JPS6028990A - Novel peptide - Google Patents

Abstract

NEW MATERIAL:The peptide of formula (Tyr is tyrosine residue; Val is valine residue). USE:The compound strongly inhibits the decomposition of proteins with protease, and is useful as a remedy for muscular dystrophy and renal hypertension. Since the compound suppresses the sprouting and rooting of dicotyledonous plants at a low dose, it is also expected to be useful as an agricultural chemical. PREPARATION:A microbial strain belonging to Streptomyces groups [e.g. Streptomyces tanabeensis (SAB-934), FERM-P No.6938)] is cultured usually in a medium under aeration and agitation, and the peptide of formula is separated from the cultured product.

Description

Detailed Description of the Invention The present invention provides a novel peptide represented by the following formula (1) (in the above formula, 15ovaleryl represents an invaleryl group, and Tyr and Vat represent residual tyrosine and valine, respectively). Regarding.

In recent years, there has been an increasing demand for highly safe and highly effective pharmaceuticals and agrochemicals, and various studies have been conducted to discover the potential of physiologically active amino acid compounds and low-molecular-weight peptide compounds.

The present inventors focused on the important role of proteases in the control mechanism of living organisms, and conducted a wide range of studies on microbial cultures with the aim of searching for protease inhibitors.
We discovered that a strain belonging to the genus Stref') produces and accumulates a substance in culture that has the effect of strongly inhibiting protease activity, isolated it, and determined its structure.

The substance of the present invention is l5ovaleryl-Tyr-V
It has an al-Arginal structure and has the effect of strongly inhibiting proteolysis by proteases of animal, plant and microbial origin, and is expected to be used as a therapeutic agent for muscular dystrophy and renal hypertension.

It suppresses the germination and rooting of seeds of dicotyledonous plants such as cucumber in small amounts, and is expected to be used as a good medicine.

The bacterium SAB-934 strain that produces the substance of the present invention is
．． It was isolated from soil in Tanabe City, Wakayama Prefecture in July 2006. S
AB-934 strain has the properties described below and is similar to the known Streptomyces garcherii, but there are slight differences in the state of aerial mycelial attachment and availability of carbon sources, and the productivity of the substance of the present invention. The inventors recognized this as a new species and called it Streptomyces tanapeensis.
) and deposited this bacterium with the Institute of Microbial Technology, Agency of Industrial Science and Technology. (Accession number Microtechnical Research Institute Bacteria No. 6938
No. FEBMP-6938) [Mycological characteristics of Streptomyces tanapeensis (SAB-934 strain)] 1. Morphology Under a microscope, a large number of slightly wavy aerial hyphae were observed extending from well-branched basal hyphae. No whorled branches are observed, and the branch is straight (Reetus).

2. Growth status in each medium In the following descriptions, the numbers in parentheses are those of Container Corporation Op America.
ation of America) (D color/
Harmony Manual (Co1or harmony
This is a color display based on the manual.

■Sucrose/nitrate agar medium (cultured at 28℃) A small amount of white (,) aerial mycelia grew on the colorless to pale yellow growth. The underside is colorless to yellow-gray. No production of soluble pigment is observed.

■Glycerin/asparagine agar medium (cultured at 28°C) A small amount of reddish-brown (5ne) aerial mycelia grew on the reddish gray growth. The back surface is reddish-gray, and the soluble dye exhibits a pale reddish-purple color under 0.05N HCt.

Does not change color due to NaOH.

■Glucose-asparagine agar medium (cultured at 28°C) Gray-white (b) aerial mycelium grows on the colorless growth, but it is extremely poor. The back side is colorless. No production of soluble pigment is observed.

■Glucose Czapek agar medium (cultured at 28°C) Colorless growth cannot be said to be poor, but neither aerial mycelial attachment nor production of soluble pigments is observed.

■Starch agar medium (cultured at 28°C) White (13 ha) aerial mycelium grew on the pale gray growth. The underside is pale ocher, and production of ocher soluble pigment is observed.

■Nutritional agar medium (cultured at 28℃) Growth is poor and colorless. Production of aerial mycelia and production of soluble pigments were not observed.

■Normal agar medium (cultured at 28℃) Growth is poor and colorless. No attachment of aerial mycelia or production of soluble pigments was observed.

■Peptone-glucose agar medium (cultured at 28°C) Colorless growth cannot be said to be poor, but neither aerial mycelial attachment nor production of soluble pigments were observed.

■Tyrosine agar medium (cultured at 28°C) Gray-white (b) aerial mycelium grows on the light brown growth. The underside is light brown with a reddish tinge. Production of a slightly brown soluble pigment is observed.

■Pareishi Tagata medium (cultured at 28℃) A well-developed light brown gray maple with sucker-shaped wrinkles. No attachment of aerial mycelia or production of soluble pigments was observed. After 8 weeks, the growth of gray-white (b) aerial mycelia was observed.

■F2 Fragment medium (cultured at 28℃) Growth is light brownish gray with sucker-shaped wrinkles. No attachment of aerial mycelia or production of soluble pigments was observed.

■Horse serum agar medium (cultured at 28℃) Growth is colorless and poor. No attachment of aerial mycelia or production of soluble pigments was observed.

■Cellulase agar medium (28℃ culture) No growth was observed during 2 weeks of observation.

■Yeast/malt agar medium (cultured at 28℃) White (,) aerial mycelia are attached to the brownish-gray growth.

The back side is brownish gray. No production of soluble pigment is observed.

■Oatmeal agar medium (cultured at 28°C) A slight white (,) aerial mycelium grows on the colorless to yellowish growth.

No production of soluble pigment is observed.

■Peptone yeast/iron agar medium (cultured at 28°C) Growth is colorless and poor, rarely with yellowish gray (2ba) aerial mycelium. The back side is colorless and no production of soluble pigment is observed.

O egg medium (cultured at 28°C) Growth is extremely poor and colorless. Neither aerial hyphae nor production of soluble pigments were observed.

■Milk (Litmus m1lk) medium (cultured at 28°C) White bacterial cells grow in the sediment, and the lidmus reagent is slightly reddish, but peptonization occurs.
Coagulation was difficult to detect in either case.

■Gelatin puncture culture (28°C culture) Although slight growth is observed, neither production of soluble pigment nor liquefaction of gelatin is observed.

■Peptone water (contains 1.0% nitrate) White to white bacterial cells grow in the sediment, and no production of soluble pigments is observed. Nitrate reduction reaction is positive.

3. Physiological Properties (1) Growth Temperature Range As a result of tests using a yeast/malt liquid medium in the range of 10 to 45°C, the optimum temperature is around 28°C. Also, from a comparison of the capacity of grown bacteria, the temperature range for growing 5 or more cells is 14-37℃, 30℃ compared to that at the optimal temperature.
% or more, the temperature was 20 to 33°C.

(2) Liquefaction of gelatin (gelatin puncture culture) No liquefaction ability was observed.

(3) Hydrolysis of starch (starch agar medium) In Lugol's solution staining, the entire plate appears reddish, indicating that the hydrolysis of amylose is widespread, but the ability to hydrolyze amylopectin is low.

(4) Coagulation and peptonization of skim milk (Lidomas milk medium) Both were not observed.

(5) Formation of melanin-like pigment Slight production is observed on tyrosine agar medium, but not on peptone yeast iron agar medium. It is also not observed in gelatin.

(6) Utilization of carbon sources (Pridham Gottlieb grows using glucose and galactose, but does not use xylose, fructose, arabinose, sucrose, inositol, rhamnose, raffinose, manniol).

To summarize the above characteristics, strain 8AB-934 belongs to the genus Streptomyces, the aerial hyphae are simply branched and linear, and the spores have smooth surfaces. For various reasons, this bacterium is a non-chromogenic bacterium.

A slight amount of melanin-like pigment is observed on the tyrosine agar medium. Liquefaction of gelatin, coagulation and liquefaction of skim milk were negative, nitrate reduction reactions were all positive, and starch hydrolysis ability was somewhat strong.

Although we searched for known bacterial species with these characteristic properties, we were unable to find any similar bacterial species. One that seems to be relatively similar is Streptomyces garcherii (Str.
eptomyces galtieri), but
Bergcy's Manual of Determinative Bacteriology
nualof Deterrnative Ba
cteriology) 8th edition, and Waksman (W
The Actinomycetes (TheAc
tinomycete@) Volume 2, page 215 (1961
), the properties are compared with those of SAB-934 as follows.

The above differences clearly go beyond the scope of intraspecific variation.

[Production method of the substance of the present invention]

The medium used in producing the substance of the present invention 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 the microorganisms producing the substance of the present invention grow and the substance of the present invention is accumulated in the medium. That is, carbon sources include, for example, glucose, lactose, glycerin, starch, sucrose, dextrin, organic acids, etc., and nitrogen sources include, for example, protein hydrolysates such as pegtone and casamino acids, meat extracts, and yeast. Extracts, soybean meal, corn stable liquor, amino acids, ammonium salts, nitrates, and various other organic or inorganic nitrogen compounds are used. As inorganic salts, 11+7 phosphates, magnesium sulfate, and sodium chloride may be added, and vitamins, nucleic acid-related compounds, etc. may be added for the purpose of promoting the growth of madder.

In some cases, addition of an antifoaming agent such as silicone, polyzorolene gellicol derivative, soybean oil, etc. to the medium may be effective in increasing the amount of the substance of the present invention accumulated.

When culturing, it is preferable to inoculate the culture medium with the culture obtained by performing a small-scale preculture in advance, in case of main culture. Conditions such as culture temperature, culture period, culture liquid, etc. are appropriately selected and adjusted so as to maximize the accumulation of the substance of the present invention.
Cultivation at ~35°C for 3 to 7 days is sufficient;
It is best to keep it at H4-7.

By culturing in this manner, the substance of the present invention is produced and accumulated in the culture. When cultured using a liquid medium,
Since the target substance is mainly accumulated in the liquid part, it is preferable to separate it from the liquid or supernatant liquid after removing the bacterial cells by filtering or centrifuging the culture. It is also possible to separate the target substance directly from the culture medium without removing the body. 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. Dissolution, removal of impurities by treatment with hexane, etc., dull filtration with Sephadex,
Ion exchange chromatography using various ion exchangers such as ion exchange resin, ion exchange cellulose, and ion exchange Sephadex, adsorption chromatography using adsorbents such as activated carbon, alumina, silica gel, Amberlite XAD-1, 2, and HP-20, etc. By using a suitable combination of these means, the substance of the present invention can be isolated in powder form. However, methods other than these can be used as appropriate as long as they effectively utilize the properties of the substance of the present invention.

[Physicochemical properties of the substance of the present invention]

1. Elemental analysis value (as C25H4oN605/4H20) 23 Molecular weight: 504.6 (as C25H4oN605) 3. Melting point: 184℃-188℃ (decomposition) 4. Specific optical rotation: [α
-10,9° (C=o, 137 in H2O)
5. Ultraviolet absorption spectrum: Absorption specific to tyrosine is observed at 278 nm.

6, FD-MS: rrv'Z 504 (M+)7,
Infrared absorption spectrum 2950 (CH) 1630 (amide l, guanidlni
um) 1540 (amiden), 1500 (phe
nyl) 8, nuclear magnetic resonance spectrum 0.80 ppm (CHs, d, J = 6.5 Hz) 0-
83 ppm (c!!3.d, J~6.5
Hz) 0-90 ppm (C5, d, J ~
6.5 Hz) 0-94 ppm (C5, d, J
~6-5 Hz) 1,50 ppm (CH, m
) 1.70-1.90ppm (5H, m) 2.09pp
m (2H, m) 2.94ppm (2H, m) 3.15~4.12ppm (3H, m) 4, 50~4
．． 65 ppm (2H, m)4, 96 ppm (0
,5H,d, J=4Hz) 5.42ppm(0
,5H,d, J=3Hz)6.80ppm (I
H, d, J=7.3Hz)6.82ppm (I
H, d, J=7.3Hz) 7, llppm (I
H, d, J = 7.3 Hz) 7.13 ppm (IH, d
, J=7.3Hz) 9. Solubility in various solvents: Easily soluble in water, methanol, ethanol, soluble in butanol, benzene, ether, petroleum ether, chloroform,
It is sparingly soluble in carbon tetrachloride, hexane, and ethyl acetate.

10. Color reaction: Positive for Sakaguchi reaction and Tollen's test, negative for ninhydrin and 2,4-dinitrophenyl hydrazine.

11. Components D Amino acid analysis includes tyrosine, valine, and valine.

Amino acid analysis results N moles Molar ratio Number of amino acids Glycine 0.110 0.014 0 Cystay O
, 1070,0140 Palin 7.748 1.004 1 Iso-4-7 0.239 0.031 0-4-4 7.717
1.0 1 [Hitachi Amino Acid Analyzer Model 835] Comprehending the above physicochemical analysis results, the chemical structure of the substance of the present invention is l
5ovaleryl-(Tyr, Val)-A
The structure was determined to be rgininal.

Amino acid sequence: This substance 1~ is dissolved in 1-sodium bicarbonate o, IWLl,
Add 0.051 V of chymotrigsin to this and hold at 40°C.
, and reacted for 1 hour. Take 2 μt of this reaction solution and add 0.0 μt.
Add 2 μt of 25 thidansyl chloride and heat at 40℃ for 20 minutes.
After reacting for a minute, the mixture was dried under reduced pressure, 6 volumes of constant hydrochloric acid was added, and the mixture was hydrolyzed under vacuum at 105°C for 24 hours. After drying the hydrolyzate under reduced pressure, it was dissolved in a 50-tipyridine solution, and this was secondarily developed using a polyamide thin layer with a 1.5% aqueous formic acid solution and benzene-acetic acid (9:1). When this was irradiated under an ultraviolet lamp, the DN attached as a marker on the back side
It matched with S-Valine.

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

H [Physiological activity of the substance of the present invention] ■9 Method for measuring enzyme inhibitory activity of the substance of the present invention Mix the substance of the present invention and an enzyme solution, pre-incubate at 30°C for 5 minutes, and then mix the substrate solution to initiate the reaction. Let it start. 0.5% casein was used as a substrate, and in addition, 5mM calcium chloride, 10mM cysteine, and 50mM
Add mM) Lis-HCl buffer (pH 7,5) and incubate at 30°C.
Let it react for 30 minutes. Then add 6.5%) to the reaction solution.
') The reaction was stopped by adding chloroacetic acid, and the trichloroacetic acid soluble fraction of casein was hydrolyzed by enzymes. Lowy-Folin (Lowy-Folin)
lin ) method, and the inhibitory ability is determined from comparison with a control solution.

Results: 0.06μy of this substance increases the papain activity of 1.25μy by 5
Inhibited by 0% or more. Similarly, calpain I and calpain I [[Calcium and Ce1l Funct
lon (W, Y.

Cheung, ed.) Vol. 3, Ac.
academic Press New York (19
83):] showed 25% and 60% activity inhibition at 1 μg, respectively.

Based on the above, the substance of the present invention has a stronger inhibitory effect on no4pain and calpain ■ than leupeptin, an existing grotease inhibitor, although it has a lower inhibitory effect on calpain ■, and is a characteristic grotease inhibitor. It is.

■1 Test method for the action of the substance of the present invention on seeds A petri dish is moistened with F paper (
9Qn diameter), place 10 seeds each of barley (monocot) and cucumber (dicot), and heat them to 30°C.
Incubate for 84 hours and compare the germination and rooting states of each seed.

Water is used as a control.

The concentration of the aqueous solution of the substance of the present invention is 25 and 50.

100.500 μp.

Results: In barley, no difference was observed in each concentration compared to the control plot.

In cucumbers, at concentrations of 25, 50, 100 μg and above, root growth was poor, the development of root hairs was also suppressed, and no germination was observed. Furthermore, neither germination nor rooting was observed at a concentration of 500 μfl/ml.

The above results demonstrate the possibility of using the substance of the present invention as a selective herbicide.

[Production example of the product of the present invention]

Synthetic medium consisting of glucose, peptone and yeast extract (
YM medium) SAB − to 60001d (pH 5, 8)
A pure culture of strain 934 was inoculated and incubated at 28°C in a small jar.
After culturing for 2 hours, the culture was centrifuged and the supernatant was transferred to a Diaio7 HP-20 column (140 mn x 200 Wm
), and the column was washed with 2 ml of water and 1 ml of water, and then eluted with 10,000 ml of methanol. The eluate was concentrated and dried under reduced pressure, and then poured into 1,000 ml of water.
and passed through a column (60+a+X100ai) packed with DEAE cellulose (H type). Add this to 1 part of water
000mA! This was combined with the above elueate and concentrated under reduced pressure to a volume of 200 d. The concentrate was adjusted to pH 5 with hydrochloric acid.
0 and adsorbed onto a column (60+mnX 1000va) of Ampanite CG-50H+ (ion exchange resin).

(7) Power: 7 mu, water: 1,000 d, and 0.2N acetic acid: 30
0ml! After washing with
After concentrating, the dried solids were dissolved in 50% methanol solution containing 0.5% acetic acid and loaded onto a Sephadex Ll (-20 column (30m + x 1850m). The elution was fractionated, and the active fractions were collected.
~ Appeared in the 79th thing.・Concentrate and dry the active fractions (total of 110 fractions), dissolve in 2ml of water, and apply this to a Sephadex G-25 column (20ml).
s+X470mm), washed with water 600d,
．． It was eluted and fractionated with 2% acetic acid 600d. There are 10 active parts in total.
It was the 25th to 27th fractions in the middle of the 0 fractions. By concentrating and drying this active fraction, almost pure substance 3711417 of the present invention was obtained as a colorless amorphous substance.

Kazuo Wakasugi, Commissioner of the Patent Office1. Display of the case 1982 Patent Application No. 116616 2, Name of the invention New penotide 3, Relationship with the person making the amendment Patent applicant address 2-1-40 Dojimahama, Kita-ku, Osaka Name (19)
0) Suntory Ltd. Representative Keizo Saji 4, Agent Address Suntory Ltd. Patent Office, 1-2-3 Motoakasaka, Minato-ku, Tokyo (Telephone: 470-1131) 6. Contents of Amendment (1) "Patent" in the specification "Scope of Claims" has been revised as shown in the attached sheet.

(2) Page 1, bottom 2nd line of the specification: "Indicates residual presence." is changed to "Indicates residue."

(3) Page 7, line 13 of the specification: 0 pezoton water (1
, 0% nitrate content)" is replaced with "pezoton water (1, 0% nitrate content)"
(4) Page 9, lines 15-16 of the specification: ``All nitrate reduction reactions are positive'' has been changed to ``Nitrate reduction reactions are positive.'' It was changed to ``Positive.''

(5) In the table on page 11 of the specification, the term "yellow to light yellow" in the color of growth section has been changed to "yellow to light gray."

(6) Page 12, line 14 of the specification: "Corn Stable Liquor" shall be changed to "Corn Stable Liquor."

(7) Page 13, line 18 of the specification: "From culture" is changed to "from culture solution."

(8) Page 13, line 20 of the specification to page 14, line 2 of the specification: ``i.e., precipitation by addition of a precipitating agent such as ammonium sulfate, n-butanol, etc.'' is replaced with ``
That is, for example, n-butanol, etc."

(9) Page 15 of the specification, line 10 from the bottom: rFn-M
S: m/Z 504 (M+) J and l'
-FD-MS: mHz 505 (M+1)+J.

αG Specification, page 16, lines 19-20: “Positive for Sakaguchi reaction and Tollens test, ninhydrin and 2,4
- For dinitrophenyl hydrazine" has been changed to "Positive for Sakaguchi reaction, plagiarism reaction, and Tollens test, for ninhydrin."

0 ■ Page 21, line 2 of the specification: [From glucose, peptone, and yeast extract] is changed to "From pudu sugar, peptone, malt extract, and yeast extract."

(b) Page 21, line 10 of the specification: r DEAE
``Cellulose (H type)'' has been changed to ``r DEAE cellulose (OH type)''.

Page 21 of the specification, lines 14 to 15, 2 “Amparite CG-50H+ (ion exchange resin) column (6
0s +

a< Page 21, line 18 of the specification: ``Elute with r10oml,'' has been changed to ``Elute with rlooooml,''.

7. List of attached documents (1) 1 attached sheet [Attachment] 2. Claims Structural Formula (In the above formula, l5ovalery+ represents an invaleryl group, and Tyr and Val are tyrosine and valine residues, respectively. A novel peptide represented by

Claims (1)

[Scope of Claims] A novel peptide represented by the structural formula (in the above formula, l5ovaleryl Id represents an invaleryl group, and Tyr and Val represent residual tyrosine and valine, respectively).