CA1053167A - Desacyl-pepsidine from bacillus pumilus - Google Patents
Desacyl-pepsidine from bacillus pumilusInfo
- Publication number
- CA1053167A CA1053167A CA241,153A CA241153A CA1053167A CA 1053167 A CA1053167 A CA 1053167A CA 241153 A CA241153 A CA 241153A CA 1053167 A CA1053167 A CA 1053167A
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- CA
- Canada
- Prior art keywords
- pepsidine
- acyl
- pentapeptide
- bacillus pumilus
- desacyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/02—Linear peptides containing at least one abnormal peptide link
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Crystallography & Structural Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Desacyl-pepsidine which are useful as pepsin-inhibitor and intermediates for the preparation of N-acyl-pentapeptide homologues. These compounds are prepared by contacting N-acyl-pentapeptide with a bacterium which belongs to Bacillus pumilus.
Desacyl-pepsidine which are useful as pepsin-inhibitor and intermediates for the preparation of N-acyl-pentapeptide homologues. These compounds are prepared by contacting N-acyl-pentapeptide with a bacterium which belongs to Bacillus pumilus.
Description
The present invelltion relates -to desacyl-pepsidine (hereinafter xeferred to DA-pepsidine).
The DA-pepsidine of the present invention is a pentapep- ~
tide, i.e. valyl-valyl-4-amino-3-hydroxy-6-methyl-heptanoyl ~ ;
alanyl-4-amino-3-hydroxy-6-methyl-heptanoic acid having the formula (I):
\ / \ / (I) H3C\ /H3 H3C / H3 CH fH
CH CH CH OH CH CH OH I ~ -~ I 1 2 1 1 3 1 2 ~
DA-pepsidine has a strong pepsin-inhibitory activity and thus may be useful for treating a patient suffering from a gastric ulcer. .
The present invention also provides a process for the ~ `
: :, .,. ~ .
preparation of DA-pepsidine which comprises contacting at least one M-acyl-pentapeptide with a bacterium belonging to Bacil;lus pumilus The N-acyl-pentapeptide used as the starting material -~ in the present invention is a pentapeptide, i.e. N-acyl-valyl-valyl-4-amino-3-hydroxy-6-methyl-heptanoyl-alanyl-4-amino-3-hydroxy-6-methyl-heptanoic acid having the formula (II):
I H3C CH3 H3C CH3 , ~ ~, H3C ~H3 H~C /CH3 \CH ~ CH (II) ~ ;
fH ; CH eH CI~3 CH2 fH
R-NH-C~I-CO-NH-CH-CO-NH-CH--CH-CH2-CO-NH-OH-CO-NH-CH--CH-CH2-COOH .`,~
wherein R is an acyl group.
'~ As N-acylpentapeptides of formula (II), more than ~;
~.- . :: .
ten compounds are known. The compound wherein R is acetyl group, -~
~ -:
~3~67 `~-for ex~mple, has beerl disclosed by N. Kuwana et al, and described in U.S. Patent No. 3,819,486, 3,878,185 and Japa~ese Patent Laid-open Publication No. 125,583/74, wherein the said compound has been referred to "S-PI" or "Pepsidine C". In Japa~ese Patent Laid-open Publication No. 125,583/74 compounds are disclosed wherein R is butyryl and propionyl group, as "Pepsidine A" and "Pepsidine B", respectively. N~acyl-pentapeptides wherein R
is isovaleryl group or straight or branched aliphatic acyl gr~up having 5 to 16 carbon atoms have been disclose~ by Umezawa et al in Japanese Patent Publication No. 8996/72, Japanese Patent Laid-open No. 29582/72 and Japanese Patent I.aid-open No. 41590/74, wherein the said compounds have been referred to "Pepstatin".
These known N-acyl-peptapeptides are known to have a strong pepsin-inhibitory activity.
N-acyl-pentapeptides are provided ~y cultivation of various Actinomycetes. The aforementioned pepsi~ines, for~
exampler are provided by cultivation of Streptomyces nahiwaensis EF 44-201 strain. The pepstatins are provided, for example, by cultivation of Streptomyces testaceus. However, N-acyl-penta-.
;~ 20 peptides provided by the cultivation of these Actinomycetes are .... i: : :
usually not homogeneous, but a mixture of more than ten N-acyl-pentapeptides havlng various acyl moieties (R) and similar prop-erties. Thus, in order to provide particular N-acyl-pentapep-tide, there are required very complicated processes for separat-ing them, whereby the yield of the desired product may be con-sid~rably reduced. ~ -DA-pepsidine having free N-terminal has the fundamental structure in common with the aforesaid N-acyl-pen~apeptide homo~
~. :
logues. Thus, when DA-pepsidine is first prepared according to the process of the present invention using the mixture of various ~ -N-acyl-pentapeptides obtained by the cultivation of above Streptomyces strain, the resulting DA-pepsidine may be acylated . ~ ~
The DA-pepsidine of the present invention is a pentapep- ~
tide, i.e. valyl-valyl-4-amino-3-hydroxy-6-methyl-heptanoyl ~ ;
alanyl-4-amino-3-hydroxy-6-methyl-heptanoic acid having the formula (I):
\ / \ / (I) H3C\ /H3 H3C / H3 CH fH
CH CH CH OH CH CH OH I ~ -~ I 1 2 1 1 3 1 2 ~
DA-pepsidine has a strong pepsin-inhibitory activity and thus may be useful for treating a patient suffering from a gastric ulcer. .
The present invention also provides a process for the ~ `
: :, .,. ~ .
preparation of DA-pepsidine which comprises contacting at least one M-acyl-pentapeptide with a bacterium belonging to Bacil;lus pumilus The N-acyl-pentapeptide used as the starting material -~ in the present invention is a pentapeptide, i.e. N-acyl-valyl-valyl-4-amino-3-hydroxy-6-methyl-heptanoyl-alanyl-4-amino-3-hydroxy-6-methyl-heptanoic acid having the formula (II):
I H3C CH3 H3C CH3 , ~ ~, H3C ~H3 H~C /CH3 \CH ~ CH (II) ~ ;
fH ; CH eH CI~3 CH2 fH
R-NH-C~I-CO-NH-CH-CO-NH-CH--CH-CH2-CO-NH-OH-CO-NH-CH--CH-CH2-COOH .`,~
wherein R is an acyl group.
'~ As N-acylpentapeptides of formula (II), more than ~;
~.- . :: .
ten compounds are known. The compound wherein R is acetyl group, -~
~ -:
~3~67 `~-for ex~mple, has beerl disclosed by N. Kuwana et al, and described in U.S. Patent No. 3,819,486, 3,878,185 and Japa~ese Patent Laid-open Publication No. 125,583/74, wherein the said compound has been referred to "S-PI" or "Pepsidine C". In Japa~ese Patent Laid-open Publication No. 125,583/74 compounds are disclosed wherein R is butyryl and propionyl group, as "Pepsidine A" and "Pepsidine B", respectively. N~acyl-pentapeptides wherein R
is isovaleryl group or straight or branched aliphatic acyl gr~up having 5 to 16 carbon atoms have been disclose~ by Umezawa et al in Japanese Patent Publication No. 8996/72, Japanese Patent Laid-open No. 29582/72 and Japanese Patent I.aid-open No. 41590/74, wherein the said compounds have been referred to "Pepstatin".
These known N-acyl-peptapeptides are known to have a strong pepsin-inhibitory activity.
N-acyl-pentapeptides are provided ~y cultivation of various Actinomycetes. The aforementioned pepsi~ines, for~
exampler are provided by cultivation of Streptomyces nahiwaensis EF 44-201 strain. The pepstatins are provided, for example, by cultivation of Streptomyces testaceus. However, N-acyl-penta-.
;~ 20 peptides provided by the cultivation of these Actinomycetes are .... i: : :
usually not homogeneous, but a mixture of more than ten N-acyl-pentapeptides havlng various acyl moieties (R) and similar prop-erties. Thus, in order to provide particular N-acyl-pentapep-tide, there are required very complicated processes for separat-ing them, whereby the yield of the desired product may be con-sid~rably reduced. ~ -DA-pepsidine having free N-terminal has the fundamental structure in common with the aforesaid N-acyl-pen~apeptide homo~
~. :
logues. Thus, when DA-pepsidine is first prepared according to the process of the present invention using the mixture of various ~ -N-acyl-pentapeptides obtained by the cultivation of above Streptomyces strain, the resulting DA-pepsidine may be acylated . ~ ~
2 -' ~'`'', by a desired acylating agent to readily produce a desired single N-acyl-pentapeptide.
The DA-pepsidine may also be used as an intermediate for the synthesis of other novel N-acyl-pentapeptide homologues.
A strain of Bacillus pumilus preferable for use in the process of the present invention is Baclllus EF 49-210 ~nov. sp.) strain, which has been separated from the soil at Kawaguchiko machi, Yamanashi Prefecture, Japan. The microbiolog-` ical properties of the strain are as follows:
~1] Microscopic observation (bouillon agar nutrient medium) 1. Morphologies: Rods with rounded ends, occuring singly or ~ -in pairs or on rare occasions in threes. ;-~
~` 2. Size: 0.4 to 0.7 by 1.0 to 4.0 microns. ~ ;
The DA-pepsidine may also be used as an intermediate for the synthesis of other novel N-acyl-pentapeptide homologues.
A strain of Bacillus pumilus preferable for use in the process of the present invention is Baclllus EF 49-210 ~nov. sp.) strain, which has been separated from the soil at Kawaguchiko machi, Yamanashi Prefecture, Japan. The microbiolog-` ical properties of the strain are as follows:
~1] Microscopic observation (bouillon agar nutrient medium) 1. Morphologies: Rods with rounded ends, occuring singly or ~ -in pairs or on rare occasions in threes. ;-~
~` 2. Size: 0.4 to 0.7 by 1.0 to 4.0 microns. ~ ;
3. Motility: Motile.
-- 4. Flagella: Peritrichous 5. Spores: Formed 6. Gram stain: Positive 7. Acid fast stain: Negative [23 Cultivation view ~ ;~
1. Bouillon agar slant medium (at 30C for one day) ,. . . .
Growth spreading flat, milky yellowish to slightly yellowish, and weakly lustered~ No change in color of nutrient medium. :;~
2. Bouillon agar flat medium ~at 30C for 1 to 7 days) Growth spreading flat, dendroid, milky yellowish; slightly ~ :;
yellowish~ smooth colonyj weakly lustered and translucent to opaque. No soluble pigment. ~;~
3. Broth medium (at 30C for 2 days) Formed pellicle on surface. Turbidity is weak and uniformO
i 30 4. Gelatin medium (at 20C, 27C and 30C for 3 days) `~
Growth weakly, but liquefies the gelatin. It is not clear -;
by stab culture.
; .
.,. -: . ... ,. .,. , - ,.. . .. . - . .... ....... , .... , .... . . . , ~
5. Litmus milk (at 30C for 6 days) ; `
Rather ~cidic, and weakly liquefied af~er 6 day cultivation. ~`
No coagulation. ;
6. Potato (at 30C for 2 days) Growth wet and spreading slimy. Potato darkened-[III] Physicological properties.
1. Nitrate reduction property : Negative 2. Denitrogen reaction : Negative 3. Methyl red test : Positive
-- 4. Flagella: Peritrichous 5. Spores: Formed 6. Gram stain: Positive 7. Acid fast stain: Negative [23 Cultivation view ~ ;~
1. Bouillon agar slant medium (at 30C for one day) ,. . . .
Growth spreading flat, milky yellowish to slightly yellowish, and weakly lustered~ No change in color of nutrient medium. :;~
2. Bouillon agar flat medium ~at 30C for 1 to 7 days) Growth spreading flat, dendroid, milky yellowish; slightly ~ :;
yellowish~ smooth colonyj weakly lustered and translucent to opaque. No soluble pigment. ~;~
3. Broth medium (at 30C for 2 days) Formed pellicle on surface. Turbidity is weak and uniformO
i 30 4. Gelatin medium (at 20C, 27C and 30C for 3 days) `~
Growth weakly, but liquefies the gelatin. It is not clear -;
by stab culture.
; .
.,. -: . ... ,. .,. , - ,.. . .. . - . .... ....... , .... , .... . . . , ~
5. Litmus milk (at 30C for 6 days) ; `
Rather ~cidic, and weakly liquefied af~er 6 day cultivation. ~`
No coagulation. ;
6. Potato (at 30C for 2 days) Growth wet and spreading slimy. Potato darkened-[III] Physicological properties.
1. Nitrate reduction property : Negative 2. Denitrogen reaction : Negative 3. Methyl red test : Positive
4. Voges-Proskauer reaction ; Positive ;
5. Formation of indole : Negative ;
6. Formation of hydrogen sulfide : Negative
7. Hydrolysis of starch : Negative
8. Utilization of citric acid : Positive in Koser's medium Negative in Symmons' medium
9. Utilization of the source of inorganic nitrogen~
Weakly positive with potassium nitrate.
~, ~eakly positive with ammonium sulfate.
Weakly positive with potassium nitrate.
~, ~eakly positive with ammonium sulfate.
10. Formation of pigment : Negative
11. Urease : Negative -` 12. Oxidase : Positive 13. Catalase : Positive 14. Range of growth:
a) pH (brot~ nutrient medium at 30C for 2 days with ~i shaking) Growth in sterilized medium at pH 5 to 9.8.
~ b) Temperature (broth nutrient medium for 2 days with ~ ~-`' shakingj ~-Growth at 20C to 45C.
c) Concentration of salt (broth nutrient medium for ~ ;
2 days with shaking) ;~ -.
4 ~ -~
Growth within one day, in the concentration of0-5 -~ ?
to 7.0~ of NaCl. Growth within two days, in the concentration of 10~ of NaCl.
15. Oxygen requirement: Facultative anaerobic.
16. O-F test by Hugh-Leifson method;
Acid is formed in both aerobic and anaerobic. No gas formed. ~-17. Fermentation test and acid formation with carbohydrates are as follows:
~cid formation ~ -~ : .
No. Carbohydrates aer _ iC anaerobic .. ;~
-~ 1 arabinose - +
2 xylose + +
3 glucose + +
4 mannose + +
;-; . ...
fructose + +
. - , . . 6 galactose + +
7 maltose - +
8 sucrose + +
9 lactose - +
trehalose + +
11 sorbitol - + ;
a) pH (brot~ nutrient medium at 30C for 2 days with ~i shaking) Growth in sterilized medium at pH 5 to 9.8.
~ b) Temperature (broth nutrient medium for 2 days with ~ ~-`' shakingj ~-Growth at 20C to 45C.
c) Concentration of salt (broth nutrient medium for ~ ;
2 days with shaking) ;~ -.
4 ~ -~
Growth within one day, in the concentration of0-5 -~ ?
to 7.0~ of NaCl. Growth within two days, in the concentration of 10~ of NaCl.
15. Oxygen requirement: Facultative anaerobic.
16. O-F test by Hugh-Leifson method;
Acid is formed in both aerobic and anaerobic. No gas formed. ~-17. Fermentation test and acid formation with carbohydrates are as follows:
~cid formation ~ -~ : .
No. Carbohydrates aer _ iC anaerobic .. ;~
-~ 1 arabinose - +
2 xylose + +
3 glucose + +
4 mannose + +
;-; . ...
fructose + +
. - , . . 6 galactose + +
7 maltose - +
8 sucrose + +
9 lactose - +
trehalose + +
11 sorbitol - + ;
12 mannitol - +
13 inositol + + '
14 glycerol + +
; 15 starch - +
.
;l No gas formed in any of the carbohydrate.
Acid (+), Sight acid (+), No acid (~
By comparing the data of the above mentioned properties of the bacterium with those described in Bergey's Manual of Determinative Bacteriology, the seventh edition, this bacterium is considered to be a variant of Bacillu pumilus, and the said ~ 5 ~
.' .,., .. .,......... .. , ... ' bacterium was nominated as Bacillus ~ llus ~ chi.
This bacterium EF 49-210 (ATCC No. 31132) strain has been deposi-ted to Agency of Industrial Science and Technology, Fermentation Research Institute in Japan as FERM-P No. 2677. It was also recognized that there is suitably used for the process of ' ; the present invention the known bacterium B~clllus pumilus IFO-12092 and IFO-12110 which were stored in Institute of ~;
Fermentation Osaka in Japan.
In the cultivation of the microorganisms to be used in ~ ~' the process of the present in~ention, there may be used any comp~
osition of the nutrient medium and any conditions for the culti~
vation, provided that the microorganisms can grow, and fully exhibit desired activities. There may be selected, a medium containing for example, any organic or inorganic nitrogen sources such as peptone, meat extract, corn steep liquor, soy-bean hydro- ' . ~ .
lyzate, soy-bean extract, yeast extract and inorganic ammonium salts; any carbon sources such as molasses, dextrose, starch ~' ' and hydrolysis products thereof; and inorganic salts. The cultivation is generally carried out with shaking or aerating . ...
at a temperature ranging from 20~C to 45~C for a period of time from one to se~en days.
It was found that the deacylating activity of the Bacillus pumilus exists both intra- and extra- cellularly. Thus, ' :' :
in the process of the present invention, there may be used cuIture broth, culture ~iltrate, ~hole cells, dry cells and/or enzyme preparations obtained therefrom. ` `
The reactant N-acyl-pen~apeptide, is not always ' '~ ' required to use in a purified form. There may be used, for example, a mixture of several N-~acyl-pentapeptides which may ,~. . .
be obtained in various purification stages such as culture filtrate of the microorganism which produces N-acyl-pentapep~
tides, and products salted out from said culture filtrate.
~'~, ' ' ~
i3~
Products obtained by extracting the culture fil.~rate with an ~ ~;
organic solvent and then removing the solvent may also be used.
In the process of the present invention, the reaction conditions such as concentration, temperature and pH, are not critical and can be varied over broad range depending upon the kinds of the strain of microorganisms used and the N-acyl- ;
pentapeptide selected as starting material. When deacylation of N-acyl-pentapeptide is carried out using Bacillus pumilus Kawaguchi EF 49-210 strain, for example, it is preferable to use ~ ~
conditions of pH 4 to 10 and at temperature of about 30 - 50C. . ;
A reaction time of from 3 to 60 hours is suitable. By addition `
of a Co compound such as CoC12, and CoSO4 the yield of DA~
pepsidine may be increased.
Separation and purifica-tion of DA-pepsidine from the :
~ reaction mixture can be carried out using any con~entional proce- :
- aure such as solvent extraction, column chromatography, fraction~ `~
al crystallization and recrystalli~ation. `
To detect the formation of DA-pepsidine thin layer chromatography can be used as follows:
Sample solution is spotted on a thi.n layer plate of -~
Silica Gel G (a trademark of Merck A. G.), and developed with a : mixed solvent (I) containing n-butyl alcohol, acetic acid and water in the ratios by volume of 3 : 1 : 1, or a mixed solvent (II) containing n butyl alcohol, acetic acid, water and n-butyl-: acetate in the ratios by vo].ume of 4 : 1 : 1 : 4. DA-pepsidine ;
is detected as a spot which is positive in the ninhydrin reaction and also Rydon-Smith reaction, at Rf - 0~48 in the case of the use of the mixed solvent (I). ~ ~-Alternatively, it is detected by Casein plate method :
as follows:
After the de~eloped and dried thin layer plate mentioned above is transferred to a flat board of Casein-containing agar, ~-, ~i ' ;
a filter paper impreynated with pepsin solution is placed on the board. Reaction is then effected at 30C for overnight, to detect a non-decomposed casein.
DA-pepsidine of the present invention has the following physicochemical properties:
(i) Appearance:
White needles (ii) Solubility~
Readily soluble in acetic acid and methanol; soluble -:
in ethanol, n-hu~anol and pyridine; and slightly soluble in acetone and ethylether.
(iii) Coloring reaction:
Positive in both ninhydrin reaction and Rydon-Smith reaction.
(i~) Ultra~iolet absorption spectrum:
0.1% methanol solution exhibits only the end absorption due to the peptide bond, but no maximum absorption is shown in the region from 250 m~ to ` 20 (v)~ Infrared absorption spectrum: ~ ;
; as shown in Fig. 1 of the accompanying drawings : . :...
(vi) Composition of amino acid: ~:
After hydrolysis with 6N HCL for 72 hours at 110C, ~
the sample was analyzed by means of amino acid `
a~alyzer.~ A molar ratio of alanine and valine of , 1 : 2 was confirmed.
(~ii) Molecular weight and structural formula: ~,~
Sample was acetylated by acid chIorids method, ;`~ followed by methyl-esterification by diazo methane method. The resulting compound was subjected to ;~
...
mass spectrum analysis and determined to M+ = 657.
Thus, the molecular weight of the sample was . ~
i i, . : ~
^l:
~3~7 identical with the calculation value 601 of DA- ~ -pepsidine. The peak of fragment was also identical with the calculation value derived from the struc~
; tural formula (I) of DA-pepsidine. In addition, aforementioned acetylated product of the sample ~-was identical with pepsidine C on thin layer chrom~
atography, and the esterified product was also identical with methyl ester of pepsidine C.
(viii) Pepsin-inhibitory activity: I
Pepsin-inhibitory activity oE DA-pepsidine was determined using the method~reported by S. Murao and S. Satoi in Agr. Biol. Chem. ~apan 34 (8)~1265 1 - 7 (1970).
. .
, It was shown that the amount of DA-pepsidine which gives 50% inhibition against 100 ~g of pepsin in 0.82 ~g. While, that of pepsidine C is 0.86 ~g.
The present invention~will be further il~lustrated by way of~the following Examples.
Example l Into 0.5 litre Sakaguchi flask, was charged 0.1 litre 1 . . . .
of liquid medium (pH 7) containing I% of meat extract, 1~ of pep~
`; tone and 0.5% of NaCl, and the medium was sterilized at 120C for 10 minutes.
Each 20 flask prepared as above was inoculated respect~
lvely by l ml~of~the broth: of~3aaillus pumilus ~ ~EF 49 210 strain which was cultivated previously on the similax nutrient ~;~
medium at the temperature~;of 30C~for 24 hours , and~cultivated .
~ith shaking at~30C for~ 72~hours.
After completion of the~cultivation, the cells ~ere remo~ed from the broth~by;centrifugatlon, and to be combined supernatant 4 litres of~cold acetone~were added dropwise with ;~
cooling. The resulting preclpitate was suspended in di tilled water to provide 50 ml o~ suspension.
10 g of N-acyl-pentapeptide mlxture (pepsidine C, pepsidine B and pepsidine A in the weight ratio of 94 : 4 : 2) were dissolved into water and 6N NaOH was added to neutralize, thereby pro~iding 500 ml of aqueous solution. Into the aqueous solution, 10 ml of the suspension of aforementioned acetone precipitate were added, and the mixture was stirred in pH 8.5 at 37C for 5 hours.
The resulting reaction solution was extracted three times with each 500 ml of n-butyl alcohol. These n-butyl alcohol layers w~re combined, evaporated to dryness under reduced pressure to obtain 9.8 g of the residue. The residue was dissolved in 90 ml of solvent mixture containing n-butyl alcohol, acetic acid, water and n-butyl acetate in the volume ratio of 4 ~ 4.
The resulting solution was charyed in a column filled with 500 g of silica gel, and t-hen subjected to column chromatography by developing with the solvent mixture as above mentioned.
About 0.75 Iitres of the main fraction were e~apor- `
ated to dryness under reduced pressure to obtain 0.7 g of - ~ .
residue. The residue was crystallized from ethyl alcohol to ;-obtain 0.3 g of crude crystals. Said crude crystals were further ., ~
recrystallized from ethyl alcohol to obtain 0.17 g of DA-pepsidine in a form of white n edles.
Melting point: Decomposed at 193C to 199C, and clear melting point was not observed.
[a¦20 = -S7 to -60 (C = l; methanol) Elementary analysis:for C29H55N5O8 H2O
C% ~% N% _ Calculated: 56.19 9.26 11.29 Found: 56.39 8.99 11.27 Example 2 : Into 30 1itre jar-fermentor, were charged 15 litres of ;-liquid nutrient medium (pH 7~ containing 1% of meat extract, ~;
1% of peptone and 0.5% of NaCl, and then the medium was , sterilized at 120C ~or 10 minutes.
The broth of EF 49-210 strain was prepared by cultivat-ing the bacterium at 30C for 24 hours with shaking on the similar nutrient medium as afore-mentioned. :~
Into said jar-fermentor, 0.2 litres of the broth of said EF 49-210 strain were inoculated and the cultivation was carried out under the following conditions:
Time: 24 hours Tamperature: 30C
Aeration: 15 litres per mlnute Stirring: 350 r.p.m.
After -the cultivation, the microorganisms were removed from the broth by centrifugation. Into 12 litres of the super-natant, ammonium sulfate was added to 0.8 saturation, and the mixture was placed in refrigerator for three days. The resulting precipitates were suspended in distilled water to obtain 750 ml of suspension. ~"
A mixture of 100 ml of the suspension, 400 ml of the neutraliæed aqueous solution containîng 8 g of pepsidine C~ 292 ml of 1/15 M phosphate buffer (pH 5.5) and 8 ml of the solution of CoC12 ~5 x 10 2M) was incubated for 40 hours at 37C. The reaction ~`
mixture was extracted 3 times with 800 ml of n-butyl alcohol, ~`
respectively. The combined extracts were evaporated to dryness. `
The residue was dissolved in a minimum volume of the solvent mixture containing n-butyl alcohol, acetic acid, water and n~
butyl acetate in the volume ratios of 4 : 1 : 1 4, and was ~ ;
subjected to silica gelcolumn chromatography with the similar solvent mixture as developing solvent.
Two litres of main fractions were combined and evapor- -ated to dryness to give 6 g of white powder. The powder was crystallized twice from ethanol to give 2 g of DA-pepsidine in a form of white needles.
3~
Example 3 100 Mg of DA-pepsidine obtained by the process of Example 1 were dissolved in 10 ml of pyridine. 1.2 Ml of the 20-fold diluted acetylchloride solution in acetone were dropped into the above pyridine solution with ice-cooling. The mixture was allowed to stand over-night. An aliquot of this reaction mixture ;~
~ was subjected to silica gel thin layer chromatography by using ; the developing solvent conta~ning n-butanol, acetic acid, water ;
and n-butyl acetate in the volume ratios of 4 ~ 4, follow~
ed by the dete~tion of ninhydrin reaction, Rydon-Smith reaction, and casein plate method, whexeby the resulting product exhibited Rf = 0.49, and it was identified with authentic pepsidine C.
The reaction solution was evaporated to dryness. The residue was dissolved in 10 ml of methanol. Into the resulting solution, 17 ml of ether solution of diazomethane were added.
The solution was allowed to stand at room temperature for three hours, and then evaporated to dryness. The residue was dissolved in methanol. By the addition of ether, 3~ mg of methylester of ` ~;
; pepsidine C. were crystallized. -Example 4 The procedure in Example 3 was repeated except that ~
isovaleryl-chloride was used in place of acetyl chloride. The - ;;
product exhibits Rf = 0.64 on the thin layer chromatography -~
identified with authentic pepstatin A~
Example 5 ;~
One milliliter of the culture broth of EF 49-210 strain cultivated by the similar procedure as Example 1 was mixed with `
one milliliter of aqueous solution which was prepared by~`
dissolving 10 mg of pepsidine C into water and neutralized by ~,;;
6N NaOH solution. The resulting solution was shaked at 37C for 5 hours `~
The reaction solution was subjected to the silica gel 'i - 12 -thin layer chromatography by developing with the solvent contain-ing n-butanol, acetic acid and water in the ratlos by volume of 3 : 1 : 1. The product exhibits Rf = 0.48, ~nd is identified with authentic DA-pepsidine.
Example 6 The procedure in Example 5 was repeated except that , ~'~
pepsidine C was replaced with pepsidine B or pepsidine A.
' Formation of DA-pepsidine was detected with thin layer chromato- '~;
graphy in the respective cases.
Example 7 ~' ~` The procedure in Example 5 was repeated except that N-,, isovaleryl pentapeptide ~pepstatin A) was substituted for pepsidine C. There was also detected the formation of DA-pepsidine ~, with thin layer chxomatography.
,~ Example 8 '~
~,~ The procedure in Example 5 was repeated, except that ~ ' ';~ N-n-hexanoyl-pentapeptide or N-n-decanoyl-pentapeptide was replaced with pepsidine C. The production of DA-pepsidine was '~
also confirmed with thin layer chromatography, in the respective ~ ', 20 ~ cases. ~' s~ Example 9 ~ The procedure in Example 5 was repeated except that ;, "~
", Bacil'lus'pumi'lus' Kawaguchi EF 49-210 was replaced with B'ac'illus ~;;`~' . . _ . :
'~i, pumilus IFO 12092 or Bacillus''pumi'lus IFO 12110. Formation ~ of DA-pepsidine was detected with thin layer chromatography, ' '; in each case.
'?
Example 10 :~i The procedure in Example 5 was repeated except that , ~' ' one milliliter of the culture broth of EF 49-210 strain was "~i 30 replaced 10 mg of acetone-dried cells of the bacterium. The ~', " formation of DA-pepsidine was detected with thin layer chromato- '~ ' r~ graphy.
, . .~ ', ~,',' ~ ~ 13 ~
; 15 starch - +
.
;l No gas formed in any of the carbohydrate.
Acid (+), Sight acid (+), No acid (~
By comparing the data of the above mentioned properties of the bacterium with those described in Bergey's Manual of Determinative Bacteriology, the seventh edition, this bacterium is considered to be a variant of Bacillu pumilus, and the said ~ 5 ~
.' .,., .. .,......... .. , ... ' bacterium was nominated as Bacillus ~ llus ~ chi.
This bacterium EF 49-210 (ATCC No. 31132) strain has been deposi-ted to Agency of Industrial Science and Technology, Fermentation Research Institute in Japan as FERM-P No. 2677. It was also recognized that there is suitably used for the process of ' ; the present invention the known bacterium B~clllus pumilus IFO-12092 and IFO-12110 which were stored in Institute of ~;
Fermentation Osaka in Japan.
In the cultivation of the microorganisms to be used in ~ ~' the process of the present in~ention, there may be used any comp~
osition of the nutrient medium and any conditions for the culti~
vation, provided that the microorganisms can grow, and fully exhibit desired activities. There may be selected, a medium containing for example, any organic or inorganic nitrogen sources such as peptone, meat extract, corn steep liquor, soy-bean hydro- ' . ~ .
lyzate, soy-bean extract, yeast extract and inorganic ammonium salts; any carbon sources such as molasses, dextrose, starch ~' ' and hydrolysis products thereof; and inorganic salts. The cultivation is generally carried out with shaking or aerating . ...
at a temperature ranging from 20~C to 45~C for a period of time from one to se~en days.
It was found that the deacylating activity of the Bacillus pumilus exists both intra- and extra- cellularly. Thus, ' :' :
in the process of the present invention, there may be used cuIture broth, culture ~iltrate, ~hole cells, dry cells and/or enzyme preparations obtained therefrom. ` `
The reactant N-acyl-pen~apeptide, is not always ' '~ ' required to use in a purified form. There may be used, for example, a mixture of several N-~acyl-pentapeptides which may ,~. . .
be obtained in various purification stages such as culture filtrate of the microorganism which produces N-acyl-pentapep~
tides, and products salted out from said culture filtrate.
~'~, ' ' ~
i3~
Products obtained by extracting the culture fil.~rate with an ~ ~;
organic solvent and then removing the solvent may also be used.
In the process of the present invention, the reaction conditions such as concentration, temperature and pH, are not critical and can be varied over broad range depending upon the kinds of the strain of microorganisms used and the N-acyl- ;
pentapeptide selected as starting material. When deacylation of N-acyl-pentapeptide is carried out using Bacillus pumilus Kawaguchi EF 49-210 strain, for example, it is preferable to use ~ ~
conditions of pH 4 to 10 and at temperature of about 30 - 50C. . ;
A reaction time of from 3 to 60 hours is suitable. By addition `
of a Co compound such as CoC12, and CoSO4 the yield of DA~
pepsidine may be increased.
Separation and purifica-tion of DA-pepsidine from the :
~ reaction mixture can be carried out using any con~entional proce- :
- aure such as solvent extraction, column chromatography, fraction~ `~
al crystallization and recrystalli~ation. `
To detect the formation of DA-pepsidine thin layer chromatography can be used as follows:
Sample solution is spotted on a thi.n layer plate of -~
Silica Gel G (a trademark of Merck A. G.), and developed with a : mixed solvent (I) containing n-butyl alcohol, acetic acid and water in the ratios by volume of 3 : 1 : 1, or a mixed solvent (II) containing n butyl alcohol, acetic acid, water and n-butyl-: acetate in the ratios by vo].ume of 4 : 1 : 1 : 4. DA-pepsidine ;
is detected as a spot which is positive in the ninhydrin reaction and also Rydon-Smith reaction, at Rf - 0~48 in the case of the use of the mixed solvent (I). ~ ~-Alternatively, it is detected by Casein plate method :
as follows:
After the de~eloped and dried thin layer plate mentioned above is transferred to a flat board of Casein-containing agar, ~-, ~i ' ;
a filter paper impreynated with pepsin solution is placed on the board. Reaction is then effected at 30C for overnight, to detect a non-decomposed casein.
DA-pepsidine of the present invention has the following physicochemical properties:
(i) Appearance:
White needles (ii) Solubility~
Readily soluble in acetic acid and methanol; soluble -:
in ethanol, n-hu~anol and pyridine; and slightly soluble in acetone and ethylether.
(iii) Coloring reaction:
Positive in both ninhydrin reaction and Rydon-Smith reaction.
(i~) Ultra~iolet absorption spectrum:
0.1% methanol solution exhibits only the end absorption due to the peptide bond, but no maximum absorption is shown in the region from 250 m~ to ` 20 (v)~ Infrared absorption spectrum: ~ ;
; as shown in Fig. 1 of the accompanying drawings : . :...
(vi) Composition of amino acid: ~:
After hydrolysis with 6N HCL for 72 hours at 110C, ~
the sample was analyzed by means of amino acid `
a~alyzer.~ A molar ratio of alanine and valine of , 1 : 2 was confirmed.
(~ii) Molecular weight and structural formula: ~,~
Sample was acetylated by acid chIorids method, ;`~ followed by methyl-esterification by diazo methane method. The resulting compound was subjected to ;~
...
mass spectrum analysis and determined to M+ = 657.
Thus, the molecular weight of the sample was . ~
i i, . : ~
^l:
~3~7 identical with the calculation value 601 of DA- ~ -pepsidine. The peak of fragment was also identical with the calculation value derived from the struc~
; tural formula (I) of DA-pepsidine. In addition, aforementioned acetylated product of the sample ~-was identical with pepsidine C on thin layer chrom~
atography, and the esterified product was also identical with methyl ester of pepsidine C.
(viii) Pepsin-inhibitory activity: I
Pepsin-inhibitory activity oE DA-pepsidine was determined using the method~reported by S. Murao and S. Satoi in Agr. Biol. Chem. ~apan 34 (8)~1265 1 - 7 (1970).
. .
, It was shown that the amount of DA-pepsidine which gives 50% inhibition against 100 ~g of pepsin in 0.82 ~g. While, that of pepsidine C is 0.86 ~g.
The present invention~will be further il~lustrated by way of~the following Examples.
Example l Into 0.5 litre Sakaguchi flask, was charged 0.1 litre 1 . . . .
of liquid medium (pH 7) containing I% of meat extract, 1~ of pep~
`; tone and 0.5% of NaCl, and the medium was sterilized at 120C for 10 minutes.
Each 20 flask prepared as above was inoculated respect~
lvely by l ml~of~the broth: of~3aaillus pumilus ~ ~EF 49 210 strain which was cultivated previously on the similax nutrient ~;~
medium at the temperature~;of 30C~for 24 hours , and~cultivated .
~ith shaking at~30C for~ 72~hours.
After completion of the~cultivation, the cells ~ere remo~ed from the broth~by;centrifugatlon, and to be combined supernatant 4 litres of~cold acetone~were added dropwise with ;~
cooling. The resulting preclpitate was suspended in di tilled water to provide 50 ml o~ suspension.
10 g of N-acyl-pentapeptide mlxture (pepsidine C, pepsidine B and pepsidine A in the weight ratio of 94 : 4 : 2) were dissolved into water and 6N NaOH was added to neutralize, thereby pro~iding 500 ml of aqueous solution. Into the aqueous solution, 10 ml of the suspension of aforementioned acetone precipitate were added, and the mixture was stirred in pH 8.5 at 37C for 5 hours.
The resulting reaction solution was extracted three times with each 500 ml of n-butyl alcohol. These n-butyl alcohol layers w~re combined, evaporated to dryness under reduced pressure to obtain 9.8 g of the residue. The residue was dissolved in 90 ml of solvent mixture containing n-butyl alcohol, acetic acid, water and n-butyl acetate in the volume ratio of 4 ~ 4.
The resulting solution was charyed in a column filled with 500 g of silica gel, and t-hen subjected to column chromatography by developing with the solvent mixture as above mentioned.
About 0.75 Iitres of the main fraction were e~apor- `
ated to dryness under reduced pressure to obtain 0.7 g of - ~ .
residue. The residue was crystallized from ethyl alcohol to ;-obtain 0.3 g of crude crystals. Said crude crystals were further ., ~
recrystallized from ethyl alcohol to obtain 0.17 g of DA-pepsidine in a form of white n edles.
Melting point: Decomposed at 193C to 199C, and clear melting point was not observed.
[a¦20 = -S7 to -60 (C = l; methanol) Elementary analysis:for C29H55N5O8 H2O
C% ~% N% _ Calculated: 56.19 9.26 11.29 Found: 56.39 8.99 11.27 Example 2 : Into 30 1itre jar-fermentor, were charged 15 litres of ;-liquid nutrient medium (pH 7~ containing 1% of meat extract, ~;
1% of peptone and 0.5% of NaCl, and then the medium was , sterilized at 120C ~or 10 minutes.
The broth of EF 49-210 strain was prepared by cultivat-ing the bacterium at 30C for 24 hours with shaking on the similar nutrient medium as afore-mentioned. :~
Into said jar-fermentor, 0.2 litres of the broth of said EF 49-210 strain were inoculated and the cultivation was carried out under the following conditions:
Time: 24 hours Tamperature: 30C
Aeration: 15 litres per mlnute Stirring: 350 r.p.m.
After -the cultivation, the microorganisms were removed from the broth by centrifugation. Into 12 litres of the super-natant, ammonium sulfate was added to 0.8 saturation, and the mixture was placed in refrigerator for three days. The resulting precipitates were suspended in distilled water to obtain 750 ml of suspension. ~"
A mixture of 100 ml of the suspension, 400 ml of the neutraliæed aqueous solution containîng 8 g of pepsidine C~ 292 ml of 1/15 M phosphate buffer (pH 5.5) and 8 ml of the solution of CoC12 ~5 x 10 2M) was incubated for 40 hours at 37C. The reaction ~`
mixture was extracted 3 times with 800 ml of n-butyl alcohol, ~`
respectively. The combined extracts were evaporated to dryness. `
The residue was dissolved in a minimum volume of the solvent mixture containing n-butyl alcohol, acetic acid, water and n~
butyl acetate in the volume ratios of 4 : 1 : 1 4, and was ~ ;
subjected to silica gelcolumn chromatography with the similar solvent mixture as developing solvent.
Two litres of main fractions were combined and evapor- -ated to dryness to give 6 g of white powder. The powder was crystallized twice from ethanol to give 2 g of DA-pepsidine in a form of white needles.
3~
Example 3 100 Mg of DA-pepsidine obtained by the process of Example 1 were dissolved in 10 ml of pyridine. 1.2 Ml of the 20-fold diluted acetylchloride solution in acetone were dropped into the above pyridine solution with ice-cooling. The mixture was allowed to stand over-night. An aliquot of this reaction mixture ;~
~ was subjected to silica gel thin layer chromatography by using ; the developing solvent conta~ning n-butanol, acetic acid, water ;
and n-butyl acetate in the volume ratios of 4 ~ 4, follow~
ed by the dete~tion of ninhydrin reaction, Rydon-Smith reaction, and casein plate method, whexeby the resulting product exhibited Rf = 0.49, and it was identified with authentic pepsidine C.
The reaction solution was evaporated to dryness. The residue was dissolved in 10 ml of methanol. Into the resulting solution, 17 ml of ether solution of diazomethane were added.
The solution was allowed to stand at room temperature for three hours, and then evaporated to dryness. The residue was dissolved in methanol. By the addition of ether, 3~ mg of methylester of ` ~;
; pepsidine C. were crystallized. -Example 4 The procedure in Example 3 was repeated except that ~
isovaleryl-chloride was used in place of acetyl chloride. The - ;;
product exhibits Rf = 0.64 on the thin layer chromatography -~
identified with authentic pepstatin A~
Example 5 ;~
One milliliter of the culture broth of EF 49-210 strain cultivated by the similar procedure as Example 1 was mixed with `
one milliliter of aqueous solution which was prepared by~`
dissolving 10 mg of pepsidine C into water and neutralized by ~,;;
6N NaOH solution. The resulting solution was shaked at 37C for 5 hours `~
The reaction solution was subjected to the silica gel 'i - 12 -thin layer chromatography by developing with the solvent contain-ing n-butanol, acetic acid and water in the ratlos by volume of 3 : 1 : 1. The product exhibits Rf = 0.48, ~nd is identified with authentic DA-pepsidine.
Example 6 The procedure in Example 5 was repeated except that , ~'~
pepsidine C was replaced with pepsidine B or pepsidine A.
' Formation of DA-pepsidine was detected with thin layer chromato- '~;
graphy in the respective cases.
Example 7 ~' ~` The procedure in Example 5 was repeated except that N-,, isovaleryl pentapeptide ~pepstatin A) was substituted for pepsidine C. There was also detected the formation of DA-pepsidine ~, with thin layer chxomatography.
,~ Example 8 '~
~,~ The procedure in Example 5 was repeated, except that ~ ' ';~ N-n-hexanoyl-pentapeptide or N-n-decanoyl-pentapeptide was replaced with pepsidine C. The production of DA-pepsidine was '~
also confirmed with thin layer chromatography, in the respective ~ ', 20 ~ cases. ~' s~ Example 9 ~ The procedure in Example 5 was repeated except that ;, "~
", Bacil'lus'pumi'lus' Kawaguchi EF 49-210 was replaced with B'ac'illus ~;;`~' . . _ . :
'~i, pumilus IFO 12092 or Bacillus''pumi'lus IFO 12110. Formation ~ of DA-pepsidine was detected with thin layer chromatography, ' '; in each case.
'?
Example 10 :~i The procedure in Example 5 was repeated except that , ~' ' one milliliter of the culture broth of EF 49-210 strain was "~i 30 replaced 10 mg of acetone-dried cells of the bacterium. The ~', " formation of DA-pepsidine was detected with thin layer chromato- '~ ' r~ graphy.
, . .~ ', ~,',' ~ ~ 13 ~
Claims (10)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of desacyl-pepsidine which comprises contacting at least one N-acyl-pentapeptide having the formula (II):
(II) wherein R is an acyl group, with a bacterium belonging to Bacillus pumilus.
(II) wherein R is an acyl group, with a bacterium belonging to Bacillus pumilus.
2. The process as claimed in claim 1 wherein the reac-tion time ranges from about 3 to 60 hours.
3. The process as claimed in claim 1 wherein the bacterium is Bacillus pumilus kawaguchi FE 49-210 (ATCC No. 31132).
4. The process as claimed in claim 1 wherein the bac-terium is Bacillus pumilus IFO-12092.
5. The process as claimed in claim 1 wherein the bac-terium is Bacillus pumilus IFO-12110.
6. The process as claimed in claim l wherein the bac-terium is used in the form of its culture broth, culture filtrate, whole cell, dry cell or enzyme preparation thereof.
7. The process as claimed in claim 1 wherein the N-acyl-pentapeptide is an N-acyl-pentapeptide- containing material selected from the group consisting of culture filtrate of the microorganism which produces N-acyl-pentapeptide, products salted out from the culture filtrate, and dried products of an extract thereof with an organic solvent.
8. The process as claimed in claim 1 wherein Co++ com-pound is added.
9. The process as claimed in claim 1 wherein CoCl2 or CoSO4 is added.
10. Desacyl-pepsidine having the formula:
when prepared by the process as claimed in claim 1, 2 or 3 or an obvious chemical equivalent thereof.
when prepared by the process as claimed in claim 1, 2 or 3 or an obvious chemical equivalent thereof.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP49138862A JPS5167791A (en) | 1974-12-05 | 1974-12-05 | Desuashiruu oyobi desuashirubariruupepushijinno seizoho narabini saiashirukaho |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1053167A true CA1053167A (en) | 1979-04-24 |
Family
ID=15231860
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA241,153A Expired CA1053167A (en) | 1974-12-05 | 1975-12-05 | Desacyl-pepsidine from bacillus pumilus |
Country Status (8)
Country | Link |
---|---|
JP (1) | JPS5167791A (en) |
CA (1) | CA1053167A (en) |
CH (1) | CH620244A5 (en) |
DE (2) | DE2554636C2 (en) |
FR (1) | FR2293215A1 (en) |
GB (1) | GB1530921A (en) |
NL (1) | NL7514241A (en) |
SE (1) | SE423710B (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2640420C2 (en) * | 1975-09-12 | 1985-01-10 | Sumitomo Chemical Co., Ltd., Osaka | Peptides designated as procidins, processes for their production and agents containing these compounds |
JPS56164153A (en) * | 1980-05-20 | 1981-12-17 | Eisai Co Ltd | Novel desacylvalypepsidin derivative and its preparation |
JPS56164152A (en) * | 1980-05-20 | 1981-12-17 | Eisai Co Ltd | Novel desacylpepsidin derivative and its preparation |
JPS57189651U (en) * | 1981-05-27 | 1982-12-01 | ||
JPS5956948A (en) * | 1982-09-28 | 1984-04-02 | Nippon Kokan Kk <Nkk> | Mold for horizontal continuous casting machine |
JPS60213347A (en) * | 1984-04-06 | 1985-10-25 | Kawasaki Heavy Ind Ltd | Billet drawing device for continuous casting |
-
1974
- 1974-12-05 JP JP49138862A patent/JPS5167791A/en active Granted
-
1975
- 1975-12-04 DE DE2554636A patent/DE2554636C2/en not_active Expired
- 1975-12-04 DE DE2560238A patent/DE2560238C2/en not_active Expired
- 1975-12-04 SE SE7513671A patent/SE423710B/en not_active IP Right Cessation
- 1975-12-04 FR FR7537098A patent/FR2293215A1/en active Granted
- 1975-12-04 CH CH1581875A patent/CH620244A5/en not_active IP Right Cessation
- 1975-12-05 NL NL7514241A patent/NL7514241A/en not_active Application Discontinuation
- 1975-12-05 GB GB49988/75A patent/GB1530921A/en not_active Expired
- 1975-12-05 CA CA241,153A patent/CA1053167A/en not_active Expired
Also Published As
Publication number | Publication date |
---|---|
FR2293215B1 (en) | 1978-07-28 |
CH620244A5 (en) | 1980-11-14 |
SE7513671L (en) | 1976-06-08 |
JPS5339514B2 (en) | 1978-10-21 |
DE2554636C2 (en) | 1983-02-17 |
FR2293215A1 (en) | 1976-07-02 |
JPS5167791A (en) | 1976-06-11 |
DE2560238C2 (en) | 1984-07-19 |
GB1530921A (en) | 1978-11-01 |
SE423710B (en) | 1982-05-24 |
DE2554636A1 (en) | 1976-06-10 |
NL7514241A (en) | 1976-06-09 |
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