JPH0378877B2 - - Google Patents
Info
- Publication number
- JPH0378877B2 JPH0378877B2 JP61243233A JP24323386A JPH0378877B2 JP H0378877 B2 JPH0378877 B2 JP H0378877B2 JP 61243233 A JP61243233 A JP 61243233A JP 24323386 A JP24323386 A JP 24323386A JP H0378877 B2 JPH0378877 B2 JP H0378877B2
- Authority
- JP
- Japan
- Prior art keywords
- substance
- kbs2
- amino acid
- ether
- absorption spectrum
- 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.)
- Expired - Lifetime
Links
- 239000000126 substance Substances 0.000 claims description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 8
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 238000000862 absorption spectrum Methods 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000004380 ashing Methods 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 2
- 238000000921 elemental analysis Methods 0.000 claims description 2
- 239000000194 fatty acid Substances 0.000 claims description 2
- 229930195729 fatty acid Natural products 0.000 claims description 2
- 150000004665 fatty acids Chemical class 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 2
- 239000003208 petroleum Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims 2
- 239000002244 precipitate Substances 0.000 description 10
- 239000012153 distilled water Substances 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000012043 crude product Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000000843 anti-fungal effect Effects 0.000 description 2
- 239000003125 aqueous solvent Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Compounds Of Unknown Constitution (AREA)
Description
本発明は抗真菌活性を有する物質KBS2−P104
及びその製造法に関するものである。
本発明者らは、バチルス属菌の生産する抗菌性
低分子物質を探索中バチルス属の一菌株が抗真菌
活性物質を生産することを知り、更に、該物質が
新規物質であることを確認し、これを物質KBS2
−P104と命名し、本発明を完成するに到つた。
本発明で使用する菌は物質KBS2−P104を生産
するものであればいずれでもよいが、例示として
はバチルス(Bacillus)KB−S2があげられる。
バチルスKB−S2は微工研にFERM P−8985と
して寄託されており、また菌学的性質の概要は次
の通りである。
グラム染色 +
胞子染色 +
カタラーゼテスト +
オキシダーゼテスト +
物質KBS2−P104生産菌の培養培地は、資化で
きる炭素源、窒素源、ビタミン等を含有する栄養
料等適宜含有したもので、物質KBS2−P104を生
産蓄積する培地であればいずれの培地でもよい。
バチルスKB−S2の培養に適した培地は例えば
次のものがあげられる。
ポリペプトン 30g
酵母エキス 5g
NaCl 5g
脱イオン水 1
(PH7.0)
培養は25〜30℃で、通気、撹拌、振とう等好気
的に4日間程度行なわれる。
得られた培養液は濾過して濾液を得、これに
6N HClを添加し、PH=3.0に調整し沈澱物を得
る。沈澱物は水に溶解し、NaHCO3を加え、PH
=7.0とし、次いでエタノールを80%まで加える。
得られた上清液を真空乾燥し、乾燥物を蒸留水に
溶解し、再び6N HClを加えてPH=3.0に調整し、
沈澱物を得る。得られた沈澱物を蒸留水に溶解
し、NaHCO3を加え、PH=7.0とし、蒸留水に対
して透析する。透析内液を凍結乾燥することによ
つて物質KBS2−P104含有粗精製物を黄色粉末と
して得る。
得られた黄色粉末を水に溶解し、アセトニトリ
ル系で高速液体クロマトにかけ、これを単離し、
凍結乾燥することによつてほぼ純品の物質KBS2
−P104を白色無定形粉末として得る。
得られた物質KBS2−P104の理化学的性質は次
の通りである。
1 元素分析値
C:49.23%
H:6.98%
N:11.08%
Ash:1.56%
2 灰化点
180〜182℃
3 物質の色及び性状:白色粉体
4 紫外線吸収スペクトル:第1図に示す通り。
5 赤外線吸収スペクトル:第2図に示す通り。
6 溶剤に対する溶解性:水、メタノール、エタ
ノール、アセトニトリルに可溶。
n−プロピルアルコール、アセトン、酢酸エ
チル、エーテル、クロロホルム、ジエチルエー
テル、石油エーテルに不溶。
7 呈色反応
ニンヒドリンテスト −
8 アミノ酸組成:アミノ酸分析機によるアミノ
酸組成は次の通りである。
The present invention provides a substance KBS2-P104 having antifungal activity.
and its manufacturing method. While searching for antibacterial low-molecular-weight substances produced by bacteria of the genus Bacillus, the present inventors discovered that a strain of the genus Bacillus produces an active antifungal substance, and further confirmed that the substance was a new substance. , this is the substance KBS2
-P104 and completed the present invention. The bacteria used in the present invention may be any species as long as it produces the substance KBS2-P104, and an example thereof is Bacillus KB-S2.
Bacillus KB-S2 has been deposited with the Microtech Institute as FERM P-8985, and the outline of its mycological properties is as follows. Gram staining + spore staining + catalase test + oxidase test + The culture medium for the substance KBS2-P104 producing bacteria contains appropriate nutrients such as assimilable carbon sources, nitrogen sources, vitamins, etc. Any medium may be used as long as it produces and accumulates. Examples of suitable media for culturing Bacillus KB-S2 include the following. Polypeptone 30g Yeast extract 5g NaCl 5g Deionized water 1 (PH7.0) Cultivation is carried out aerobically for about 4 days at 25-30°C with aeration, stirring, shaking, etc. The obtained culture solution was filtered to obtain a filtrate, and this
Add 6N HCl to adjust the pH to 3.0 to obtain a precipitate. The precipitate was dissolved in water, added NaHCO3 , and the PH
= 7.0 and then add ethanol to 80%.
The obtained supernatant liquid was vacuum dried, the dried product was dissolved in distilled water, and 6N HCl was added again to adjust the pH to 3.0.
Obtain a precipitate. The resulting precipitate is dissolved in distilled water, NaHCO 3 is added to adjust the pH to 7.0, and the solution is dialyzed against distilled water. A crude product containing substance KBS2-P104 is obtained as a yellow powder by freeze-drying the dialysis fluid. The obtained yellow powder was dissolved in water, subjected to high performance liquid chromatography using acetonitrile system, and isolated.
Almost pure substance KBS2 by freeze-drying
- P104 is obtained as a white amorphous powder. The physical and chemical properties of the obtained substance KBS2-P104 are as follows. 1 Elemental analysis values C: 49.23% H: 6.98% N: 11.08% Ash: 1.56% 2 Ashing point 180-182°C 3 Color and properties of substance: White powder 4 Ultraviolet absorption spectrum: As shown in Figure 1. 5 Infrared absorption spectrum: As shown in Figure 2. 6 Solubility in solvents: Soluble in water, methanol, ethanol, and acetonitrile. Insoluble in n-propyl alcohol, acetone, ethyl acetate, ether, chloroform, diethyl ether, petroleum ether. 7 Color reaction Ninhydrin test - 8 Amino acid composition: The amino acid composition determined by an amino acid analyzer is as follows.
【表】
9 物質1分子あたり構造未決定の脂肪酸が1分
子存在する。
10 本物質は真菌に対して強い抗菌性を示す。
11 精製法:PH=3.0程度で沈澱させ、沈澱物を
PH=7.0で溶解する工程をくりかえし、最後に
高速液体クロマトにかけることによつてほぼ純
品を得ることができる。高速液体クロマトにお
けるÅ220による吸光度曲線は第3図に示す通
りである。矢印が物質KBS2−P104を示してい
る。
なお、高速液体クロマトはカラムサイズφ1.0
×20cm、ゲルは日立ゲル3053(シリカ系)、溶出
条件は初発アセトニトリル40%、トリフルオロ
酢酸0.1%の水性溶媒で、最終(40分後)アセ
トニトリル60%、トリフルオロ酢酸0.1%の水
性溶媒になる直線濃度勾配によつて行つた。
12 抗菌スペクトラム:物質KBS2−P104の抗菌
活性は次の表1に示される。[Table] 9 There is one molecule of undetermined fatty acid molecule per molecule of substance. 10 This substance exhibits strong antibacterial properties against fungi. 11 Purification method: Precipitate at pH=3.0 and remove the precipitate.
By repeating the dissolution process at pH=7.0 and finally applying it to high performance liquid chromatography, an almost pure product can be obtained. The absorbance curve at Å220 in high performance liquid chromatography is shown in FIG. The arrow indicates substance KBS2-P104. For high-performance liquid chromatography, the column size is φ1.0.
×20cm, the gel is Hitachi Gel 3053 (silica-based), the elution conditions are an initial aqueous solvent of 40% acetonitrile and 0.1% trifluoroacetic acid, and a final (after 40 minutes) aqueous solvent of 60% acetonitrile and 0.1% trifluoroacetic acid. A linear concentration gradient was used. 12 Antibacterial spectrum: The antibacterial activity of substance KBS2-P104 is shown in Table 1 below.
【表】
次に本発明の実施例を示す。
実施例 1
バチルスKB−S2、FERM P−8985を次の培
地に接種して、27℃で4日間振とう培養した。
ポリペプトン 30g
酵母エキス 5g
NaCl 5g
脱イオン水 1
(PH7.0)
得られた培養液を濾過して、培養濾液とし、こ
れを6N HClでPH=3.0とし、しばらく放置する
と沈澱物が得られるので、得られた沈澱物を蒸留
水に溶解し、これにNaHCO3を加え、PH=7.0と
し、更にエタノールを80%まで加え、濾過する。
得られた上清を真空乾燥し、乾固物を得る。この
乾固物を蒸留水に溶解し、6H HClでPH=3.0と
し、しばらく放置し、沈澱物を得る。この沈澱物
を蒸留水に溶解し、NaHCO3でPH=7.0とし、こ
の溶液を蒸留水に対して透析する。
得られた透析液を凍結乾燥し、物質KBS2−
P104含有の粗精製物を黄色粉末で得る。
得られた黄色粉末を高速液体クロマトにかけ、
第3図に矢印で示す物質KBS2−P104該当部分を
とり、これを凍結乾燥することにより、ほぼ純品
の物質KBS2−P104を白色無定形粉末として得
た。[Table] Next, examples of the present invention are shown. Example 1 Bacillus KB-S2, FERM P-8985 was inoculated into the following medium and cultured with shaking at 27°C for 4 days. Polypeptone 30g Yeast extract 5g NaCl 5g Deionized water 1 (PH7.0) Filter the obtained culture solution to obtain a culture filtrate, adjust the pH to 3.0 with 6N HCl, and leave it for a while to obtain a precipitate. The obtained precipitate is dissolved in distilled water, NaHCO 3 is added thereto to adjust the pH to 7.0, ethanol is further added to 80%, and the mixture is filtered.
The obtained supernatant is vacuum dried to obtain a dried product. Dissolve this dry solid in distilled water, adjust the pH to 3.0 with 6H HCl, and leave for a while to obtain a precipitate. The precipitate is dissolved in distilled water, the pH is brought to 7.0 with NaHCO 3 and the solution is dialyzed against distilled water. The resulting dialysate was lyophilized and the substance KBS2−
A crude product containing P104 is obtained as a yellow powder. The obtained yellow powder was subjected to high performance liquid chromatography.
A corresponding portion of the substance KBS2-P104 indicated by the arrow in FIG. 3 was taken and freeze-dried to obtain an almost pure substance KBS2-P104 as a white amorphous powder.
第1図は物質KBS2−P104の紫外線吸収スペク
トルを示す図で、第2図は同じく赤外線吸収スペ
クトルを示す図で、第3図は物質KBS2−P104含
有粗精製物の高速液体クロマトによるÅ220によ
る吸光度曲線を示す図である。矢印は物質KBS2
−P104を示す。
Figure 1 shows the ultraviolet absorption spectrum of the substance KBS2-P104, Figure 2 shows the infrared absorption spectrum, and Figure 3 shows the absorbance at Å220 measured by high performance liquid chromatography of the crude product containing the substance KBS2-P104. It is a figure showing a curve. Arrow indicates substance KBS2
- Indicates P104.
Claims (1)
P104。 1 元素分析値 C:49.23% H:6.98% N:11.08% Ash:1.56% 2 灰化点 180〜182℃ 3 物質の色及び性状:白色粉体 4 紫外線吸収スペクトル:278nmに吸収あり。 5 赤外線吸収スペクトル:1200、1660、2860、
2930、3320cm-1に吸収あり。 6 溶剤に対する溶解性:水、メタノール、エタ
ノール、アセトニトリルに可溶。 n−プロピルアルコール、アセトン、酢酸エ
チル、エーテル、クロロホルム、ジエチルエー
テル、石油エーテルに不溶。 7 呈色反応 ニンヒドリンテスト − 8 アミノ酸組成:アミノ酸分析機によるアミノ
酸組成は次の通りである。 【表】 9 物質1分子あたり構造未決定の脂肪酸が1分
子存在する。 2 バチルス属に属する物質KBS2−P104生産菌
を培養し、物質KBS2−P104を採取することを特
徴とする物質KBS2−P104の製造法。[Claims] 1. A substance having the following physical and chemical properties KBS2-
P104. 1 Elemental analysis values C: 49.23% H: 6.98% N: 11.08% Ash: 1.56% 2 Ashing point 180-182°C 3 Color and properties of substance: White powder 4 Ultraviolet absorption spectrum: Absorption at 278 nm. 5 Infrared absorption spectrum: 1200, 1660, 2860,
There is absorption at 2930 and 3320 cm -1 . 6 Solubility in solvents: Soluble in water, methanol, ethanol, and acetonitrile. Insoluble in n-propyl alcohol, acetone, ethyl acetate, ether, chloroform, diethyl ether, petroleum ether. 7 Color reaction Ninhydrin test - 8 Amino acid composition: The amino acid composition determined by an amino acid analyzer is as follows. [Table] 9 There is one molecule of fatty acid whose structure has not been determined per molecule of substance. 2. A method for producing the substance KBS2-P104, which comprises culturing a substance KBS2-P104-producing bacteria belonging to the genus Bacillus and collecting the substance KBS2-P104.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61243233A JPS6399087A (en) | 1986-10-15 | 1986-10-15 | Kbs2-p104 substance and production thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61243233A JPS6399087A (en) | 1986-10-15 | 1986-10-15 | Kbs2-p104 substance and production thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS6399087A JPS6399087A (en) | 1988-04-30 |
JPH0378877B2 true JPH0378877B2 (en) | 1991-12-17 |
Family
ID=17100815
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61243233A Granted JPS6399087A (en) | 1986-10-15 | 1986-10-15 | Kbs2-p104 substance and production thereof |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6399087A (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0601187A4 (en) * | 1992-03-31 | 1994-09-21 | Higeta Shoyu Kk | Novel antibiotic and production and use thereof. |
-
1986
- 1986-10-15 JP JP61243233A patent/JPS6399087A/en active Granted
Also Published As
Publication number | Publication date |
---|---|
JPS6399087A (en) | 1988-04-30 |
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