JPS5938220B2 - peptide - Google Patents

Description

[Detailed Description of the Invention] The present invention is based on the formula Mushi 1 Todoroki Tv▲↓ VV↓1▲▲ ν↓1! ▲
1 ShiJ4Zshit (hereinafter referred to as Leu-X-Ala-X).

) and its production method, namely VA-Leu-Leu-X-Ala-X (1) and IVA-VAl-Leu-X-Ala-X () (wherein IVA is isofernic acid, Leu is leucine, Ala is alanine, VaI is valine, X is the amino acid of the formula,
Each amino acid has a peptide bond, and isofernic acid and leucine or valine have an amide bond). It is characterized by contacting with a culture of microorganisms belonging to the genus Bacillus, Brevibacterium, Microbacterium, Micrococcus, Alcaligenes, or Aeromonas that can be cleaved or a processed product thereof (in the formula, Leu, Al
(a,

The acyl peptides represented by formulas (g) and (3) are strong acidic protease inhibitors produced by microorganisms.In particular, their ability to inhibit pepsin activity is extremely strong, and their toxicity is weak, so pepsin is one of the causative factors. It is expected to be developed as a preventive or therapeutic agent for gastric and intestinal ulcers, which are thought to be common. The present inventors conducted various studies in order to produce other useful compounds using the acyl peptides (1) and () as raw materials.

As a result, certain types of microorganisms contain the acyl peptides (1).
The peptide bonds between the two leucine residues in () and between the valine and leucine residues in (
) was found to produce a new peptide (l]). and the peptide itself is (1),
It has pepsin inhibitory activity similar to (...), (1), (H
) was found to be able to be used for the same purpose. The present invention was completed based on this knowledge and as a result of further research. In the method of the present invention, Bacillus species capable of cleaving the peptide bond between two leucine residues or between a valine residue and a leucine residue of the above-mentioned acyl peptides,
Microorganisms belonging to the genus Brevibacterium, Microbacterium, Micrococcus, Alcaligenes, or Aeromonas are appropriately used.

Such strains can be obtained by culturing bacteria isolated using an ordinary agar medium for bacterial isolation in a liquid medium, and then combining the culture or cultured product with acyl peptides (
The peptide (111) produced after contacting with 1) or () was subjected to thin layer chromatography and then detected by ninhydrin reaction, and the produced isofernic acid was extracted with ether and then detected by gas chromatography. selected from among the strains that showed high production ability. The mycological properties of the representative strain SCl7l2 will be described below.

The SCl7l2 strain is a strain isolated from soil. The classification criteria is based on Bergey's Manual of Determinative Bacteriology (BergeyOsMa
nualOfDeterminativeBacte
riOlOgy) 8th edition, the description of colors follows the ``Color Standards'' of the Japan Color Research Institute.

To summarize the sexual characteristics of 2θ and 3θ, the SCl7l2 strain is aerobic, Gram-positive, forms spores with short bacilli, and is catalase-positive, so it is considered to be Bacillus.
belongs to

Furthermore, the properties of this strain are described in Bergey's Manual of Determinative Bacteriology.
! S,ManualOfDeterminative
BacteriOlOgy) 8th edition, the SCl7l2 strain is Bacillus pasturi (Bacillus pasturi).
lluspasteuri). Although it is most similar to Bacillus sphaericus, it differs from the former in nitrate reduction, gelatin liquefaction, starch decomposition, etc., and also differs from the latter in starch decomposition, so it is considered a new bacterial species.
It was named lussphaericusSCl7l2.

The SCl7l2 strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under "Microorganism Accession No. 3727." In addition, bacteria that are recognized to be advantageously used include:
For example, Bacillus subtilis
btlllsIFO3O37), Bacillus megateriumIFOl2
lO8), Bacillus cereus
reusIFO3466), Bacillus circulans (NRRLB7l)
7), Bacillus sp.
haericusNRRLB1876), Brevibacterium deivaricatum (Brevibacterium
mdivaricatum ATCCl4O2O), Brevibacterium linens (Brevibacteri
umllnensIFOl2l4l), LeucOnOstOcdeXt
raniCLrIlNRRLBll46), LeucOnOstOc
mesenterOidesNRRLB5l2), Microbacterium ammoniaphilum (MicrOba
cteriumITT]10niaPhi1UmAT
CC15354), Micrococcus 0 glutamicus (M
icrOcOccusglutamicusATCCl
3O32), Micrococticus rhizodichteicus (M
icrOcOccuslysOdelkticusIF
O3333), Serratia marcescens (Serra
tiamarcescens IFOl2648), Achromobacter liquifaciens (AhrOmOb
actesliquefaciensFOl26O5)
, Aerobacterium aerogenes
raerOgenesI [FO'2059), Sarcina lutea (Sarcina lutea IFOl27O8)
), Alcaligenes faecalis (Alcallge
nesfaecallsIFOl2669), Bacterium cadaveris
risIFO7373l), Aeromonas liquefaciens (AerOmOnusriquefacien)
sIFOl2978), PediOcoccuscervisiaeIFO
l2229), etc.

The method of the present invention is carried out by contacting a culture of such a microorganism or a treated product thereof with an acyl peptide (1) or (2) and converting it into a peptide (111).

The cultured product or treated product referred to herein refers to the culture product itself obtained by culturing microorganisms in a medium, and the culture product itself that has been subjected to appropriate processes such as centrifugation, filtration, drying, polishing, and extraction to favor the reaction. Live bacterial cells, dried bacterial cells, and crushed bacterial cells obtained through treatment, or cultured liquid, bacterial cell extract, etc., obtained by salting out, organic solvent precipitation, gel filtration, chromatography, etc. acyl peptides (1) and (H) to produce peptide (1I).

Furthermore, it is also possible to use insolubilized enzymes or immobilized microbial cells by techniques such as insolubilization of enzymes and immobilization of microbial cells. 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, shakerose, dextrin, molasses, organic acids, and hydrocarbons; nitrogen sources include, for example, proteins such as peptone, casamino acids, and N-Z-amines. Hydrolysates, yeast spoilage, soybean meal, corn steep 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 for the purpose of promoting bacterial growth.
Vitamins, compounds related to the acid, etc. may be added. Furthermore, depending on the culture method and culture conditions, adding an antifoaming agent such as silicone, polypropylene glycol derivatives, soybean oil, etc. to the medium may be effective in increasing the production amount 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 liquid nature of the medium will vary depending on the type of microorganism used and the composition of the medium, but should be adjusted appropriately to maximize the final production of the enzyme system. If selected and regulated, in most cases under aerobic conditions about 2
It is preferable to culture at 5 to 35° C. for about 1 to 4 days, and maintain the pH of the medium around 4 to 8 during this period. Examples of the acyl peptides (1) and () used as starting materials include, for example, Japanese Patent Application No. 50-112387 and Japanese Patent Application No. 50-112388.
In other words, by culturing acyl peptide (1) or/and () producing bacteria belonging to the genus Streptomyces, acyl peptide (1) or/and () is produced in the culture. In addition to purified specimens, culture products, cultured fluids, and partially purified specimens obtained by processing these can all be used in the present invention.

Furthermore, in formulas (1), (), and (11), the amino acid represented by the terminal X may be a free acid, an ester thereof, 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 acyl peptide (1), () in the reaction system as high as possible;
It is usually advisable to add about 0.1 to 2%. In some cases, acyl peptide (1
), () can also be added separately. The reaction may be carried out by standing, shaking, or stirring, and the reaction temperature is usually about 10 to 60°C.

The degree 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) and (), the reaction style or reaction conditions, etc. Therefore, the reaction time is selected appropriately. After the reaction is completed, the peptide (11) produced in the reaction solution can be separated from the reaction solution by conventional separation means.

In other words, it is better to separate these from the bottom liquid or supernatant liquid after removing solids by filtering or centrifuging the reaction liquid, but it is also possible to separate the target substance directly from the reaction liquid without removing solids. It 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 the peptide. That is, for example, extraction with an organic solvent such as n-butanol that is not arbitrarily mixed with water and can dissolve the peptide, dissolution in a highly polar solvent such as methanol or ethanol, ethyl acetate,
Removal of impurities by treatment with hexane etc., gel filtration with Cephadex, ion exchange chromatography using various ion exchangers such as ion exchange resin, ion exchange cellulose, ion exchange Cephadex, activated carbon, alumina, silica gel or synthetic adsorption. Adsorption chromatography using a resin or adsorbent such as Amberlite XAD-1-1 or -2 can be effectively used.
It goes without saying that in addition to these, any purification means based on the characteristics of peptide (111) can be used as appropriate. By using a suitable combination of these means, peptide (111) can be isolated from the reaction solution. Next, peptide (11) obtained by the method of Example 1
The properties are as follows.

7 Solubility in solvents Easily soluble in methanol, ethanol, and butanol, slightly soluble in benzene, ether, chloroform, ethyl acetate, and hexane.

8 Color reaction: Positive for ninhydrin reaction, Lydon, Smith reaction, and potassium permanganate reaction.

As a result of analysis of the nine constituent amino acids, leucine, alanine, and 4-amino-3-hydroxy-6-methylheptanoic acid were found to have a molar ratio of 1:
Detected at a ratio of 1:2.

The 10-mass vector peptide (111) was methylated with diazomethane in methanol and then crystallized from methanol to yield the peptide (111).
The methyl ester of 111) was prepared.

In the mass spectrum, the molecular ion peak is m/153
It was 0. By integrating the above physicochemical analysis results, we found that the structure of peptide (10 is leucyl-4-amino-3-hydroxy-6-methylheptanoyl-alanyl-4-amino-3-hydroxy-6-methylheptanoic acid). Peptide (I) determined to have the ability to inhibit pepsin activity.

The method used to measure the ability to inhibit pepsin activity is as follows. The substrate casein (Wako Pure Chemical, Hammersten Casein) was 0.6% in 0.08M lactic acid buffer (PH2.2).
1.0ml of casein solution dissolved in 0.02N hydrochloric acid
0.02M potassium chloride buffer (PH2.2) 0.7m
1 and peptide (111) sample solution 0.2ml
40μ9/! to 1.9ml of reaction solution consisting of 1! Add 0.1 ml of crystalline pepsin (Sigma) solution with a concentration of 37
After reacting at ℃ for 30 minutes, 1.7M perchloric acid solution 2
．． Add 0ml to stop the reaction.

After leaving it at room temperature for another hour, it was centrifuged and the absorbance (A) at 280 mμ of the supernatant was measured.
Absorbance (B) was measured in the same manner for a control using only a buffer solution without peptide blood), and the inhibition rate was calculated as (B
-A)/BXlOO.

When measured using this method, the activity of crystalline pepsin 4μ9 was
The amount of peptide ([11) required for 0% inhibition (.
1Y). The value) was 9.0It9/ml. Next, the method of the present invention will be specifically described with reference to examples, but the present example is merely an example specifically describing the method used in the present invention.
It goes without saying that the method of this embodiment does not limit the content of the present invention in any way. Example 1 Bacillus sphaericus S
C-1712 strain with 0.5% meat scratches and 1.5% peptone.
, atrium chloride 0.50! ), glucose 1.0%,
Sodium monohydrogen phosphate 0.5%, magnesium sulfate 0
．． 1%, PH7. The cells were inoculated into a 22-volume sloped flask containing 500 ml of O liquid medium, and cultured with reciprocal shaking at 30°C for 44 hours.

This culture solution was centrifuged to collect the bacterial cells, and the resulting wet bacterial cells (13.89) were added to 0.1M phosphate buffer (PH7.O).
Suspended in 500d, acyl peptide (1) 1.09
was dissolved in 50ml of methanol and reacted at 37°C for 68 hours. Hydrochloric acid was added to this reaction solution to adjust the pH to 5.0, and the mixture was heated at 8000 rpm. p. The supernatant obtained by centrifugation at m for 20 minutes was extracted twice with an equal volume of n-butanol, concentrated to dryness, dissolved in 200 ml of 30 (!) methanol water, and passed through a 120 ml activated carbon column. After adsorbing the target substance and washing with 30(f) methanol water 500N,
The target substance was eluted by gradually increasing the methanol concentration.

After concentrating the eluate containing the target substance to dryness, add 100 m of 20% methanol water containing 0.01N sodium hydroxide.
1 and passed through a 100 d Amberlite XAD-2 column (Rohm and Haas Co., Ltd. 100-200 mesh) to adsorb the target substance. The target substance was eluted by washing with the same solvent 11 and gradually increasing the methanol concentration of the solvent from 20% to 60%. Elution fraction containing the target substance 1.
52 to PH5. The eluate was concentrated to dryness to obtain 305 mv of white powder of peptide Qll, which showed a single spot on thin layer chromatography. Example 2
Bacillus sphaecus
ricus NRRLl876) strain was inoculated into the same medium 22 as in Example 1, and cultured with shaking for 48 hours, the washed bacterial cells were collected using a centrifuge, suspended in 100 ml of cooled 0.01N phosphate buffer PH7O, and cultured using a French press. Crush the bacterial cells and incubate for 10,000 r. p. A cell-free extract was obtained by centrifugation at m for 20 minutes.

Powdered ammonium sulfate 56.19 was added to this extract 10011Le, and the resulting precipitate was mixed with a small amount of 0.01M phosphate buffer pH7.
It was dissolved in O and dialyzed and desalted against the same buffer. The dialysate was diluted with 0,
Gel filtration was performed with the same buffer containing 2M sodium chloride,
The active fraction was freeze-dried to obtain a crude enzyme solution. Add 50 liters of acyl peptide (1) to 50 ml of the obtained crude enzyme solution and add 3
The reaction was carried out at 7°C for 5 hours.

This reaction solution was purified in the same manner as in Example 1 to obtain 21m9 of white powder as the target substance. Example 3 Bacillus cereus I
FO3466) stock with 0.5% meat scratches and 1.5 peptone
%, glucose 1.0%, sodium chloride 0.5%, sodium monohydrogen phosphate 0.5%, magnesium sulfate 0.
1%, PH7. 500 m containing 100 ml of liquid medium of O
It was inoculated into a 1-volume Sakalo flask and cultured with shaking at 30°C for 48 hours.

This culture solution was centrifuged to collect washed bacterial cells, and after freeze-drying, 500 mv of dried bacterial cells were suspended in 3 ml of 0.85% physiological saline and acrylamide monomer 0.759
,N-N'-methylenebisacrylamide 40m7,5
%β-dimethylaminopropionitrile 0.5ml 1,2
．． 0.5 ml of 5% potassium persulfate was added and mixed in a mixer, allowed to stand at 37°C for 24 hours to gel, and then crushed to obtain granular insolubilized enzyme.

Packed onto the obtained insolubilized enzyme column (φ = 10 x 200 m0, 0.01% concentration of acyl peptide (1) 5
0 m9 was passed at 37°C. This reaction solution was collected and purified in the same manner as in Example 1, resulting in a white powder of the target substance 13.
1m7 was obtained. Example 4 Brevibacterium divaricatum
umATCCl4O2O) strain with glucose 1.0%, peptone 1.0%, meat scratches 0.5 (f), sodium chloride 0.301), sodium monohydrogen phosphate 0.1%, magnesium sulfate 0.1% PH7 ．． O liquid medium 500m
1 was inoculated into a 22-capacity slope flask, and cultured with shaking at 30°C for 48 hours.

This culture solution was centrifuged, and the obtained wet bacterial cells were suspended in 300 ml of 0.1M phosphate buffer, and acyl peptide (
1) 50mv was added and reacted at 37°C for 24 hours. After the reaction is completed, hydrochloric acid is added to adjust the pH to 2. O, 8000r. p
．． m. The supernatant obtained by centrifugation for 20 minutes was transferred to a Dowex 50WX2 (H type) column (φ = 20X100m).
After adsorption with 0 and washing with water, elution was performed with 2(:f) aqueous ammonia.

The eluted fraction containing the target substance was concentrated to dryness, dissolved in a small amount of n-butanol, and silica gel equilibrated in advance with a solvent of n-butanol:acetic acid:water:butyl acetate (4:1:1:4). After adsorption on a column (φ=20x300mm),
Elution was performed with the same solvent. 1 elution fraction containing the target substance
After adsorption/desorption treatment with 0 ml of activated carbon, the mixture was concentrated to dryness to obtain 22 ml of white powder of peptide (11). Example 5 Bacillus subtilis IFO3O37, Microbacterium ammoniaphilum ATCCl5354, Micrococcus glutamicus ATCCl3O32, Alcaligenes faecalis FO12669, Aeromonas
When each strain of Liquifaaciens IFOl 2978 was cultured and the bacterial cells were treated according to the method described in Example 4, the yields of peptides shown in Table 1 were obtained. 6 Bacillus sphaericus SC-1712 strain 1.0% glucose, 1.5% peptone, 0.5% meat scratches, 0.3% sodium chloride, 0.1% sodium monohydrogen phosphate
, magnesium sulfate 0.1%, PH7O liquid medium 50
A 21-volume slope flask containing 0 ml was inoculated and incubated at 30°C for 4
Shaking culture was carried out for 8 hours.

This culture solution was centrifuged, and the obtained wet bacterial cells were suspended in 300 ml of 0.1M phosphate buffer (PH7.O), and 50% of acyl peptide () was added. was added and reacted at 37°C for 24 hours. After the reaction was completed, purification was performed according to the method described in Example 4 to obtain 12 my of white powder of peptide ().

[Brief explanation of drawings]

Figure 1 shows leucyl-4-amino-3 obtained in Example 1.
-Hydroxy-6-methylheptanoylalanyl-4
Figure 2 shows the ultraviolet absorption spectrum of -amino-3-hydroxy-6-methylheptanoic acid and its infrared absorption spectrum (KBr method).

Claims (1)

[Claims] 1. A novel peptide having the following structure. ▲There are mathematical formulas, chemical formulas, tables, etc.▼2 IVA-Leu
-Leu-X-A1a-X and IVA-Val-Le
u-X-Ala-X (in the formula, IVA is iso-acid, L
eu is leucine, Ala is alanine, Val is valine,
X represents the amino acid of the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼, each amino acid has a peptide bond, and acyl peptides have a structure shown by iso■ herbal acid and leucine or valine have an amide bond), Microorganisms belonging to the genus Bacillus, Brevibacterium, Microbacterium, Micrococcus, Alcaligenes, or Aeromonas that can cleave the peptide bond between the two leucine residues or between the leucine and valine residues. L characterized by contacting with a culture or a treated product thereof
A method for producing a novel peptide represented by eu-X-Ala-X (wherein, Leu, Ala, and X all have the same meanings as above, and each amino acid is peptide bonded).