JPS581917B2 - Peptide noseizoho - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the general formula R-Val-Val-X-Ala-X (1) (wherein R is an aliphatic carboxylic acid, Val is valine, Ala is alanine, and X is Nocardia and Aspergillus species that can cleave the peptide bond between the two valine residues of acyl peptides represented by amino acids (each amino acid has a peptide bond, and carboxylic acid and valine have an amide bond) , Val-X-Ala-X (
II) (In the formula, Val, Ala, and X all have the same meanings as above, and each amino acid is peptide bonded.)
The present invention relates to a method for producing a novel peptide shown in

The acyl peptides represented by the formula (1) are powerful acidic protease inhibitors produced by microorganisms, and their ability to inhibit pepsin activity is particularly strong, and their toxicity is weak, so pepsin is thought to be one of the causes. It is expected to be developed as a preventive or therapeutic agent for gastric and duodenal ulcers.

The present inventors conducted various studies in order to produce other useful compounds using the acyl peptides (1) as raw materials.

As a result, certain microorganisms cleave the peptide bond between the two valine residues of the acyl peptides (1), and the above formula (
It was discovered that a novel peptide (II) represented by n) was produced.

It was also found that the peptide itself has vepsin inhibitory activity similar to (1), and can be used for the same purpose as (1).

The present invention was completed based on such knowledge and as a result of extensive research.

In the method of the present invention, acyl peptides belonging to the genus Nocardia, Aspergillus, Cephalosporium, Mucor, Microascus, or Emericelopsis that can cleave the peptide bond between two valine residues of the above-mentioned acyl peptides are used. Microorganisms can be used as appropriate, but examples of bacteria that are recognized to be particularly advantageous include Nocardia, Asteroides ATCC 3308 (IFO 3584)
, Aspergillus whetidius IFO4031, Aspergillus awamori IFO4122, Microascus desmosporus IFO7022, Microascus desmosporus IFO7022
Trigonosporus IFO7026, Emeriselopsis
Humicola IFO8518, Cephalosborium sclerodeigenum IFO8385, Cephalosborium
Collemioides IFO 8579, Mucor Ambigus IFO 6742, Mucor Betlinslaris IF
O6751, Mukor Silnoku Takes IFO675
3, Mucor Rufussence IFO 7102, Mucor Recurvus IF0 8093, etc.

The high production ability of such strains can be determined by assaying the peptide (It) produced by contacting the culture or cultured product with the acyl peptides (1), for example, by the casein-pepsin agar plate method described below. selected from among the strains that showed

That is, first, thin layer chromatography is performed using an appropriate solvent system selected from the various solvent systems listed below, and then the bath medium adhering to the thin layer is evaporated and a dry thin layer is obtained. .

On the other hand, an agar bath solution in which 3% agar was added in advance and dissolved by heating, and a 0.4% Hammerstein casein solution (pH 2).

0) in a ratio of 1=1, cooled to 50℃,
A crystalline pepsin solution is added in an amount of 2 to 100 solutions per 100 solutions of the mixed solution, stirred quickly, poured into a petri dish, and left to stand for a while to prepare a casein-pepsin agar plate with a thickness of 2 to 3 cm.

The dry thin layer obtained above was pasted onto this agar plate and kept in a cool place for about 10 minutes to allow the active substance in the thin layer to permeate onto the agar. After peeling off the thin layer, the agar plate was heated to 37°C. -Leave it overnight.

The agar plate becomes transparent due to the proteolytic action of pepsin, but if a substance that inhibits pepsin activity is present, the agar remains cloudy, making it easy to detect the peptide (It) that has the ability to inhibit pepsin activity.

The Rf values of the reaction product peptide (n) measured by thin layer chromatography in various solvent systems using this casein-pepsin agar plate method were as follows.

A silica gel thin layer manufactured by Merck & Co. (Product No. 5721, layer thickness 0.25 mm) was used as the tazo thin layer, and the development was carried out at room temperature.

Methanol 0.74, ethanol 0.16, n-propanol 0.11, n-butanol 0.02, water-saturated phenol 0.51, ethyl acetate 0, n-butanol-acetic acid monohydrate (4:1:2:0. 51, n-butanol-acetic acid monohydrate-n-butyl monoacetate (20:1:1:20) 0.03,
Ethyl acetate-methanol-acetic acid (80:20:1)0.
03, ethyl acetate-acetone-, methanol-acetic acid (6
0: 20: 20: 1) 0.09, chloroform-methanol-acetic acid (86:12:2) O, 04,
Benzene-acetone-methanolacetic acid (60:20:2
0:l) 0.12.

Peptide (II) on the thin layer can also be detected by ninhydrin reaction or Riddon-Smith reaction.

The method of the present invention is carried out by contacting a culture of such a microorganism or a treated product thereof with an acylbebutide (1) and converting it into a peptide (n).

The culture or its processed product referred to herein refers to the culture product itself obtained by culturing microorganisms in a medium, as well as the culture product itself, which is subjected to centrifugation, filtration, washing, drying, grinding, extraction, etc., in order to be advantageous for the reaction. Specimens containing an enzyme system involved in the reaction of hydrolyzing acyl peptides (1) to produce peptide (II), such as live bacterial cells, dried bacterial cells, and crushed bacterial cells obtained by appropriate treatment of refers to quality.

In culturing the microorganism, the medium may be liquid or solid, but shaking culture or aerated agitation culture using a liquid medium is usually convenient.

Any medium may be used as long as these microorganisms can grow and produce the enzyme system.

That is, carbon sources include, for example, glucose, lactose, glycerin, starch, sucrose, dextrin, molasses, organic acids, and hydrocarbons, and nitrogen sources include, for example, peptone, casamino acid (manufactured by Difco), N
-Z-Amine A (Protein hydrolyzate (manufactured by Sheffield), yeast extract, soybean meal, corn stave liquor, amino acids, various ammonium salts, various nitrates, and other organic or inorganic nitrogen compounds are used.

Various phosphates, magnesium sulfate, sodium chloride, etc. may be added as inorganic salts, and vitamins, nucleic acid-related compounds, etc. may be added for the purpose of promoting bacterial growth.

In addition, depending on the culture method and culture conditions, silicon,
Adding antifoaming agents such as polypropylene glycol derivatives, soybean oil, etc. to the culture medium may be effective in increasing the production of the enzyme system.

When culturing, it is desirable to inoculate the medium with a culture solution obtained by performing small-scale preculture in advance.

Culture conditions such as culture temperature, culture period, and medium liquid properties vary depending on the type of microorganism used and the composition of the medium, but ultimately they should be selected appropriately to maximize the production amount of the enzyme system. In many cases, it is preferable to culture the culture under aerobic conditions at about 20 to 40°C for about 1 to 7 days, and maintain the pH of the medium at about pH 4 to 8 during this period.

As starting material acyl peptides (1), aliphatic carboxylic acids represented by R having 2 to 19 carbon atoms are known.
, JP-A No. 47-29582, No. 47-43290, No. 4
8-99387, 49-26485, 49-415
90, Agricultural and Biological
Chemistry, vol. 35, pp. 1482-1487 (197
It can be produced by the method described in 1999), and any purified preparation, culture, culture P solution, partially purified preparation obtained by processing these, etc. can be used in the present invention.

Furthermore, in formulas (1) and (n), the amino acid represented by the terminal X may be a free acid or a salt thereof.

The contact between the acyl peptide (1) and the culture of the microorganism or its treated product is preferably carried out in a liquid medium, and such a medium is usually an aqueous medium.

It goes without saying that it is advantageous to increase the concentration of the acyl peptide (1) in the reaction system as high as possible, but it is usually advisable to add the acyl peptide (1) at a concentration of about 0.1 to 2%.

In some cases, acyl peptides (1) at regular intervals
can also be added separately.

The reaction may be carried out by standing still, shaking or stirring, and the reaction temperature is usually about 10 to 50°C.

The rate of progress of the reaction varies depending on the type of microorganism used, the amount of enzyme contained in the culture or its processed material, the type and concentration of (1), the mode of reaction, the reaction conditions, etc., so the reaction time is selected as appropriate.

After the reaction is completed, the peptide (n) produced in the reaction solution can be easily separated from the reaction solution by conventional separation means.

In other words, it is better to separate these from the liquid or supernatant liquid after removing solids by filtering or centrifuging the reaction liquid, but it is better to separate them from the liquid or supernatant liquid after removing solids from the reaction liquid. may be separated.

Separation and purification of the target product from the reaction solution can be easily carried out by appropriately combining various methods based on the chemical properties of peptide (II).

That is, for example, extraction with an organic solvent that does not mix with water and can dissolve the peptide (n) such as n-butanol, dissolution in a highly polar solvent such as methanol or ethanol, or treatment with ethyl acetate, hexane, etc. Removal of impurities, gel filtration with Sephadex,
Ion exchange chromatography using various ion exchangers such as ion exchange resin, ion exchange cellulose, and ion exchange Sephadex, activated carbon, alumina, silica gel, or synthetic adsorption resins such as Amberlite XAD-1
,-2 (Rohm and Haas) Diaion HP-10
Adsorption chromatography using an adsorbent such as Mitsubishi Kasei (Mitsubishi Kasei) is effectively used.

It goes without saying that in addition to these, any purification means based on the characteristics of peptide (II) can be used as appropriate.

By using a suitable combination of these means, peptide (It) can be isolated from the reaction solution.

Next, the properties of peptide (II) obtained by the method of Example 1 are as follows.

1-, melting point 142-146°C (decomposition) 2. [α), 35-47.1° (0.56% methanol] 3. Elemental analysis value C 5 4.3 7, H9.02
, N10.554. Ultraviolet absorption spectrum 210-4
No maximum absorption is observed at 00 mμ.

5. Infrared absorption spectrum (Figure 1) 6. Amino acid analysis valine, alanine, 4-amino-3
-Oxy-6-methylheptanoic acid is detected in a molar ratio of 1:1:2.

7. Mass spectrum Peptide (II) was methylated with diazomethane in methanol and then crystallized from methanol to obtain peptide (II).
The methyl ester of was prepared.

Elemental analysis values C58.28, H9.36, N 10.7
2.

In the mass spectrum, the molecular ion peak is m/e
It was 516.

Combining the above physicochemical analysis results, the structure of the peptide (It) obtained in Example 1 is valyl-4-amino-3
-Oxy-6-methylheptanoylalanyl-4-amino-3-oxy-6-methylhebutanoic acid.

Peptide (II) has the ability to inhibit pepsin activity.

The method used to measure the ability to inhibit pepsin activity is as follows.

0 substrate casein (Merkuhammersten casein)
．． Casein solution dissolved at 0.6% in 08M lactic acid1.
0ml, 0.02N hydrochloric acid-0.02M potassium chloride buffer icPH2.0) 0.7m/! ' and peptide (
II) Sample solution containing 0. Reaction solution consisting of 2 ml 1.
Add 0.1 ml of crystalline pepsin (Sigma) solution with a concentration of 50 μg/ml to 9 ml, react at 37° C. for 30 minutes, and then add 2.0 ml of 1.7 M perchloric acid solution to stop the reaction.

After leaving it at room temperature for another 30 minutes, strain it on a paper towel.
The absorbance (A) of the filtrate at 280 mμ was measured.

On the other hand, absorbance (B) was measured in the same manner for a control using only a buffer solution that does not contain peptide (II), and A and B correspond to A and B, respectively, from the standard curve created at the same time showing the relationship between pepsin concentration and absorbance. The residual pepsin concentrations C and D in the reaction solution were determined, and the protease inhibitory ability of the sample was calculated from the following formula.

When measured using this method, the activity of 5 μg of crystalline pepsin was 5 μg.
The amount of peptide (II) required for 0% inhibition (I
D50 value) was approximately 4 μg.

The method for producing peptide (1) using a microbial enzyme system, which was newly completed by the present inventors, has been described in detail above.

In addition, as described in the specification of Japanese Patent Application No. 49-35029 filed by the present applicant, when producing raw material acyl peptides, acyl peptides in which alanine is replaced with serine in general formula (1) are produced as by-products. However, the method of the present invention can also be applied to such peptides.

Next, the method of the present invention will be described in detail with reference to Examples. However, this Example is only an example specifically describing the method used in the present invention, and the method of this Example does not limit the content of the present invention in any way. Needless to say, don't do it.

Example 1 Aspergillus awamori IFO 4122 strain was mixed with 2% glucose, 0.7% meat extract, and 1.0% polypeptone.
, sodium chloride 0.3% pH 6. 8 liquid medium 3
5 200 ml Erlenmeyer flasks containing 0 ml
The culture solution obtained by inoculating the cells and culturing with shaking at 28°C for 3 days was transferred to 5 Sakaguchi flasks containing 500ml of the same medium as above, and cultured with reciprocating shaking for 3 days at 28°C. .

Approximately 2.5 liters of this culture solution was filtered through Okikamizawa, and approximately 270 g of wet bacterial cells obtained were ground together with sea sand, and 1.3 tons of 0.01 g of wet bacterial cells were ground with sea sand. 1M
Suspended in phosphate buffer.

To this suspension, 5 g of pepsinostreptin-P (in the structural formula (1), R is propionic acid) dissolved in 1.3 liters of water was added, and the mixture was reacted at 37° C. for 24 hours.

After the reaction is complete, add hydrochloric acid to adjust the pH to 5. The mixture was filtered with suction through a Buchner funnel using Hypro Super Cell (Johns Manville) as a filtering agent to obtain about 2.5 liters of spasmodic fluid.

This filtrate was passed through a 300 ml activated carbon column to adsorb the target substance, and after washing with 1 liter of 40% methanol water,
The target substance was eluted with 75% methanol/water.

The eluate was concentrated to dryness, then dissolved in a 20% methanol solution containing 0.0 IN sodium hydroxide and 0.1% sodium chloride, and passed through a 450 ml XAD-2 (Rohm and Haas 100-200 mesh) column. After adsorbing the target substance and washing with the same solvent, the target substance is eluted while gradually increasing the methanol concentration from 20% to 65%.

Collect the elution fractions containing the target substance, add hydrochloric acid to
H5. 0 and pass through a 200 ml activated carbon column to adsorb the target substance.

After washing the column with 600 ml of 30% methanol water, the target substance was eluted while gradually increasing the methanol concentration from 30% to 80%, and a thin layer chromatogram (n-
Butanol-acetic acid monohydrate (4:1:2) was used to obtain 400 m of both parts giving a single spot.

This eluate was concentrated to dryness to obtain 150 mg of a white powder of peptide (lI).

Example 2 200 mg of white powder of peptide (II) was obtained from 5 g of a mixture of pepsinostreptin and pepsinostreptin-P by the method shown in Example 1 using Cephalosporium colemioides UFO8579 strain.

Example 3 The yields of peptide (II) shown in Table 1 were obtained using the strains Microascus trigonosporus UFO 7026, Emericelopsis humicola IFO 8518, Mucor rufuescens IFO 7102, and Nocardia asteroides ATCC 3308.

The culture conditions of the microorganism used, the treatment of the obtained bacterial cells, the substrate and the purification method of peptide (II) are shown in Table 1.

[Brief explanation of the drawing]

Figure 1 shows the baryl-4-amino-3 obtained in Example 1.
-Oxy-6-methylheptanoyl-alanyl-4-amino-3-oxy-6-methylheptanoic acid shows an infrared absorption spectrum.

Claims (1)

[Claims] 1 General formula R-Val-Val-X-Ala. -X (1)(
In the formula, R is an aliphatic carboxylic acid, Val is valine, and A1a
is alanine, X is an amino acid in the formula, each amino acid has a peptide bond, and carboxylic acid and valine have an amide bond), and the peptide bond between the two valine residues is cleaved. Va 1 -X-Ala -, which is characterized by contacting with a culture of a microorganism belonging to the genus Urunocardia, Asbergillus, Cephalosporium, Mucor, Microascus, or Emeryseropsis or a treated product thereof. X (II
) (wherein Va l , Ala , and X all have the same meanings as above, and each amino acid is peptide bonded).