JP4113923B2 - Novel alkaloids urubactin D, E, and methods for producing them - Google Patents
Novel alkaloids urubactin D, E, and methods for producing them Download PDFInfo
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- JP4113923B2 JP4113923B2 JP4949297A JP4949297A JP4113923B2 JP 4113923 B2 JP4113923 B2 JP 4113923B2 JP 4949297 A JP4949297 A JP 4949297A JP 4949297 A JP4949297 A JP 4949297A JP 4113923 B2 JP4113923 B2 JP 4113923B2
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- 0 C=COc(cccc1)c1C(*C1)=*C1C(*CC1C2OCC3*22)*12C3=O Chemical compound C=COc(cccc1)c1C(*C1)=*C1C(*CC1C2OCC3*22)*12C3=O 0.000 description 1
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- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
Description
【0001】
【発明の属する技術分野】
本発明は、紫外線吸収物質として有用な新規化合物、および微生物を用いたその製造法に関する。
【0002】
【従来の技術】
従来、化粧品などには紫外線吸収剤としてベンゾフェノン誘導体、パラアミノ安息香酸誘導体、パラメトキシ桂皮酸誘導体、サリチル酸誘導体などの化学合成品が主に使用されてきた。しかし、化学合成品に対する拒否反応も伴って、天然物の中から安全性が高い新規紫外線吸収物質を探索し、それを応用、利用しようとする研究が行われている。天然の紫外線吸収物質としては海藻、海底動物、微細藻、カビ類などが生産するマイコスポリン様アミノ酸類が知られている(ボータニカマリーナ(Bot. Mar.)16, 23 (1974)、マリーンバイオロジー(Mar. Biol.)108, 157 (1991)、ジャーナルオブプランクトンリサーチ(J. Plankton Res.)12, 909 (1990)、プラントディジーズレポーター(Plant Dis. Reptr.)57, 760 (1973))。
【0003】
【発明が解決しようとする課題】
しかしながら、これらの物質は水溶性であるためその応用範囲が限られている。このため、天然物に由来し、脂溶性の紫外線吸収物質が求められていた。
本発明は、かかる技術的背景の下になされたものであり、その目的は、新規な紫外線吸収物質およびその製造方法を提供することにある。
【0004】
【発明を解決するための手段】
本発明者は、アルテロモナス属に属する菌株の培養液から得られた化合物が、従来まったく知られていない新規な化合物であることを見出し、本発明を完成した。
即ち、本発明は、下記の式(I)又は(II)
【0005】
【化3】
【0006】
【化4】
【0007】
で表わされる化合物である。
【0008】
また、本発明は、アルテロモナス属に属し、上記記載の化合物を生産する能力を有する微生物を培地に培養し、培養物中に上記記載の化合物を生成蓄積させ、該培養物から上記記載の化合物を採取することを特徴とする上記記載の化合物の製造法である。
【0009】
【発明の実施の形態】
以下に本発明を詳細に説明する。
(1)本発明の化合物の性質
本発明の一群の新規な化合物は、下記の式(I)又は(II)で表わされる。
【0010】
【化5】
【0011】
【化6】
【0012】
本発明者は、これらの新規化合物のうち、式(I)で表わされる化合物を「ウルバクチン D(Ulbactin D)」、式(II)で表わされる化合物を「ウルバクチン E(Ulbactin E)」と命名した。以下に各化合物の理化学的性質を示す。
【0013】
<ウルバクチン D>
(1) 物質の色:無色
(2) 物質の形状:油状
(3) 分子量:379
(4) 分子式:C16H17N3 O2 S3
(5) 質量分析:高分解FABMS 実測値 380.0566 〔M+H〕+
計算値 380.0561 (C16H18N3 O2 S3 )
(6) 比旋光度:〔α〕D 25+257° (c 0.0033 、クロロホルム中)
(7) 紫外線吸収スペクトル
(メタノール中):λmax 316nm (log ε3.68)、253(4.09)、212(4.44)
(8) 赤外線吸収スペクトル (KBr)
νmax 3442、2934、1659、1622、1597、1491、1230 cm -1
【0014】
(9)1H−NMR(重クロロホルム中で測定、400MHz)
δppm 2.91(dd, J=9.6, 4.8Hz 1H), 3.03(dd, J=11.2, 9.6Hz, 1H),
3.13(dd, J=10.7, 6.4Hz, 1H), 3.28(dd, J=11.2, 8.0Hz, 1H),
3.36(dd, J=11.2, 8.0Hz, 1H), 3.50(d, J=10.7Hz, 1H),
3.97(dd, J=11.2, 4.8Hz, 1H), 4.50(d, J=6.4Hz, 1H),
4.90(qd, J=8.0Hz, 1H), 5.14 (s, 1H), 5.62(d, J=8.0Hz, 1H),
6.86(ddd, J=7.8, 7.6, 1.2Hz, 1H), 6.99 (dd, J=8.4, 1.2Hz, 1H)
7.34(ddd, J=8.4, 7.6, 1.6Hz, 1H), 7.39 (dd, J=7.8, 1.6Hz, 1H)
【0015】
(10)13C−NMR(重クロロホルム中で測定、125MHz)
δppm 34.1 (t), 34.3 (t), 40.4 (t), 61.3 (d), 65.1 (d), 65.3 (d),
69.2 (d),81.0 (d), 116.2 (s), 117.2 (d), 118.9 (d), 130.7 (d), 133.4 (d),159.2 (s), 167.9 (s), 174.2 (s)
(11)溶解性 :メタノール、エタノール、アセトン、酢酸エチルおよびクロロホルムに可溶、水には難溶
(12)呈色反応:塩化第二鉄発色陽性
【0016】
(13)薄層クロマトグラフィー:シリカゲル薄層 (Kieselgel 60 F 254 Art.5554. Merck社製) クロロホルム:メタノール (10:1)展開溶媒でのRf値は0.48
(14)高速液体クロマトグラフィー:Develosil 充填カラムODS-HG-5 4.6mmφ、25cm、野村化学社製、アセトニトリル:水(0〜5分:20%アセトニトリル、5〜6分:20〜36%アセトニトリル、6〜35分:36%アセトニトリル、35〜36分:36〜60%アセトニトリル、36〜50分:60%アセトニトリル)、流速1.0ml/minでの保持時間は44.8分。
【0017】
<ウルバクチン E>
(1) 物質の色:黄色
(2) 物質の形状:油状
(3) 分子量:264
(4) 分子式:C12H12N2 OS2
(5) 質量分析:高分解FABMS 実測値 265.0489 〔M+H〕+
計算値 265.0469 (C12H13N2 OS2 )
(6) 比旋光度:〔α〕D 24 -19 ° (c 0.015、クロロホルム中)
(7) 紫外線吸収スペクトル
(メタノール中):λmax 315nm (log ε3.72)、252(4.10)、213(4.41)
(8) 赤外線吸収スペクトル (KBr)
νmax 3446、1622、1595、1491、1255、1222 cm -1
【0018】
(9)1H−NMR(重クロロホルム中で測定、500MHz)
δppm 3.27(dd, J=10.8, 9.0Hz, 1H), 3.30(dd, J=10.8, 9.0Hz, 1H),
3.98(dd, J=4.3, 1.3Hz, 2H), 5.38(td, J=9.0, 4.0Hz, 1H),
6.37(tdd, J=4.3, 4.0, 2.4Hz, 1H),
6.87(td, J=7.6, 1.2Hz, 1H), 6.99(dd, J=8.2, 1.2Hz, 1H),
7.35(ddd, J=8.2, 7.6, 1.6Hz, 1H), 7.39 (dd, J=7.6, 1.6Hz, 1H)
7.58(dt, J=2.4, 1.3Hz, 1H)
【0019】
(10)13C−NMR(重クロロホルム中で測定、125MHz)
δppm 32.0 (t), 44.6 (t), 80.3 (d), 87.3 (d), 116.1 (s), 117.1 (d),
118.8 (d), 130.7 (d), 133.2 (d), 159.2 (s),162.5 (d), 173.2 (s)
(11)溶解性 :メタノール、エタノール、アセトン、酢酸エチルおよびクロロホルムに可溶、水には難溶
(12)呈色反応:塩化第二鉄発色陽性
【0020】
(13)薄層クロマトグラフィー:シリカゲル薄層 (Kieselgel 60 F 254 Art.5554. Merck社製) クロロホルム:メタノール (10:1)展開溶媒でのRf値は0.63
(14)高速液体クロマトグラフィー:Develosil 充填カラムODS-HG-5 4.6mmφ、25cm、野村化学社製、アセトニトリル:水(0〜5分:20%アセトニトリル、5〜6分:20〜36%アセトニトリル、6〜35分:36%アセトニトリル、35〜36分:36〜60%アセトニトリル、36〜50分:60%アセトニトリル)、流速1.0ml/minでの保持時間は43.5分。
【0021】
(2)本発明の化合物の製造
本発明の化合物は、微生物を培地に培養し、培養物中に本発明の化合物を生成蓄積させ、該培養物から本発明の化合物を採取することにより製造することができる。ここで用いる微生物としては、アルテロモナス属に属し、本発明の化合物を生産する能力を有するものであれば特に限定されず、例えば、アルテロモナス sp. D-12株を挙げることが出来る。また、前記菌株の変異株であって、本発明の化合物を生産する能力を有する菌株も使用することができる。
【0022】
アルテロモナス sp. D-12株は、自然界から新たに単離した株であり、その細菌学的性質については下記の通りである。なお、形態観察は、培地にアルテロモナス sp.D-12株を植菌し、30℃で3〜5日間培養したのちに光学顕微鏡及び透過型電子顕微鏡を用いて行った。培地は、マリンアガー(Bacto Marine Agar 2216, Difco社製)あるいはマリンブロス(Bacto Marine Broth 2216, Difco社製)を用いた。なお、本菌株は培地中の塩化ナトリウム濃度が1%以下では菌の生育が認められないことから、生化学的性状の試験は75%人工海水を用いて培地を作製するか、あるいは培地中に3%塩化ナトリウムを添加することによって行った。
【0023】
a. 形態
1) 細胞の形および大きさ:桿菌、1.6-2.5×0.8-1.1μm
2) 細胞の多形性の有無:無し
3) 運動性の有無、鞭毛の着生状態:有り、極毛。
4) 胞子の有無:無し
5) グラム染色性:陰性
6) 抗酸性の有無:無し
【0024】
【0025】
【0026】
【0027】
【0028】
上記の菌学的性質をバージーズ・マニュアル・オブ・システマティック・バクテリオロジー第8版の分類基準に従って公知の菌種と比較した。本菌株は好気性グラム陰性桿菌で極鞭毛を有し、ブドウ糖を酸化的に分解、オキシダーゼ、カタラーゼが陽性であった。さらに、ゼラチンを液化し、DNAを加水分解し、GC含量が50%以下であることからアルテロモナス属に所属すると考えられ、アルテロモナス スピーシーズ(Alteromonas sp.)と同定した。アルテロモナスsp. D-12株は、平成8年7月12日付けで工業技術院生命工学工業技術研究所にFERM P-15733として寄託されている。
【0029】
本発明に用いる微生物の培養は、通常の微生物の培養方法が用いられる。培地としては、資化可能な炭素源、窒素源、無機物および必要な生育、生産促進物質を程よく含有する培地であれば合成培地、天然培地いずれでも使用可能である。炭素源としてはグルコース、澱粉、デキストリン、マンノース、フラクトース、シュクロース、ラクトース、キシロース、アラビノース、マンニトール、糖蜜などを単独または組み合わせて用いられる。さらに、菌の資化能によっては炭化水素、アルコール類、有機酸なども用いられる。窒素源としては塩化アンモニウム、硫酸アンモニウム、硝酸アンモニウム、硝酸ナトリウム、尿素、ペプトン、肉エキス、酵母エキス、乾燥酵母、コーン・スチープ・リカー、大豆粉、カザミノ酸などが単独または組み合わせて用いられる。そのほか、必要に応じて食塩、塩化カリウム、硫酸マグネシウム、炭酸カルシウム、燐酸二水素カリウム、燐酸水素二カリウム、硫酸第一鉄、塩化カルシウム、硫酸マンガン、硫酸亜鉛などの無機塩類を加える。さらに使用菌の生育やウルバクチン Dおよびウルバクチン Eの生産を促進する微量成分を適当に添加することができる。培養法としては、液体培養法が最も適している。培養温度30〜35℃が適当であり、培養中の培地のPHは7〜8に維持することが望ましい。液体培養で通常3〜5日間培養を行うと、目的物質が培養液中に生成蓄積される。培養物中の生成量が最大に達した時に培養を停止する。
【0030】
培養物からウルバクチン Dおよびウルバクチン Eの単離精製は、微生物代謝生産物をその培養物から単離精製するために常用される方法に従って行われる。例えば培養物を濾過や遠心分離により培養瀘液と菌体に分け、菌体を含水メタノール、含水エタノールなどで抽出する。ついで、抽出液と培養瀘液とを合わせて濃縮し、シリカゲルによるカラムクロマトグラフィー、ゲル濾過、高速液体クロマトグラフィーなどにより、ウルバクチン Dおよびウルバクチン Eを得る。
なお、培養、精製操作中のウルバクチン Dおよびウルバクチン Eの動向は、高速液体クロマトグラフィーによる紫外線吸収を指標として追跡することができる。
【0031】
(3)本発明の化合物の用途
本発明の化合物は、紫外線吸収能が強いので、化粧品などの紫外線吸収成分として利用することができる。
以下に本発明を実施例により具体的に説明する。ただし、本発明はこれら実施例によりその技術的範囲が限定されるものではない。
【0032】
【実施例】
種菌としてアルテロモナスsp. D-12株を用いた。該菌株を、50mlの昆布茶液体培地(昆布茶:10g、グルコース:3.0g、ブイヨン:1.0g、炭酸カルシウム:2.0g、ろ過海水:500ml、蒸留水:500ml、pH=7.4)を入れた300ml三角フラスコ中で、30℃、24時間振盪(100rpm.)培養し、前々培養液を得た。培養中、培地のpHは特に制御しなかった。この前々培養液12mlを、300mlの昆布茶液体培地を入れた1L三角フラスコに植菌し、30℃、8時間振盪(100rpm.)培養し、前培養液を得た。この前培養液全量を3Lの昆布茶液体培地を入れた5Lジャーファーメンターに植菌し、30℃、200rpm、通気量0.2L/minで5日間本培養を行った。
【0033】
このようにして得られた培養液3.3Lを遠心分離し、得られた上澄み液を、酢酸エチル(6.6L)で抽出する。酢酸エチル抽出部を40℃以下で減圧濃縮し、得られた濃縮液を逆相高速液体クロマトグラフィー(Develosil 充填カラムODS-HG-5 20mmφ、25cm、野村化学社製、水:アセトニトリル=80:20〜40:60へ段階的に変化させた)により分画し、2つの画分を得た。1つ目の画分はシリカゲルカラムクロマトグラフィー(Sep-Pak(登録商標)Vac 12cc 、Waters製、ヘキサン-アセトン=10:1)により精製し、ウルバクチン Dを0.6mg を得た.2つ目の画分は、シリカゲルカラムクロマトグラフィー(Sep-Pak(登録商標)Vac 12cc 、Waters製、ヘキサン-酢酸エチル=10:1)により精製し、ウルバクチン Eを0.8mg を得た。
ここで得たウルバクチン Dおよびウルバクチン Eは、前記した理化学的性質を示した。
【0034】
【発明の効果】
本発明は、新規な化合物および該物質の微生物を用いた製造法を提供する。本発明の化合物は、脂溶性で、紫外線吸収能が強いので、化粧品の成分等として利用できる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel compound useful as an ultraviolet absorber and a method for producing the same using a microorganism.
[0002]
[Prior art]
Conventionally, chemical synthetic products such as benzophenone derivatives, paraaminobenzoic acid derivatives, paramethoxycinnamic acid derivatives, salicylic acid derivatives have been mainly used as cosmetics and the like as ultraviolet absorbers. However, with the rejection of chemically synthesized products, research is underway to search for new UV-absorbing substances with high safety from natural products, and to apply and use them. As natural UV-absorbing substances, mycosporine-like amino acids produced by seaweeds, marine animals, microalgae and fungi are known (Botanica Marina (Bot. Mar.) 16, 23 (1974), Marine Biology (Mar. Biol.) 108, 157 (1991), Journal of Plankton Res. 12, 909 (1990), Plant Dis. Reptr. 57, 760 (1973)).
[0003]
[Problems to be solved by the invention]
However, since these substances are water-soluble, their application range is limited. For this reason, a fat-soluble UV-absorbing substance derived from a natural product has been demanded.
The present invention has been made under such a technical background, and an object thereof is to provide a novel ultraviolet absorbing material and a method for producing the same.
[0004]
[Means for Solving the Invention]
The present inventor has found that a compound obtained from a culture solution of a strain belonging to the genus Alteromonas is a novel compound that has not been known so far, and has completed the present invention.
That is, the present invention provides the following formula (I) or (II)
[0005]
[Chemical 3]
[0006]
[Formula 4]
[0007]
It is a compound represented by these.
[0008]
The present invention also relates to a microorganism belonging to the genus Alteromonas and capable of producing the above-described compound, cultivating the medium in the medium, producing and accumulating the above-described compound in the culture, and obtaining the above-described compound from the culture. The method for producing a compound as described above, wherein the compound is collected.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
(1) Properties of the Compound of the Present Invention A group of novel compounds of the present invention is represented by the following formula (I) or (II).
[0010]
[Chemical formula 5]
[0011]
[Chemical 6]
[0012]
The present inventor named the compound represented by the formula (I) among these novel compounds as “Ulbactin D” and the compound represented by the formula (II) as “Ulbactin E”. . The physicochemical properties of each compound are shown below.
[0013]
<Urubactin D>
(1) Material color: colorless
(2) Shape of substance: oily
(3) Molecular weight: 379
(4) Molecular formula: C 16 H 17 N 3 O 2 S 3
(5) Mass spectrometry: High resolution FABMS measured value 380.0566 [M + H] +
Calculated value 380.0561 (C 16 H 18 N 3 O 2 S 3 )
(6) Specific rotation: [α] D 25 + 257 ° (c 0.0033 in chloroform)
(7) UV absorption spectrum
(In methanol): λ max 316nm (log ε3.68), 253 (4.09), 212 (4.44)
(8) Infrared absorption spectrum (KBr)
ν max 3442, 2934, 1659, 1622, 1597, 1491, 1230 cm -1
[0014]
(9) 1 H-NMR (measured in deuterated chloroform, 400 MHz)
δppm 2.91 (dd, J = 9.6, 4.8Hz 1H), 3.03 (dd, J = 11.2, 9.6Hz, 1H),
3.13 (dd, J = 10.7, 6.4Hz, 1H), 3.28 (dd, J = 11.2, 8.0Hz, 1H),
3.36 (dd, J = 11.2, 8.0Hz, 1H), 3.50 (d, J = 10.7Hz, 1H),
3.97 (dd, J = 11.2, 4.8Hz, 1H), 4.50 (d, J = 6.4Hz, 1H),
4.90 (qd, J = 8.0Hz, 1H), 5.14 (s, 1H), 5.62 (d, J = 8.0Hz, 1H),
6.86 (ddd, J = 7.8, 7.6, 1.2Hz, 1H), 6.99 (dd, J = 8.4, 1.2Hz, 1H)
7.34 (ddd, J = 8.4, 7.6, 1.6Hz, 1H), 7.39 (dd, J = 7.8, 1.6Hz, 1H)
[0015]
(10) 13 C-NMR (measured in deuterated chloroform, 125 MHz)
δppm 34.1 (t), 34.3 (t), 40.4 (t), 61.3 (d), 65.1 (d), 65.3 (d),
69.2 (d), 81.0 (d), 116.2 (s), 117.2 (d), 118.9 (d), 130.7 (d), 133.4 (d), 159.2 (s), 167.9 (s), 174.2 (s)
(11) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate and chloroform, hardly soluble in water
(12) Color reaction: ferric chloride color positive [0016]
(13) Thin layer chromatography: Silica gel thin layer (Kieselgel 60 F 254 Art.5554. Merck) Chloroform: Methanol (10: 1) Rf value in developing solvent is 0.48
(14) High-performance liquid chromatography: Develosil packed column ODS-HG-5 4.6 mmφ, 25 cm, manufactured by Nomura Chemical Co., Ltd., acetonitrile: water (0-5 minutes: 20% acetonitrile, 5-6 minutes: 20-36% acetonitrile, 6 to 35 minutes: 36% acetonitrile, 35 to 36 minutes: 36 to 60% acetonitrile, 36 to 50 minutes: 60% acetonitrile), and the retention time at a flow rate of 1.0 ml / min is 44.8 minutes.
[0017]
<Urubactin E>
(1) Material color: Yellow
(2) Shape of substance: oily
(3) Molecular weight: 264
(4) Molecular formula: C 12 H 12 N 2 OS 2
(5) Mass spectrometry: High resolution FABMS measured value 265.0489 [M + H] +
Calculated value 265.0469 (C 12 H 13 N 2 OS 2 )
(6) Specific rotation: [α] D 24 -19 ° (c 0.015 in chloroform)
(7) UV absorption spectrum
(In methanol): λ max 315 nm (log ε3.72), 252 (4.10), 213 (4.41)
(8) Infrared absorption spectrum (KBr)
ν max 3446, 1622, 1595, 1491, 1255, 1222 cm -1
[0018]
(9) 1 H-NMR (measured in deuterated chloroform, 500 MHz)
δppm 3.27 (dd, J = 10.8, 9.0Hz, 1H), 3.30 (dd, J = 10.8, 9.0Hz, 1H),
3.98 (dd, J = 4.3, 1.3Hz, 2H), 5.38 (td, J = 9.0, 4.0Hz, 1H),
6.37 (tdd, J = 4.3, 4.0, 2.4Hz, 1H),
6.87 (td, J = 7.6, 1.2Hz, 1H), 6.99 (dd, J = 8.2, 1.2Hz, 1H),
7.35 (ddd, J = 8.2, 7.6, 1.6Hz, 1H), 7.39 (dd, J = 7.6, 1.6Hz, 1H)
7.58 (dt, J = 2.4, 1.3Hz, 1H)
[0019]
(10) 13 C-NMR (measured in deuterated chloroform, 125 MHz)
δppm 32.0 (t), 44.6 (t), 80.3 (d), 87.3 (d), 116.1 (s), 117.1 (d),
118.8 (d), 130.7 (d), 133.2 (d), 159.2 (s), 162.5 (d), 173.2 (s)
(11) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate and chloroform, hardly soluble in water
(12) Color reaction: Ferric chloride color positive [0020]
(13) Thin layer chromatography: Silica gel thin layer (Kieselgel 60 F 254 Art.5554. Merck) Chloroform: Methanol (10: 1) Rf value in developing solvent is 0.63
(14) High-performance liquid chromatography: Develosil packed column ODS-HG-5 4.6 mmφ, 25 cm, manufactured by Nomura Chemical Co., Ltd., acetonitrile: water (0-5 minutes: 20% acetonitrile, 5-6 minutes: 20-36% acetonitrile, 6 to 35 minutes: 36% acetonitrile, 35 to 36 minutes: 36 to 60% acetonitrile, 36 to 50 minutes: 60% acetonitrile), and the retention time at a flow rate of 1.0 ml / min is 43.5 minutes.
[0021]
(2) Production of the compound of the present invention The compound of the present invention is produced by culturing a microorganism in a medium, producing and accumulating the compound of the present invention in the culture, and collecting the compound of the present invention from the culture. be able to. The microorganism used here is not particularly limited as long as it belongs to the genus Alteromonas and has the ability to produce the compound of the present invention, and examples thereof include the Alteromonas sp. D-12 strain. Moreover, the strain which is a mutant of the said strain and has the capability to produce the compound of this invention can also be used.
[0022]
The Alteromonas sp. D-12 strain is a newly isolated strain from nature, and its bacteriological properties are as follows. Morphological observation was performed using an optical microscope and a transmission electron microscope after inoculating Alteromonas sp. D-12 strain in the medium and culturing at 30 ° C. for 3 to 5 days. As the medium, marine agar (Bacto Marine Agar 2216, manufactured by Difco) or marine broth (Bacto Marine Broth 2216, manufactured by Difco) was used. Since the bacterial strain does not grow at a sodium chloride concentration of 1% or less in this medium, the biochemical property test should be carried out using 75% artificial seawater or in the medium. This was done by adding 3% sodium chloride.
[0023]
a. Form
1) Cell shape and size: Neisseria gonorrhoeae, 1.6-2.5 × 0.8-1.1μm
2) Presence or absence of cell polymorphism: None
3) Presence of motility, flagellar state: Yes, polar hair.
4) Presence or absence of spores: None
5) Gram staining: Negative
6) Presence or absence of acidity: None [0024]
[0025]
[0026]
[0027]
[0028]
The above bacteriological properties were compared with known bacterial species according to the classification criteria of the Virgie's Manual of Systematic Bacteriology 8th edition. This strain was an aerobic gram-negative gonococcus, had polar flagella, oxidatively degraded glucose, and was positive for oxidase and catalase. Furthermore, gelatin was liquefied, DNA was hydrolyzed, and since the GC content was 50% or less, it was considered to belong to the genus Alteromonas and identified as Alteromonas sp. Alteromonas sp. D-12 was deposited as FERM P-15733 at the Institute of Biotechnology, Institute of Industrial Science and Technology on July 12, 1996.
[0029]
For culturing the microorganism used in the present invention, an ordinary microorganism culturing method is used. As the medium, any of a synthetic medium and a natural medium can be used as long as it contains moderately assimilable carbon sources, nitrogen sources, inorganic substances and necessary growth and production promoting substances. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses and the like are used alone or in combination. Furthermore, hydrocarbons, alcohols, organic acids and the like are also used depending on the assimilation ability of the bacteria. As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean flour, casamino acid and the like are used alone or in combination. In addition, inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, and zinc sulfate are added as necessary. Furthermore, trace components that promote the growth of the bacteria used and the production of urubactin D and urubactin E can be appropriately added. As the culture method, a liquid culture method is most suitable. A culture temperature of 30 to 35 ° C. is appropriate, and it is desirable to maintain the pH of the medium during culture at 7 to 8. When culturing is usually performed for 3 to 5 days in liquid culture, the target substance is produced and accumulated in the culture solution. The culture is stopped when the production amount in the culture reaches the maximum.
[0030]
Isolation and purification of wolvactin D and wolbactin E from the culture are carried out according to a commonly used method for isolating and purifying microbial metabolic products from the culture. For example, the culture is divided into culture broth and cells by filtration or centrifugation, and the cells are extracted with water-containing methanol, water-containing ethanol, or the like. Subsequently, the extract and the culture broth are combined and concentrated, and wolvactin D and wolbactin E are obtained by column chromatography using silica gel, gel filtration, high performance liquid chromatography, and the like.
In addition, the trend of wolbactin D and wolbactin E during culturing and purification operations can be traced using ultraviolet absorption by high performance liquid chromatography as an index.
[0031]
(3) Use of the compound of the present invention Since the compound of the present invention has a strong ultraviolet absorbing ability, it can be used as an ultraviolet absorbing component for cosmetics and the like.
Hereinafter, the present invention will be described specifically by way of examples. However, the technical scope of the present invention is not limited by these examples.
[0032]
【Example】
Alteromonas sp. D-12 strain was used as an inoculum. 50 ml of kelp tea liquid medium (kombu tea: 10 g, glucose: 3.0 g, bouillon: 1.0 g, calcium carbonate: 2.0 g, filtered seawater: 500 ml, distilled water: 500 ml, pH = 7.4 ) In a 300 ml Erlenmeyer flask and shaken (100 rpm) for 24 hours at 30 ° C. to obtain a culture solution in advance. During the culture, the pH of the medium was not particularly controlled. 12 ml of the previous culture solution was inoculated into a 1 L Erlenmeyer flask containing 300 ml of kelp tea liquid medium and cultured at 30 ° C. for 8 hours with shaking (100 rpm) to obtain a preculture solution. The total amount of this preculture was inoculated into a 5 L jar fermenter containing 3 L of kelp tea liquid medium, and main culture was performed at 30 ° C., 200 rpm, and aeration rate of 0.2 L / min for 5 days.
[0033]
The thus obtained culture solution (3.3 L) is centrifuged, and the obtained supernatant is extracted with ethyl acetate (6.6 L). The ethyl acetate extract was concentrated under reduced pressure at 40 ° C. or lower, and the resulting concentrate was subjected to reverse phase high performance liquid chromatography (Develosil packed column ODS-HG-5 20 mmφ, 25 cm, Nomura Chemical Co., Ltd., water: acetonitrile = 80: 20). Fractionation) to obtain two fractions. The first fraction was purified by silica gel column chromatography (Sep-Pak (registered trademark) Vac 12cc, manufactured by Waters, hexane-acetone = 10: 1) to obtain 0.6 mg of urubactin D. The second fraction was purified by silica gel column chromatography (Sep-Pak (registered trademark) Vac 12cc, manufactured by Waters, hexane-ethyl acetate = 10: 1) to obtain 0.8 mg of urubactin E.
The obtained urubactin D and urubactin E exhibited the physicochemical properties described above.
[0034]
【The invention's effect】
The present invention provides a novel compound and a method for producing the substance using a microorganism. Since the compound of the present invention is fat-soluble and has a strong ultraviolet absorbing ability, it can be used as a cosmetic ingredient.
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