JPS58198422A - Novel protein kud-pc and preparation thereof - Google Patents
Novel protein kud-pc and preparation thereofInfo
- Publication number
- JPS58198422A JPS58198422A JP57081466A JP8146682A JPS58198422A JP S58198422 A JPS58198422 A JP S58198422A JP 57081466 A JP57081466 A JP 57081466A JP 8146682 A JP8146682 A JP 8146682A JP S58198422 A JPS58198422 A JP S58198422A
- Authority
- JP
- Japan
- Prior art keywords
- kud
- protein
- reaction
- culture
- water
- 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.)
- Pending
Links
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- DEQXHPXOGUSHDX-UHFFFAOYSA-N methylaminomethanetriol;hydrochloride Chemical compound Cl.CNC(O)(O)O DEQXHPXOGUSHDX-UHFFFAOYSA-N 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229920002545 silicone oil Polymers 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical compound [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 description 1
- 235000019254 sodium formate Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 230000002311 subsequent effect Effects 0.000 description 1
- 230000019086 sulfide ion homeostasis Effects 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 125000002338 threonino group Chemical group [H]O[C@]([H])(C([H])([H])[H])[C@](N([H])[*])(C(=O)O[H])[H] 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 241001446247 uncultured actinomycete Species 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
本発明は誓1規蛋白質KUD−PCおよびその製造法に
関する。さらに膵しくは、本発明はストレットスポラ7
ギウム属に輌する蛋白質KUD−PC生産−の培養によ
って産生され、かつ抗謙瘍作中を著しく増強する作用を
有する新規蛋白質KUD−P Cおよびその製造法に関
する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a primary protein KUD-PC and a method for producing the same. More particularly, the present invention provides streptospora 7
The present invention relates to a novel protein KUD-PC which is produced by culturing KUD-PC produced in the genus Gium and which has the effect of significantly enhancing anticancer activity, and a method for producing the same.
本発明者らは、先に東京都世田谷区内の土壌より分離さ
れたストレブトスボフ/ギウム属に属するdiaPO−
357がスタフイロツカス・アウレウスおよびバチルス
・ズブチリスに対して抗菌活性を有し、かつ抗腫瘍性活
性を有する抗生物質PO−357物質(その彼スポラマ
イシンと命名された)を産生ずることを艶い出しており
、その理化学性質およびその生産菌株の菌学的性状は特
開昭53−7601号公報に記載されている。The present inventors previously discovered that diaPO-
357 has antibacterial activity against Staphylotchus aureus and Bacillus subtilis, and produces an antibiotic substance PO-357 (named sporamycin) that has antitumor activity. Its physicochemical properties and the mycological properties of its producing strain are described in JP-A-53-7601.
本発明者らは、上記スポラマイ7ン生産劇を培養して産
生ずる生理活性物質について更に研究を続けた結果、抗
歯作用を示さないこと、並びに電気泳動により明らかに
区別されることなどからスボフマイシ/とは明らかに異
なり、しかも抗11111m性物質の抗論瘍作用を増強
する作用を有する生理活性物質を新たに見い出し、該生
理活性物質を珈白質KUD−PCと称することにした。As a result of further research on the physiologically active substances produced by culturing the sporamine-7 production system, the present inventors found that subofumi-7 does not exhibit anti-dental effects and can be clearly distinguished by electrophoresis. We have discovered a new physiologically active substance that is clearly different from / and has the effect of enhancing the anticancer effect of anti-11111m substances, and we have decided to name this physiologically active substance KUD-PC.
本発明LJ上記の知見に基いて完成されたものである。The LJ of the present invention was completed based on the above findings.
本発明の新規蛋白質KUD−PCは次の理化学性質を有
する。The novel protein KUD-PC of the present invention has the following physical and chemical properties.
■元素分析;炭素、水嵩、窒素、酸素、硫黄をむ。■Elemental analysis; includes carbon, water volume, nitrogen, oxygen, and sulfur.
元素分析値;049.98%、H7,27%、N15.
23%、81.08%(100℃3時間真空乾燥後の実
測イO11)
リノ分子普;S:OS−ポリアクリルアミドケル盲気泳
動法により標準品(分子12512〜16949の標準
ポリペプチドまたは標準蛋白憤)と比較して蔦出された
本物質の分子菫は1150(1である。Elemental analysis value: 049.98%, H7.27%, N15.
23%, 81.08% (actually measured after vacuum drying at 100°C for 3 hours). The molecular violet of this substance extracted is 1150 (1).
■融点;258〜260℃(分解)
・り比旋光度;[α] −5&8° (0=1.0.
水)(り紫外線吸収スペクトル;本物質の水溶液おJび
0. I N水酸化ナトリウム水溶液の紫外1151載
l1l(スペクトルは第1図の通りであって、275.
280(肩) nm 。■Melting point; 258-260°C (decomposed) ・Specific rotation; [α] -5&8° (0=1.0.
Ultraviolet absorption spectrum of an aqueous solution of this substance and a 0.1 N sodium hydroxide aqueous solution at 1151 l1l (the spectrum is as shown in Figure 1 and 275.1 l).
280 (shoulder) nm.
i 11.1NNaθ”;294nm
aX
!赤外線吸収スペクトルHKBr法による赤外線スペク
トルは第2図の通り。i 11.1NNaθ"; 294nm aX! Infrared absorption spectrum The infrared spectrum obtained by the HKBr method is shown in Figure 2.
(誹溶媒に対する溶解性;水に可溶、アルコール、アセ
ト/、ベンゼンなどの有機溶媒に不溶。(Solubility in solvents: Soluble in water, insoluble in organic solvents such as alcohol, acetate, and benzene.
(!・呈色反応;〕Aリノローり反応、ビューレット反
応、フイドン・スミス反応に陽性、フェノール硫酸反応
、ア/スロ/硫酸反応に陰性、塩酸加水分解物はニンヒ
ドリン反応に陽性。(!・Color reaction;] Positive for A-Linoroll reaction, Biuret reaction, Huidon-Smith reaction, negative for phenol-sulfuric acid reaction, A/Suro/sulfuric acid reaction, positive for hydrochloric acid hydrolyzate for ninhydrin reaction.
[有]物質の色、形色;無色結晶。水溶液を限外−幅し
て得られる結晶は六方晶系であり、硫安ja4Frによ
る結晶は柱性晶である。[Yes] Color and shape of substance; colorless crystals. Crystals obtained by ultra-width an aqueous solution are hexagonal crystals, and crystals obtained from ammonium sulfate ja4Fr are columnar crystals.
@−基性、酸性、中性の区別二本物質の0.1%水制帽
1c+jp)iは4〜8゜
Qk ’−/ :ノ酸組成:本物+AI5■を封管中塩
酸加水分解し、液体りVマトクラノイ(化デル日立03
4−2 U )により測定し、たアミノ酸分析1′@(
μへ1)は次の荊っである。@ - Distinguish between basic, acidic and neutral 0.1% water cap 1c + jp) i is 4~8゜Qk '-/: Noic acid composition: Genuine + AI 5■ is hydrolyzed with hydrochloric acid in a sealed tube, Liquid Ri V Matokuranoi (Kadel Hitachi 03
Amino acid analysis 1'@(
1) to μ is the next step.
アスパラギアeli 0.144スレオニノ
0.258
セリフ 0.226
グルタミン酸 0.141
プロリン 0.111
グリシン 0.250
アラニy O,363
バリン 0.265
・rソロイシン 0.016
0イシン 0.117
メチオニン −
チロシy O,020
フエニルアラニア 0.079
リジ10.(189
アルギニ10.040
ヒスチジン (微量)
@電気泳動; 8D8−ポリアクリルアミドゲル電気泳
動法により分析し、クロマトスキャ、ナー(I4津08
−910)により描いた泳動ノロフィールは第3図の通
りであって、本物質は幀−である。セルロースアセテー
ト膜電気泳動t1:(セルロゲルKS、pH2,1M酢
酸−ギ酸緩衝液、2(1(IV、1時間、アミドブラッ
クIOB染色目、よる体動図は第4図の通りであつ゛C
1本物質は卑−バAドを与える。また、本発明は、スト
し]lスホラ/ギウム属に楓する蛋白質KUD−P U
生産菌を培地に培養して培養物中に蛋白質K U 1)
−P Cを蓄積せ・しめ、該培養物から蛋白質kUD−
PCを採取することを特徴とする新規蛋白質K U D
−P (:!の製造法である。Asparagia eli 0.144 threonino
0.258 Serif 0.226 Glutamic acid 0.141 Proline 0.111 Glycine 0.250 Araniy O,363 Valine 0.265 ・rSoloucine 0.016 0Isine 0.117 Methionine-Tyrosy O,020 Phenylalania 0.079 Lizzy 10. (189 Argini 10.040 Histidine (trace) @electrophoresis; Analyzed by 8D8-polyacrylamide gel electrophoresis, chromatoscanner (I4 Tsu08
The migration profile drawn by 910) is as shown in Fig. 3, and this substance is spherical. Cellulose acetate membrane electrophoresis t1: (Cellulogel KS, pH 2, 1M acetic acid-formate buffer, 2 (1 IV, 1 hour, Amido black IOB staining, body movement diagram as shown in Figure 4)
One substance gives the base-bad A. In addition, the present invention provides protein KUD-P U
The producing bacteria are cultured in a medium to produce protein K U in the culture.1)
- Accumulate and tighten PC and extract protein kUD- from the culture.
Novel protein KUD characterized by collecting PC
-P (This is the manufacturing method of:!
蛋白質KLID−PCの生産菌は、上記した通りストし
)゛倉スポラ/キウム桐に輌4−るが例えは木登1省ら
か分離したヌトレプトスボフノギウム属にM−fる菌株
PO−357は本発明に最も有効に用いらJ”+る鉋の
一例であって、本劇株の一学的性状をボすと次の通りで
ある。The producing bacteria of the protein KLID-PC are strained as described above. -357 is an example of a J''+ plane that is most effectively used in the present invention, and the chemical properties of this strain are as follows.
ユ、形態的特徴
)’0−357一体は真直にのびた気菌糸と多数の胞子
のりを形成する。胞子のうの大きさは1a径5〜lOμ
平均75μ、で、胞子のり着生気菌柄の長さは大半が3
μ前後で短がく、胞子(ま円形または楕円形で、表面は
平滑であり、大きざは直径0゜9〜1.4μで胞子の運
動性はない。Yu, morphological characteristics) '0-357 forms a straight aerial mycelium and a large number of spores. The size of the sporangium is 1a diameter 5~10μ
The average length of the spore paste is 75 μ, and the length of most of the spores is 3.
The spores are round or oval, the surface is smooth, the diameter is 0°9 to 1.4μ, and the spores are not motile.
51次の各培地における生育状卸
下記の性状はいずれも27℃において1()〜14日間
培養後の観察である。51 Growth status in each culture medium The following properties were observed after culturing at 27°C for 1 to 14 days.
培 地 生育 裏iil] シ菌糸uJ阿世
鳴シュクロース・硝酸塩寒天 弱い や−橙色 乳
白色 見られ4゛グルコース・硝酸塩寒天 弱い
や−黄味 乳白色 ・・グリセロール・アスパラギ
ン
寒天 弱い や\黄味 乳白色
リスターチ・無機塩寒天 弱い 乳白色 乳
白 リチロシン寒天 普通 や\橙色
や−橙色 ・I栄養寒天 普通〜良
橙色゛ 橙色イースト・麦芽軸寒天 良
橙色 橙色オートミル・寒天 良 暗橙
色 暗橙色C,次の各生理的性質
■生育11& ; 43℃でも生育するが、27〜30
℃位が最も発育はよい。Medium Growth Back Iil] Shihypha uJ Asonari Sucrose/Nitrate Agar Weak Medium-Orange Milky White 4゛Glucose/Nitrate Agar Weak
Ya-yellowish milky white Glycerol/asparagine agar weak Ya\yellowish milky white
Listarch/Inorganic salt agar Weak Milky white Milky white Richyrosin agar Normal Ororange Ya-orange ・I Nutrient agar Average to good
Orange ゛ Orange yeast/malt stem agar Good
Orange Orange oatmil/agar Good Dark orange Dark orange C, each of the following physiological properties ■Growth 11 &; It grows even at 43℃, but at 27~30℃
Growth is best at temperatures around ℃.
(t1ゼラチ/の液化;陽性
[有]でんぷんの加水分解:陽性
・−ノメラニ/色素の生成;チロンン哩天では陽性(、
り脱脂乳のベプト/化;陽性
■亜II4酸生成反応:陽性
■硫化水素の生成;に性
41次の各炭素源の利用性
Pridham Gottlieb寒天培地に0.05
%の割合に酵母抽出液を添加した培地Fでの試験である
。(Liquification of t1 gelatin; positive [presence] Hydrolysis of starch: positive - Nomelani/formation of pigment; positive in Chiron Nangten (,
Vept/conversion of skimmed milk; positive ■ II4 acid production reaction: positive ■ hydrogen sulfide production;
This is a test using medium F to which yeast extract was added at a ratio of %.
利JIする;/ラヒソース、キシa−ス、D−グルコー
ス、マノノース
や−利用するニアラクト−2、ラムノース、ラフィノー
ス、ガラクトース
利用L ftい:サッカロース、・イノシトールe、細
胞壁組成;
Beckerらの方法(ApploMicr 、Moi
、、 13236−■■―−■−−雫−−−■□
〜243.1965年)によって分析した結果は次の通
りである。Utilize; / Rahisose, xia-sose, D-glucose, manonose, utilize nialact-2, rhamnose, raffinose, galactose Utilize: saccharose, inositol e, cell wall composition; method of Becker et al. ,Moi
,, 13236-■■--■--Shizuku---■□ ~243.1965) The results of the analysis are as follows.
ジアミノビメリy @ m e e o型りリ″
存在しない
アラビノース
ガラクトース I/
以上の菌学的性状から、本PO−3571!Ifiはス
トレプトスボラノギウム属に属する菌株で多1イ、こと
は明らかである。そこで、既に報誌されでいるストレプ
トスポラ/イウム属に幀ずZ)菌株のうち、本菌株と類
似する菌種を比較検討1.たと、=ろ、本菌株はストレ
プトスボランギウム・シコー トブルガレエ(5tor
・+ptosporanqium pssnd vul
g「xlaOlNl 111 om 11 r a に
その形態的特徴、各種培地−Lでの生育状態、生理的性
質とも良く一致した。従−′)で、本菌株はストレプト
スポラ/イウム・/ニー・Fブルカレエに極めて類似し
た第であ61しかしながら、既知のストレプトスボラン
ギウム・ン′ユードブルカレエはそれまで抗生物質ス、
1、ラマイン/を生能することは知られていないし、ま
た本蛋白憤KUI)−PCも生産することも知られ−(
いないから、本−株°を既知のストレグトス小ラノギウ
ム・/ニー ドプルガレエと区別し、ストレグトスオう
;””l:’、’1llll:
/キウム・ンユードプルカレエP (,1−357トロ
n名した。本曲は工業技術院微生物上条研究f9目、″
4f託番号、微工研薗寄託絨3571号(1”ERM−
PNn3571)として寄託されている。Diaminobimeliy @ m e e o type reli''
Absent arabinose galactose I/ Based on the above mycological properties, this PO-3571! It is clear that Ifi is a bacterial strain belonging to the genus Streptosboranogium. Therefore, we conducted a comparative study of bacterial strains similar to this strain among the strains of the genus Streptospora that have already been reported.1. This strain is Streptosborangium sichot vulgarae (5tor).
・+ptosporanquium pssnd vul
The morphological characteristics, growth conditions on various media, and physiological properties were in good agreement with the 111 om 11 r a . 61 However, the known Streptosborangium n'eudoburcarae had not previously been treated with antibiotics,
1. It is not known to produce lamaine/, and it is also known to produce this protein (KUI)-PC.
Therefore, we distinguished this strain from the known Stregutos minor lanogium / Niedpurgae, and named it Stregutos. .This song is part 9 of the Research on Microorganisms by the Agency of Industrial Science and Technology.
4f consignment number, Microtech Kenzon consignment number 3571 (1”ERM-
It has been deposited as PNn3571).
以上蛋白質KUD−PC生産菌について説明したが、放
線菌の一般的性状として歯学上の性状は極めて変異し易
く、一定したものではなく、自然的にあるいは通常行わ
れる紫外線照射、放射線照射または変異誘起剤、例えば
N−メチル−N−二トローN−ニトロノグアニシ/、エ
チルメタンスルホネートなどを用いる人工的変異手段に
より変異することは周知の事実であり、このような人工
的変異株は勿論、自然変異株も含め、ストレプトスポラ
/イウム属に属し、蛋白質KUD−PCを生産する能力
を有する菌株はすべて本発明に使用することができる。As described above, protein KUD-PC-producing bacteria are explained, but as a general property of actinomycetes, the dental properties are extremely variable and are not constant, and are not caused by natural or normal ultraviolet irradiation, radiation irradiation, or mutagenesis. It is a well-known fact that mutations can occur by artificial mutation means using agents such as N-methyl-N-nitro-N-nitronoguanisi/, ethyl methanesulfonate, etc., and not only such artificial mutants but also natural mutants. All strains belonging to the genus Streptospora/ium and having the ability to produce the protein KUD-PC can be used in the present invention.
本発明においては、先ずストレプトスポラ/イウム箋に
属する蛋白質KUD−PC生産菌が適当な培地に培養さ
れる。重曹の培養においては通常放線菌の培養法が一般
に用いられろう培地としては微生物が同化し得る炭素源
、消化し得る窒素5.源および無機塩などを含有させた
栄養培地が使用される。同化し得る炭素源としては、ぶ
どう糖、しよ糖、糖蜜、でんぷん、デキストリン、グリ
セリン、有機酸が単独または組合せて用いられる。消化
し得る窒素源としてはペプトン、肉エキス、酵母エキス
、乾燥酵母、大豆粉、コーン・スチーブ・リカー、綿実
粕、カゼイン、大豆蛋白分解物、アミノ酸、尿素などの
有機窒素源、硝酸塩、アンモニウム塩などの無機窒素化
合物などが単独または組合せて用いられる。その他、必
要に応じ、ナトリウム塩、カリウム塩、カルシウム塩、
マグネシウム塩、リン酸塩などの無機塩類が添加される
1、また、培地には必要に応じて重曹の生育や蛋白質K
UD−PC!の生産を促進する微量栄養素、発育促進物
質を適当に添加してもよい。In the present invention, first, a protein KUD-PC-producing bacterium belonging to Streptospora spp. is cultured in an appropriate medium. In the cultivation of baking soda, the actinomycete culture method is generally used.The wax medium contains a carbon source that can be assimilated by microorganisms, and nitrogen that can be digested5. Nutrient media containing sources, inorganic salts, etc. are used. As assimilable carbon sources, glucose, sucrose, molasses, starch, dextrin, glycerin, and organic acids are used alone or in combination. Digestible nitrogen sources include peptone, meat extract, yeast extract, dried yeast, soybean flour, corn stave liquor, cottonseed meal, casein, soy protein digests, amino acids, organic nitrogen sources such as urea, nitrates, and ammonium. Inorganic nitrogen compounds such as salts are used alone or in combination. In addition, as necessary, sodium salt, potassium salt, calcium salt,
Inorganic salts such as magnesium salts and phosphates are added1, and the culture medium is supplemented with baking soda for growth and protein K as necessary.
UD-PC! Micronutrients and growth-promoting substances that promote the production of can be added as appropriate.
培養は通常振とうまたは通気攪拌培養などの好気的条件
下で行うのがよい。工業的には深部通気攪拌培養が好盪
しい。培地のpnは中性附近で培養を行うのが好ましい
。培養温度は通常27〜30℃附近に保つのが好ましい
。液体培養で通常40〜72時間培養を行うと、本物質
が培養液中に生成蓄積される。好ましくは培養液中の蓄
積蒼が最大に達したときに培養を停止すればよい。これ
らの培養組成物、培地の液性、培養側1攪拌速度、通気
1などの培養条件は使用する菌株の種類や外部の条件な
どに応じて好ましい結果が得られるように適宜選択され
ることはいうまでもない。液体培養において発泡がある
ときはシリコン油、植物油、界面活性剤などの消泡剤を
適宜使用される。Cultivation is usually carried out under aerobic conditions such as shaking or aerated agitation culture. Industrially, deep aeration agitation culture is preferred. It is preferable to culture the medium at a pn of around neutrality. It is usually preferable to keep the culture temperature around 27 to 30°C. When culture is carried out in liquid culture for usually 40 to 72 hours, this substance is produced and accumulated in the culture solution. Preferably, the culture may be stopped when the accumulated blue water in the culture solution reaches the maximum. These culture conditions, such as the culture composition, liquid properties of the medium, stirring speed on the culture side, and ventilation should be selected as appropriate to obtain favorable results depending on the type of bacterial strain used and external conditions. Needless to say. When foaming occurs in liquid culture, antifoaming agents such as silicone oil, vegetable oil, and surfactants are used as appropriate.
このようにして得られた培養物中に蓄積されたKUD−
PCは主として培養液中に含有されているので、培養物
に必要に応じ一過補助剤を加えてP遇するか筐たは遠心
分離して培養p液から蛋白質KUD−PCを採取するの
が有利である。培養V液から蛋白質KUI)−PCを分
離、精製するためには、本蛋白質KUD−PCが前述の
ように水にi’] ilであるが、アルコール、アセト
ン、べ/ゼ/などの有機溶媒には不溶の水溶性蛋白質が
あるので、これらの性質を利用した精製法が用いられる
。一般には、蛋白質およびポリペプチドを採取するのに
通常用いられる公知の分離精製方法を適宜利用すること
ができる。例えば硫安、塩安などの塩析、メタノール、
エタノール、アセト/などによる分別沈澱、合成イオン
交換樹脂、イオン交換セルロース、イオン交換セファデ
ックスなどによるイオン交換クロマトグラフィー、活性
炭、シリカゲル、アルミナ、ヒドロキクアパタイト、セ
ルロースまたはHP−10樹脂などの吸着樹脂などによ
る吸着クロマトグラフィー、セファデックス、バイオゲ
ルなどによるゲル濾過クロマトグラフィへ、電気泳動、
向流分配、透析、限外濾過もしくは濃縮などの手段を単
独あるいは任意の順序に組合せて、または反復して用い
られる。クロマトグラフィーに用いられる溶出溶媒とし
ては担体の種類によって異なるが、例えば水、含水アル
コール、含水アセト/、緩衝液あるいは無機塩または有
S塩などの水溶液などが用いられる。KUD− accumulated in the culture thus obtained.
Since PC is mainly contained in the culture solution, it is recommended to add a temporary aid to the culture as necessary to treat the culture with P, or collect the protein KUD-PC from the culture P solution by centrifugation. It's advantageous. In order to separate and purify the protein KUD-PC from the culture solution V, the protein KUD-PC is dissolved in water as described above, but in an organic solvent such as alcohol, acetone, benzene, etc. Since there are insoluble and water-soluble proteins in the protein, purification methods that take advantage of these properties are used. In general, known separation and purification methods commonly used to collect proteins and polypeptides can be used as appropriate. For example, salting out ammonium sulfate, ammonium chloride, methanol,
Fractional precipitation with ethanol, acetate/etc., ion exchange chromatography with synthetic ion exchange resins, ion exchange cellulose, ion exchange Sephadex, etc., adsorption resins such as activated carbon, silica gel, alumina, hydroquapatite, cellulose or HP-10 resin, etc. adsorption chromatography, gel filtration chromatography using Sephadex, biogel, etc., electrophoresis,
Measures such as countercurrent distribution, dialysis, ultrafiltration or concentration may be used alone or in combination in any order, or repeatedly. The elution solvent used in chromatography varies depending on the type of carrier, but for example, water, hydrous alcohol, hydrous acetate, a buffer solution, or an aqueous solution of an inorganic salt or S-containing salt are used.
次に蛋白質KUD−POの採取方法について更に詳細に
述べる。Next, the method for collecting protein KUD-PO will be described in more detail.
培養の終了した培養−を−過補助剤を加えて濾過し菌体
を除去する。培養F液に冷却下中性にて硫安を90%飽
和となるように加えて硫安塩析を行う。硫安沈澱物を濾
過管たは遠心分離により集め、水または中性緩衝液に溶
解し、その溶液を冷却下透析によるか、または半透膜を
用いる限外濾過により脱塩処理する。半透膜としては分
子量10 (10および10000以上をカットする合
成半透膜、例えばUH−1、UK−10(東洋p紙社製
)などが用いられる。After completion of cultivation, a filtering aid is added to the culture and filtered to remove bacterial cells. Ammonium sulfate is added to the culture solution F under cooling and neutral conditions to achieve 90% saturation, and salting out of ammonium sulfate is carried out. The ammonium sulfate precipitate is collected in a filter tube or centrifuged, dissolved in water or a neutral buffer, and the solution is desalted by cold dialysis or by ultrafiltration using a semipermeable membrane. As the semipermeable membrane, synthetic semipermeable membranes that cut molecular weights of 10 (10 and 10,000 or more), such as UH-1 and UK-10 (manufactured by Toyo P Paper Co., Ltd.), are used.
脱塩溶液はイオン交換セルロース、イオン交換セファデ
ックスなどのカラムに通し、カラムを水洗後、溶出溶媒
により溶出する。イオン交換セルロースとしてはDEA
E−セル【】−ス、BOTEOLA−セルロース、8B
−セルロース、CM−セルロースなどが用いられる。上
記のイオン交換セルロースおよびイオン交換セファデッ
クスは予め希釈#度の緩衝液で緩衝化しておくのが有利
であるっ溶出溶媒としては無機塩または有機塩を含有す
る緩衝液が用いられる。無機塩としては塩化ナトリウム
、塩イしカリウム、塩化アンモニウム、硫酸アンモニウ
ム、リノ鮭塩などが用いられ、有mtmとしては酢酸ナ
トリウム、ギ酸ナトリウムなどが用いられる。上記の無
機塩または有機塩の濃度は0.O1〜I M#が好まし
い。緩衝液としては9ノ@塩、酢酸塩、クエ/fs塩、
トリスヒドロキシメチルアミノメタン塩酸塩などの緩衝
液が用いられる。この緩衝液のp)(は5〜8好ましく
は中性であり、濃度はα001〜α5M/!の範囲であ
る。The desalted solution is passed through a column such as ion-exchange cellulose or ion-exchange Sephadex, and after washing the column with water, it is eluted with an elution solvent. DEA as ion exchange cellulose
E-Cell【】-S, BOTEOLA-Cellulose, 8B
-cellulose, CM-cellulose, etc. are used. It is advantageous to buffer the above-mentioned ion-exchanged cellulose and ion-exchanged Sephadex in advance with a diluted buffer solution. As the elution solvent, a buffer solution containing an inorganic salt or an organic salt is used. As the inorganic salt, sodium chloride, potassium chloride, ammonium chloride, ammonium sulfate, salmon salt, etc. are used, and as the mtm, sodium acetate, sodium formate, etc. are used. The concentration of the above inorganic salt or organic salt is 0. O1 to IM# are preferred. Buffer solutions include 9@salt, acetate, que/fs salt,
A buffer such as trishydroxymethylaminomethane hydrochloride is used. This buffer p)( is 5-8 preferably neutral and the concentration ranges from α001 to α5M/!).
上記の溶出溶媒による溶出工程においては無機塩または
有機塩の濃度ならびに緩衝液の#I#′またはpHを段
階的に変える手段もしくは連続的に変える手段が用いら
れる。In the above-mentioned elution step using the elution solvent, a means for changing the concentration of the inorganic salt or organic salt and the #I#' or pH of the buffer solution stepwise or continuously is used.
本蛋白質KUD−PCを含有する溶液の濃縮または脱塩
処理は、硫安塩析、限外濾過または透析などの手段が用
いられる。濃縮液または脱塩溶液から精製するにはゲル
濾過クロマトグラフィーによる手段が有利に用いられる
。ゲルー過剤としてはバイオゲル゛P−10,バイオゲ
ルP−30,セファデックスG−75などが用いられる
。溶出溶媒としては水または緩衝液などが用いられる。For concentrating or desalting a solution containing the present protein KUD-PC, methods such as ammonium sulfate salting out, ultrafiltration, or dialysis are used. Gel filtration chromatography is advantageously used for purification from concentrated solutions or desalted solutions. As the gelling agent, Biogel P-10, Biogel P-30, Sephadex G-75, etc. are used. Water or a buffer solution is used as the elution solvent.
このようにして高度に精製された蛋白質KUD−PCを
含有する溶液から蛋白質KUD−PCを結晶として単離
することができる。例えばイオン交換クロマトグラフィ
ーおよび(または)ゲル濾過クロマトグラフィーなどに
より高度に精製された溶出液を限外濾過による一縮処理
およq(または)硫安沈澱処理すると蛋白質KUD−轟
0は結晶化する。結晶化しない場合虹は、更にクロマト
グラフィーに附すかまたは凍結乾燥により蛋白質KUD
−PCを、単離することができる。In this manner, protein KUD-PC can be isolated as crystals from a solution containing highly purified protein KUD-PC. For example, when an eluate highly purified by ion exchange chromatography and/or gel filtration chromatography is subjected to condensation treatment by ultrafiltration and ammonium sulfate precipitation treatment, protein KUD-Todoroki 0 is crystallized. If it does not crystallize, the protein KUD can be further purified by chromatography or lyophilized.
-PC can be isolated.
次に本発明の蛋白質KUD−PCの生物学的性質を述べ
る。Next, the biological properties of the protein KUD-PC of the present invention will be described.
1)、抗菌作用
本物質はスタフィロコッカス・アウレウス、バチルス・
ズブチリス、バチルス・セレウス、サル7す・ルテアな
どの検定菌に対して抗菌作用を示さないっ
2)、細胞損傷作用
第1表の通りであって、本物質はBHK細胞に対して強
い損傷作用を有する。1), Antibacterial effect This substance has Staphylococcus aureus, Bacillus
It does not show antibacterial activity against tested bacteria such as B. subtilis, Bacillus cereus, and Lutea monkeys.2) Cell damaging effect As shown in Table 1, this substance has a strong damaging effect on BHK cells. has.
第1表
試料
蛋白質KUD−POスポラマイシン 3H−TdR3
H−I、IRProt。Table 1 Sample protein KUD-PO Sporamycin 3H-TdR3
H-I, IRProt.
Oμf/d 15U/+m 4 2] 220
5 52423
0 10 553042
0.1 区5 27−1322
1 λ5 26−2728
10 15 42 017
0.1 5 491442
1 5 34 −330
10 5 33−1223
0.1 10 84 4352
1 10 684441
10 10 42 530
3)、抗腫瘍作用
■実験方法
(イ)サルコーマ1so’、i * Ig5個EddY
−rウスの皮下に移殖し、3日目にスポラマイ7ノとK
UD−Pctを試験管の中で混ぜて静脈投与した。さら
に、4.5.6日月KUD−PCのみを静脈投与してそ
の後の砥命効果を調べた。Oμf/d 15U/+m 4 2] 220
5 52423 0 10 553042 0.1 Ward 5 27-1322 1 λ5 26-2728 10 15 42 017 0.1 5 491442 1 5 34 -330 10 5 33-1223 0.1 10 84 4352 1 10 684 441 10 10 42 530 3) Antitumor effect ■ Experimental method (a) Sarcoma 1so', i * 5 Ig EddY
-Transplanted under the skin of rus, and on the 3rd day Sporamai 7 and
UD-Pct was mixed in a test tube and administered intravenously. Furthermore, KUD-PC alone was administered intravenously on April 4, 5, and 6, and the subsequent effects on survival were investigated.
(ロ)Ehrlich腹水癌に対する効果Ehrlic
h腹水癌細胞15 X 10個をddYマウス(5週)
の腹腔に移殖し、翌日にスポラマイ7/をKUD−PC
をあらかじめ試験管の中で混ぜたものを1回投与(腹腔
内)して宿主の延命日数を調べた。(1群5匹)
O〕判定方法
(、り実験結果
本物質を動物移殖癌、Ehr11Ch腹水癌に対して抗
腫瘍剤スポラマイ7)と併用した結果は第2表の通りで
あって、本物質はスポラマイ7ノの抗腫瘍作用を著しく
増強する
□
第2表
■実験結果
4)、毒性
本物質の急性毒性L Dsoは次の通りである。(b) Effect of Ehrlich on ascites cancer
h 15 x 10 ascites cancer cells were added to ddY mice (5 weeks)
The next day, Sporamai 7/ was transplanted into the abdominal cavity of KUD-PC.
were mixed in advance in a test tube and administered once (intraperitoneally) to determine the number of days the host could survive. (5 animals per group) O] Judgment method (results) The results of using this substance in combination with the antitumor agent Sporamy 7 for animal transplantation cancer and Ehr11Ch ascites cancer are shown in Table 2. The substance significantly enhances the antitumor effect of Sporamai 7. □ Table 2 ■ Experimental results 4) Toxicity The acute toxicity L Dso of this substance is as follows.
マウス IP LD50 ”9/kyマウスIV
LD5o ”9/ki次に実施例を挙げて本発明を
具体的に説明するが、これにより本発明の方法を限定す
るものではない。Mouse IP LD50 “9/ky Mouse IV
LD5o "9/ki Next, the present invention will be specifically explained with reference to Examples, but the method of the present invention is not limited thereto.
実施例1
50〇−容坂ロフラスコに入れたA培地(グルコース1
0%、乾燥酵母(13%、ペプトン0.5%、肉エキス
α5%、炭酸カルシウム0.3%、塩化ナトリウム0.
5%を含む液体培地、pH7,0) 100−にストレ
プトスポラ/イウム・シュードプルガレエPO−357
(FItRMP−Nn3571 )の斜面培養寒天から
一白金耳を接種し、毎分170回転27℃・で72時間
往復振盪培養した。この種培養液10mで各々10本の
5001I/容坂ロフラχ
スコに入れたB培地(グルコース0.2%、デンプンL
5%、乾燥酵母0.15%、ペプトン0.25%、肉エ
キス0.3%、炭酸カルシウム0.25%を含む70回
転、27℃で48時間往復振盪培養した。Example 1 A medium (glucose 1
0%, dry yeast (13%, peptone 0.5%, meat extract α5%, calcium carbonate 0.3%, sodium chloride 0.
Liquid medium containing 5%, pH 7,0) 100- to Streptospora/Ium pseudopulgalae PO-357
(FItRMP-Nn3571) was inoculated from a loop of slant culture agar and cultured with reciprocating shaking at 170 rpm and 27° C. for 72 hours. B medium (glucose 0.2%, starch L
5% dry yeast, 0.15% dry yeast, 0.25% peptone, 0.3% meat extract, and 0.25% calcium carbonate, and cultured with reciprocating shaking at 70 rpm at 27° C. for 48 hours.
得られた第二次培讐液1)を200!容ステンレススチ
ールタ/りに仕込んだA培地13,0ノに移殖し、通気
量IQOA/分、攪拌速If 25 Or−p・m、内
圧0.5 kg / cm2.28℃で72時間通気攪
拌培養した。得られた培養物中にはセルロースアセテー
ト膜電気泳動試験法により蛋白質KUD−PCの存在が
確認された。この主培養物115ノを5℃以下に冷却し
、これにハイフロス−パーセル23に9を加え、フィル
タープレスでp遇した。菌体は水洗し、培養F液と洗液
を合せ、5℃以下に冷却し、ハイフロス−バーセル40
0fを加えた徒、90%飽和となるよう硫安82.5’
ljを〃口えた。5゜以下で1時間攪拌した後、生じた
沈澱物を枠取し、湿潤固形分900tを得た。以下の操
作はすべて5℃の暗室で行った。200% of the obtained secondary culture solution 1)! Transferred to A medium 13.0 μm in a stainless steel tank and aerated for 72 hours at aeration rate IQOA/min, stirring speed If 25 Or-p・m, internal pressure 0.5 kg/cm, and 2.28°C. Agitated culture was performed. The presence of protein KUD-PC in the obtained culture was confirmed by cellulose acetate membrane electrophoresis test method. This main culture (115 mm) was cooled to 5° C. or lower, 23 ml of Hyfloth Parcel 9 was added thereto, and filtered using a filter press. The bacterial cells were washed with water, the culture solution F and the washing solution were combined, cooled to below 5°C, and Hyfloth-Basel 40
Adding 0f, ammonium sulfate 82.5' to achieve 90% saturation.
I spoke lj. After stirring for 1 hour at a temperature of 5° or less, the resulting precipitate was collected and a wet solid content of 900 t was obtained. All the following operations were performed in a dark room at 5°C.
上記固形分を水3j&:溶かし、不溶物を一過、水洗し
た。p液と洗液を合せ(4))、これに90%飽和とな
るよう硫安15kgを加え、1時間攪拌した後、生じた
沈澱物をp取して沈澱物102tを得た。これを水45
0−に溶かし、セロファンチューブにより脱イオン水3
0jを加′えて3日間透析処理した。その間脱イオン水
は毎日取り替えた。得られた脱塩溶液1170 mを予
め0.002 M IJン酸緩1ti’?1!(pH7
,5〜&0)で緩価化したDBAE−セルロース(ブラ
ウン社製、顆紳型)のカラム((IX50cm)にチャ
ージし、カラムクロマトグラフィーを行った。ao02
Mリン@緩衝液(pH7,4)[5j、0.01Mリ
ン鍍緩衝液(p fl 7゜4)5!の順で溶出し、溶
出液は30ゴづつ分画した。各分画をフォーリンローリ
−法による蛋白質定量およびスタフィロコッカス・アウ
レウス209Pに対する抗菌活性試験を行ない、分画番
号181〜300番に蛋白質mtyD−pcが、分画番
号321〜゛400番にスポラマイシンが昭められた。The above solid content was dissolved in water 3j&:, and the insoluble matter was temporarily washed with water. The P liquid and the washing liquid were combined (4)), 15 kg of ammonium sulfate was added to the mixture to achieve 90% saturation, and after stirring for 1 hour, the resulting precipitate was collected to obtain 102 tons of precipitate. Add this to 45 ml of water
0-3 in deionized water using a cellophane tube.
0j was added and dialysis was performed for 3 days. During that time, deionized water was replaced daily. 1170 m of the obtained desalted solution was pre-mixed with 0.002 M IJ acidic acid. 1! (pH7
Column chromatography was performed by charging a column (IX50 cm) of DBAE-cellulose (manufactured by Braun, condensate type) softened with , 5 to &0). ao02
M phosphorus @ buffer (pH 7,4) [5j, 0.01M phosphorus buffer (p fl 7゜4) 5! It was eluted in this order, and the eluate was fractionated into 30 fractions. Each fraction was subjected to protein quantification using the Folin-Lowry method and an antibacterial activity test against Staphylococcus aureus 209P. The protein mtyD-pc was found in fraction numbers 181 to 300, and sporamycin was found in fraction numbers 321 to 400. was celebrated.
得られた蛋白質KUD−PCJ画分&8ノに画分全8ノ
て40%飽和とし、1時間に生じた沈澱物を戸別した。The obtained protein KUD-PCJ fraction & 8 fractions were adjusted to 40% saturation in all 8 fractions, and the precipitate formed in 1 hour was collected from house to house.
P液に再び硫安を加えて90%飽和とし、1時間攪拌し
た後、生じた沈澱物をF取した。これを水に溶かし、そ
の溶液78ゴを予め水で十分に膨潤させたバイオゲルP
−30(バイオラド・ラボラドリース社製)のカラム(
60X6251)にチャージし、ゲル濾過クロマトグラ
フィーを行った。水で溶出し、溶出液は20fづつ分画
した。各分画を前記と同様に蛋白質定量および抗菌活性
試験を行い、分画番号41〜54番の画分280 ml
を得た。これを限外濾過(東洋p紙社製、UHP−90
、限外−過膜UH−1を使用)により約10+dtで濃
縮し、−夜冷却下静置した。析出した結晶をP取し、少
量の冷水で洗浄後、乾燥して六方晶系の無色結晶の蛋白
質KUD−PCI20”9を得た。融点258〜260
℃(分解)。Ammonium sulfate was again added to the P solution to make it 90% saturated, and after stirring for 1 hour, the resulting precipitate was collected. Biogel P is prepared by dissolving this in water and pre-swelling the resulting solution with water.
-30 (manufactured by Bio-Rad Laboratories) column (
60X6251) and gel filtration chromatography was performed. Elution was carried out with water, and the eluate was fractionated into 20 f fractions. Each fraction was subjected to protein quantification and antibacterial activity tests in the same manner as above, and 280 ml of fractions Nos. 41 to 54 were collected.
I got it. This is subjected to ultrafiltration (manufactured by Toyo P Paper Co., Ltd., UHP-90).
, using an ultra-filtration membrane UH-1) to about 10+ dt, and left to stand under cooling overnight. The precipitated crystals were collected, washed with a small amount of cold water, and dried to obtain hexagonal colorless crystal protein KUD-PCI20''9. Melting point 258-260.
°C (decomposition).
この−次母液と洗液を合わせ、硫安を加えて25%飽和
とし、3日間、冷暗所で静置した。析出した結晶をP取
し、少量の冷水で洗浄後、乾燥して柱状晶の蛋白質KU
D−PO770■を得た。この二次母液と洗液を合わせ
、硫安30%飽和処理により得られた結晶は560→で
あり、史に三次母液を硫安60%飽和処理により得られ
た結晶は45(19であった。This secondary mother liquor and washing liquid were combined, ammonium sulfate was added to make the mixture 25% saturated, and the mixture was allowed to stand in a cool, dark place for 3 days. The precipitated crystals are collected, washed with a small amount of cold water, and dried to form columnar crystals of protein KU.
D-PO770■ was obtained. The number of crystals obtained by combining the secondary mother liquor and the washing liquid and treating with 30% saturation with ammonium sulfate was 560 →, and the number of crystals obtained by treating the tertiary mother liquor with saturation with 60% ammonium sulfate was 45 (19).
このようにして得られた六方晶系の無色結晶および柱状
晶はセルロースアセテート膜(CHgMgTRON社製
セルロゲルR8,5X1251)による電気泳動分析(
1M酢酸−ギ酸緩衝液、PH2,20(IV。The hexagonal colorless crystals and columnar crystals thus obtained were analyzed by electrophoresis using a cellulose acetate membrane (Cellulogel R8, 5X1251 manufactured by CHgMgTRON).
1M acetate-formate buffer, PH2,20 (IV.
1時間、アミドプラックIOB染色〕の結果、第4図の
通り、同じ流動位置に各々同一のバンドを与えた。また
、六方晶系の無色結晶は5DS−ポリアクリルアミドゲ
ル電気泳動分析し、スキャナー(高滓製作所社製、C!
8−910)により泳動プロフィールを描くと第3図の
通りであり、単一であることが確認された。As a result of amide plaque IOB staining for 1 hour, identical bands were obtained at the same flow position, as shown in FIG. In addition, hexagonal colorless crystals were analyzed by 5DS-polyacrylamide gel electrophoresis and scanned using a scanner (manufactured by Takasu Seisakusho Co., Ltd., C!
8-910), the migration profile was drawn as shown in Figure 3, and it was confirmed that it was single.
実施例2
実施例1と同様の方法で得た主培養物116jを実施例
1の前段の精製工程と同様の方法により処理して脱塩溶
液1080−を得た。これを予め0.002Mリン酸緩
衝液(pH7,,0,〜74)で酸価化したDI!:A
E−セルロースのカラム(9X50m)にチャージし、
実施例1と同様の条件でカラムクロマトグラフィーによ
り分離精製すると、蛋白質KUD−PCとスポラマイ7
ノは分離せずに混合物の分画番号121〜260番の分
画が得られた。Example 2 Main culture 116j obtained in the same manner as in Example 1 was treated in the same manner as in the first purification step of Example 1 to obtain desalted solution 1080-. This was pre-oxidized with 0.002M phosphate buffer (pH 7, 0, ~ 74) to DI! :A
Charge an E-cellulose column (9 x 50 m),
When separated and purified by column chromatography under the same conditions as in Example 1, protein KUD-PC and Sporamai 7 were separated and purified.
Fractions numbered 121 to 260 of the mixture were obtained without separation.
この分画を実施例1と同様の方法により硫安90%飽和
による沈澱処理およびバイオゲルP30カラムクロマト
クラフィーによる分離精製すると、蛋白質KUD−PC
は分画番号41〜50番に、スポラマイ//は分画番号
53〜64番に分離された。このバイオゲルP−30カ
ラムクロマトグラムを第5図に示すっ
蛋白質KUD−PC画分を実施例】と同様の方法で限外
p4および結晶化を行って、六方晶系の無色結晶730
1m9、融点258〜260℃(分解)柱状晶1.48
9および結晶0.99を得た。これらの結晶はセルロー
スアセテート膜電気泳動分析により各々同一泳動位置に
単一バンドを与えた。This fraction was separated and purified by precipitation treatment with 90% ammonium sulfate saturation and biogel P30 column chromatography in the same manner as in Example 1, resulting in protein KUD-PC.
was separated into fraction numbers 41-50, and sporamy // was separated into fraction numbers 53-64. This Biogel P-30 column chromatogram is shown in Figure 5.The protein KUD-PC fraction was subjected to ultrap4 and crystallization in the same manner as in Example], and the hexagonal colorless crystal 730 was obtained.
1m9, melting point 258-260℃ (decomposition) columnar crystals 1.48
9 and 0.99 crystals were obtained. These crystals each gave a single band at the same electrophoretic position when analyzed by cellulose acetate membrane electrophoresis.
実施例3
実施例1と同様の方法で得た主培養物110ノを実施例
1と同様の方法で分離精製してDEAR−セルロース力
ラムクロマトグラフイーの蛋白質KUD−PC画分の硫
安40〜9 (1%飽和沈澱画分を得た。Example 3 110 pieces of the main culture obtained in the same manner as in Example 1 were separated and purified in the same manner as in Example 1 to obtain ammonium sulfate of 40 ~ 9 (1% saturated precipitate fraction was obtained.
この画分を水70−に溶かし、これを予め水で十分に膨
潤させたセファデックスG−50のカラム(6X 78
5II )によりゲルー過クロマトグラフィーを行った
。水で溶出し、溶出液は20tづつ分画し、各分画を蛋
白質定量により分画番号56〜80番の蛋白質KUD−
PC!1iii分を集めた。この溶液を限外p過により
脱塩および濃縮処理し7、濃縮液100Talを凍結乾
燥して白色粉末状の蛋白質KUD−PC1,45Mを得
たつ
この粉末はセルロースアセテート膜電気泳動分析により
単一バンドを示し、その泳動位置から実施例1で得た六
方晶系無色結晶および柱状晶と同一であると認められた
。This fraction was dissolved in 70° of water, and this was placed on a Sephadex G-50 column (6X 78°) that had been sufficiently swollen with water.
Gel permeation chromatography was performed using 5II). Elution was carried out with water, the eluate was fractionated into 20t portions, and each fraction was analyzed for protein quantification.
PC! Collected 1iii minutes. This solution was desalted and concentrated by ultrapolar filtration (7), and 100 Tal of the concentrated solution was lyophilized to obtain a white powder protein KUD-PC1,45M. It was recognized that the crystals were the same as the hexagonal colorless crystals and columnar crystals obtained in Example 1 based on the migration position.
実施例°4
実施例1において200!容夕/りにおける培養時間を
96時間行って主培養物114!を得た。Example °4 200 in Example 1! After culturing for 96 hours, the main culture was 114! I got it.
この培養物中にはセルロースアセテート#電気泳動分析
により蛋白質KUD−PCの存在が確認されたが、抗菌
活性試験によりスポラマイシンの存在は確認されなかっ
た。The presence of the protein KUD-PC in this culture was confirmed by cellulose acetate electrophoresis analysis, but the presence of sporamycin was not confirmed by an antibacterial activity test.
この主培養物を実施例1と同様の方法により分解精製し
て、六方晶系の無色結晶501m9.融点258〜26
0℃(分解)および柱状晶L89fを得た。これらの結
晶はセルロースアセテート膜電気泳動分析により徹−バ
ンドを示した。This main culture was decomposed and purified in the same manner as in Example 1 to produce 501 m9 of hexagonal colorless crystals. Melting point 258-26
0°C (decomposition) and columnar crystals L89f were obtained. These crystals showed a transband when analyzed by cellulose acetate membrane electrophoresis.
第1FyJは蛋白質KDD−PCの紫外線吸収スペクト
ル、第2図は蛋白質KUD−PC!の赤外線吸収スペク
トル、第3図は蛋白質KDD−PC5D8−ポリアクリ
ルアミドゲル電気泳動法による泳動プロフィール、第4
図は蛋白質KUD−P(3のセルロースアセテート膜電
気泳動図、第5図は蛋白質KUD−PCおよびスボラマ
イシ/の)(イオゲルP−30カフムクロマトグラムを
示す。
第4図
第5図
六方r8系無色鮎晶
柱状品The first FyJ is the ultraviolet absorption spectrum of the protein KDD-PC, and the second figure is the protein KUD-PC! Figure 3 shows the migration profile of protein KDD-PC5D8-polyacrylamide gel electrophoresis, Figure 4 shows the infrared absorption spectrum of
The figure shows the cellulose acetate membrane electrophoretogram of protein KUD-P (3), and the figure 5 shows the protein KUD-PC and Sboramaycin/Iogel P-30 cuff chromatogram. Colorless sweetfish crystal columnar product
Claims (1)
)8う〇(り分子蓋; 11500 (8DS−ポリア
クリルfミドゲル電気泳動法による) ■融点、258〜260℃(分解) ■比旋光度;〔α)D−!MLs°(0=1.0、水)
■紫外線吸収スペクトル;第1図の辿り■赤外線吸収ス
ペクトル;第2図の通り■溶剤に対する溶解性;水に可
溶、アル:ノール、アセトン、ペンゼノなど の有機溶剤に不M (り呈色反応;フォリンローリ−反応、ビューレット反
応、ライドン・スミス反応に陽 性、フェノール硫酸反応、ア/スロ /(iik反応に陰性、14酸加水分解物は二/ヒドリ
ノ反応に陽性 ・り塩基性、酸性、中性の区別一本物質の0.1%水溶
液のpHは4〜8 J9物貿の色、形状;無色結晶 ■アミノIli組fiy;塩酸加水分解物のアミ〕無分
析(1(モル比)は次の通り 了スパフキン酔 0.144 スレオニア 0.258 セ リ 7 0.
226グルタミy @ O,I 41ゾロリ
y 0.111グリンy
O,250 アラニ/ 0.363 バリア0.265 イソロイシフ 0.016 Uイシ10.11 ? メチオニ/ − チロンノ0.1120 フェニルアフニ7 0.079 リジy0.089 アルギニン 0.040 ヒスチジ/ (微量) Ot電気泳動 8D8−ポリアクリルアミドゲル電気泳
動法による泳動プロフィ− ルは第3図の通り、本物質は皐− である。 2)ストレプトスボランギウム属に属する蛋白質KUD
−PC生産菌を培地に培養して培養物中に蛋白質KUD
−PCjを蓄積せしめ、該培養物から蛋白質KUD−P
Cを採取することを特徴とする新規蛋白質KUD−PC
の製造法。 3)ストレプトスボランギウム属に属する蛋白質KUD
−PC生産菌がストレプトスポラ/イウム・シュードブ
ルガレエPO−357(F、ERM−P11&1357
1)である特許請求の範囲第2項記載の製造法。[Claims] l) A novel protein having the following physical and chemical properties 1 KUl) Cj ■Elemental analysis: Contains carbon, hydrogen, nitrogen, oxygen, and ammonium sulfate 1 049.98 gourd H? , 27%, Nl & 23%, 814
)8〇(ri molecular cap; 11500 (by 8DS-polyacrylic f midgel electrophoresis) ■Melting point, 258-260℃ (decomposition) ■Specific optical rotation; [α)D-! MLs° (0=1.0, water)
■Ultraviolet absorption spectrum; as shown in Figure 1 ■Infrared absorption spectrum; as shown in Figure 2 ■Solubility in solvents: Soluble in water, insoluble in organic solvents such as al:nol, acetone, and penzeno (color reaction) ; Positive for Folin-Lowry reaction, Biuret reaction, Lydon-Smith reaction, negative for phenol-sulfuric acid reaction, a/thro/(iik reaction, positive for di/hydrino reaction for 14-acid hydrolyzate, basic, acidic, medium The pH of a 0.1% aqueous solution of one substance is 4 to 8.Color and shape of J9 Materials; colorless crystals; As follows: Ryo Puffkin Drunkness 0.144 Threonia 0.258 Seri 7 0.
226 Glutami y @ O, I 41 Zorori y 0.111 Grin y
O,250 Arani/0.363 Barrier 0.265 Isoleusif 0.016 Uishi 10.11? Methiony/-Tyronno 0.1120 Phenylafni7 0.079 Lyzyy0.089 Arginine 0.040 Histide/ (trace amount) Ot electrophoresis 8D8-Polyacrylamide gel electrophoresis shows the migration profile of this substance as shown in Figure 3. is 琐. 2) Protein KUD belonging to the genus Streptosborangium
-Culture PC-producing bacteria in a medium and produce protein KUD in the culture.
- Accumulate PCj and extract the protein KUD-P from the culture.
KUD-PC, a novel protein characterized by collecting C.
manufacturing method. 3) Protein KUD belonging to the genus Streptosborangium
-PC-producing bacteria are Streptospora/Ium pseudobulgalae PO-357 (F, ERM-P11 & 1357
1) The manufacturing method according to claim 2.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP57081466A JPS58198422A (en) | 1982-05-17 | 1982-05-17 | Novel protein kud-pc and preparation thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP57081466A JPS58198422A (en) | 1982-05-17 | 1982-05-17 | Novel protein kud-pc and preparation thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS58198422A true JPS58198422A (en) | 1983-11-18 |
Family
ID=13747171
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP57081466A Pending JPS58198422A (en) | 1982-05-17 | 1982-05-17 | Novel protein kud-pc and preparation thereof |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS58198422A (en) |
-
1982
- 1982-05-17 JP JP57081466A patent/JPS58198422A/en active Pending
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