
Microbiology) Volume 1, pages 82-88, 1975]
Each was conducted in accordance with the following. According to the above-mentioned mycological properties, the taxonomic status of the strain of the present invention (SAM-0019) was determined based on "Vergey's Manual of Determining Bacteriology" (8th edition, 1974). , Gram-positive, catalase-positive, coccus, and the G+C content of the DNA was 35.7 mol%, so this strain was identified as belonging to the genus Staphylococcus. Therefore, “The Prokaryots A Handbook
on Habitats, Isolation, and Identification of
Bacterio” (MPStarr, H. Stolp, HG
Truper, A. Baloms. and H.G. Sehlegel, eds.
Kloos, W. in Springer−Verlag.Berlin.1981).
“The Genus” by E. and K. H. Schleifer
Comparing the known species of the genus Staphylococcus and mycological properties based on the ``Staphylococcus'',
This strain is negative for coagulase and has no resistance to Novobiocin (MIC 1.6 μg/ml), but is resistant to Lysostaphin.
(MIC 100 μg/ml) Because of its resistance, Staphylococcus epidermidis (S.
It is considered to be included in the bacterial group (e.g. epidermidis). This group of bacteria includes S. epidermidis,
S. capitis, S. hominis
and S. haemolyticus 4
Contains seeds. Comparing the mycological properties of the strain of the present invention with these four species, this strain differs from S. epidermidis in that it produces acid from trehalose and mannitol on a purple agar-based medium, but does not produce acid from sucrose. , 15% NaCl
They differ in that they can grow on top. It differs from S. capitis in that it produces acid from maltose, lactose, and trehalose in purple agar-based medium, but not from sucrose. S.
It differs from Hominis in that it produces more acid than mannitol but not more than sucrose, and it also has alkaline phosphatase and can grow on 15% NaCl. Based on the above, it was concluded that it is appropriate that the strain of the present invention is a new species of the genus Staphylococcus. Therefore, the bacterial strain of the present invention (SAM-0019 strain)
named it Staphylococcus tanabensis. It is believed that production of Statucopine P-1 and P-2 can be easily carried out by those skilled in the art by known peptide synthesis methods, and products obtained by such synthesis methods are also included in the present invention, An advantageous production method is to culture bacteria capable of producing a compound of the formula belonging to Staphylococcus in an appropriate medium, and then isolate and purify the resulting culture. The medium used when producing the substance of the present invention is
Although it may be in liquid or solid form, shaking culture or aerated agitation culture using a liquid medium is usually convenient. Any medium may be used as long as the microorganisms producing the substance of the present invention grow and the substance of the present invention is accumulated in the medium. That is, as a carbon source, for example, glucose,
Lactose, glycerin, starch, sucrose, dextrin, molasses, organic acids, etc. Nitrogen sources include protein hydrolysates such as peptone, casamino acids, meat extract, yeast extract, soybean phase, corn staple liquor, amino acids. , ammonium salts, nitrates, and various other organic or inorganic nitrogen compounds are used. Various phosphates, magnesium sulfate, and sodium chloride may be added as inorganic salts, and vitamins, nucleic acid-related compounds, etc. may be added for the purpose of promoting bacterial growth. Note that adding an antifoaming agent such as silicone, polypropylene glycol derivative, soybean oil, etc. to the medium may be effective in increasing the amount of the present substance accumulated. When culturing, it is preferable to inoculate the culture medium with the culture obtained by performing small-scale preculture in advance, rather than carrying out main culture all at once. Conditions such as culture temperature, culture engine, culture liquid, etc. are appropriately selected and adjusted so as to maximize the amount of accumulation of the substance of the present invention. °C, 3
~7 days of culture is sufficient, and the liquid pH of the medium is PH4 ~
It is best to keep it at 9.5. By culturing in this manner, the substance of the present invention is produced and accumulated in the culture. When culturing using a liquid medium, the target substance is mainly accumulated in the liquid part, so it is separated from the liquid or supernatant after the culture has been filtered or centrifuged to remove bacterial cells. However, if necessary, it is also possible to separate the target product directly from the culture solution without removing the bacterial cells. Various means based on the chemical properties of the substance of the present invention may be employed to separate and purify the target product from the culture. That is, for example, precipitation by adding a precipitant such as ammonium sulfate, extraction with an organic solvent such as n-butanol that is not arbitrarily mixed with water and can dissolve the substance of the present invention, and highly polar solvent such as methanol or ethanol. removal of impurities by treatment with hexane, gel filtration with ion exchange resins, ion exchange cellulose, ion exchange chromatography with various ion exchangers such as ion exchange cellulose, activated carbon, alumina,
Adsorption chromatography using adsorbents such as silica gel, Amberlite isolated. However, methods other than these may be used as appropriate as long as they effectively utilize the properties of the substance of the present invention. Particularly preferred adsorbents include Diaion HP-20, DEAE-
Examples include a combination of cellulose, CM-cellulose and Cephadex LH-20. When purified by chromatography using an adsorbent, the substances of the present invention, statucopine P-1 and P-2, have slightly different adsorption properties depending on the presence or absence of hydroxyl groups, and statucopine P-2, which has a polar hydroxyl group,
2 is eluted slightly later. Based on this property, both can be easily separated by, for example, DEAE-cellulose column chromatography, but after separation of the two, further purification can be performed by substantially the same operation. Statucopine P-1 and P-2 of the present invention are
It exhibited inhibitory activity against cysteine proteases such as papain and calpain, and was confirmed to inhibit the decomposition of denatured casein by these enzymes, for example, as shown in the Examples below. (Example) Statucopine P-1 by culturing SAM-0019
Production of P-2 SAM-0019 in a synthetic medium (YM medium) 8 (PH5.8) consisting of grapes, peptides, and yeast extract
A pure culture of the strain was inoculated and cultured with aeration for 48 hours at 30°C in a small jar at an aeration rate of 10/min and 200 revolutions/min. Centrifuge the culture and transfer the supernatant 8 to Diaion HP
-20 (manufactured by Mitsubishi Chemical Industries) column (100 x 500 mm)
was adsorbed to. This column was washed with 10 parts of water and 10 parts of 10% methanol and then eluted with 5 parts of methanol to elute the anti-cysteine protease active fraction. After concentrating the eluate under reduced pressure and drying it, it was dissolved in 500 ml of water to form the OH form.
It was adsorbed onto a column (60 mm x 200 mm) packed with DEAE cellulose (Watmann DE23). This was eluted with water 1, and as shown in FIG. 1, it was fractionated into statucopine P-1, which eluted first, and statucopine P-2, which eluted later. Each fraction was concentrated separately and adjusted to form H.
CM cellulose (Brown) column (40 mm)
×300mm). After washing this column with 500 ml of water, it was eluted with a concentration gradient (total of 1000 ml) of O-60 mM ammonium acetate buffer (PH 5.8).
Purification was carried out. Elution results of statucopine P-
1 is shown in FIG. 2, and P-2 is shown in FIG. 3, respectively. The enzyme inhibitory activity fractions indicated by white circles in Figures 2 and 3 were collected and concentrated under reduced pressure, and the dried residue was dissolved in 10 ml of a mixed solution of 50% methanol:acetic acid (200:3) and columned on a Sephadex LH-20 column. (30mm×1400
mm) and eluted using the same solvent to recover pure statucopine P-1 and P-2, respectively.
Figures 4 and 5 show the elution patterns of stuccopine P-1 and P-2, respectively. By the above method, the substance statucopine P of the present invention can be obtained.
15 mg of P-1 and 30 mg of Statucopine P-2 were obtained as pure colorless amorphous substances. The analysis results of the obtained stuccopine are shown below.

【table】

【table】

[Table] Amino acid analysis Statucopine P-1 (2μg), Statucopine P
-2 (3 μg) was separately dissolved in 0.1 ml of 6N HCl, vacuum-sealed, and then hydrolyzed at 105° C. for 48 hours. After hydrolysis, dry under vacuum and each
0.3 ml of 0.02N HCl was added and dissolved, and 0.225 ml of the solution was subjected to amino acid analysis, and the following analysis results were obtained. Amino acid analysis results

[Table] Calculated numbers when .
From the above analysis results, statucopine P-1 and P
The structure of -2 was determined to be the above formula. Physiological Activity of Statucopine The enzyme inhibitory activity of Statucopine, a substance of the present invention, was investigated using the following measurement method. The substance of the present invention and the enzyme solution are mixed and pre-incubated at 30°C for 5 minutes, and then the substrate solution is mixed to start the reaction. 0.5% casein was used as a substrate, and 5mM calcium chloride and 10mM
Cysteine and 50mM Tris-HCl buffer (PH
7.5) and react at 30℃ for 30 minutes. Next, 6.5% trichloroacetic acid was added to the reaction solution to stop the reaction, and the amount of protein in the trichloroacetic acid soluble fraction of enzyme-hydrolyzed casein was measured by the Lowry-Folin method.
Determine the inhibitory ability by comparing with the control solution. As a result of the above test, 0.19μg of this substance is 1.25μg
papain activity was inhibited by more than 50%. Similarly, 1 μg each for calpain and calpain
It showed more than 50% activity inhibition. The results are shown in the following table as the amount of statucopine that inhibits the activity of 1.25 μg of papain or calpain or 1 μg of each by 50%.

[Table] (Effects of the Invention) Since the substance represented by the formula of the present invention has cysteine protease activity, it can be used as a therapeutic agent for inflammation, cataracts, epidermolysis bullosa, and pemphigus, or as an anti-inflammatory agent using the present compound as a ligand. The preparation of the inity column is expected to be useful for purposes such as purification of cysteine proteinase and as a reagent for research on enzyme reaction mechanisms.

[Brief explanation of drawings]

Figure 1 shows the production of stuccopine in an example.
FIG. 2 is a graph showing the elution pattern of DEAE-cellulose column chromatography, and FIGS. 2 and 3 are graphs showing the elution pattern of CM-cellulose column chromatography of stutucopine P-1 and P-2 in the production of stutucopine in the example. FIGS. 4 and 5 are graphs showing the elution patterns of Statucopine P-1 and P-2 in Sephadex LH-20 column chromatography, respectively, and FIGS. The figure shows statucopine P-1 and P
Figures 8 and 9 are IR spectra of Statucopine P-1 and P-2, respectively.
FIG. 1 is a ¹ H-NMR spectrum diagram.

Claims (1)

[Claims] Primary formula: (In the formula, R represents a hydrogen atom or a hydroxyl group.) A peptide compound represented by: 2. Culture bacteria that belong to the genus Staphylococcus and have the ability to produce a peptide represented by the following formula, and add the following formula to the culture solution: (In the formula, R represents a hydrogen atom or a hydroxyl group.) Producing and accumulating a peptide compound represented by
A method for producing at least one peptide compound represented by the above formula by purifying the culture solution. 3. The method according to claim 2, wherein Staphylococcus tanavensis SAM0019 [FERM P-7891] is used as the bacterium belonging to Staphylococcus. Quaternary formula: (In the formula, R represents a hydrogen atom or a hydroxyl group.) Using a peptide compound represented by
A method for inhibiting cysteine-protease activity in vitro.