JPH0235081A - Arginine deiminase and use thereof - Google Patents
Arginine deiminase and use thereofInfo
- Publication number
- JPH0235081A JPH0235081A JP63184556A JP18455688A JPH0235081A JP H0235081 A JPH0235081 A JP H0235081A JP 63184556 A JP63184556 A JP 63184556A JP 18455688 A JP18455688 A JP 18455688A JP H0235081 A JPH0235081 A JP H0235081A
- Authority
- JP
- Japan
- Prior art keywords
- arginine
- arginine deiminase
- deiminase
- enzyme
- solution
- 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.)
- Granted
Links
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Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、新規なアルギニン・デイミナーゼおよびその
製造法並びにその用途に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel arginine deiminase, its production method, and its use.
アルギニン・デイミナーゼ（酵素分類番号３゜５．３．
６）は、Ｌ−アルギニンと水からし一シトルリンとアン
モニアを生成する酵素反応を触媒する酵素であり、動物
、細菌、酵母類にその存在が知られている〔酵素ハンド
ブック、６０２頁。Arginine deiminase (enzyme classification number 3゜5.3.
6) is an enzyme that catalyzes the enzymatic reaction that produces L-arginine, water mustard, citrulline, and ammonia, and its existence is known in animals, bacteria, and yeasts [Enzyme Handbook, p. 602].
朝食書店、 １９８４年版、第１版第３刷〕、これらの
酵素のうち、精製されたと報告のあるものには、シュー
ドモナス・プチダ（Ｐｓｅｕｄｏｎ＋ｏｎａｓ ｐｕｔ
ｉｄａ）マイコプラズマ・ホミニス（Ｍｙｃｏｐｌａｓ
ｍａ ｈｏｍｉｎｉｓ）。Breakfast Shoten, 1984 edition, 1st edition, 3rd printing] Among these enzymes, those that have been reported to have been purified include Pseudomonas putida (Pseudomonas putida).
ida) Mycoplasma hominis
ma hominis).
マイコプラズマ・アルスリティディス（Ｍｙｃｏｐｌａ
ｓｍａ ａｒｔｈｒｉｔｉｄｉｓ）がある〔カキモト、
ティー、ら（Ｋａｋｉｍｏｔｏ、 Ｔ、ｅｔａｌ） フ
ェブス レター（ＦＥＢＳ Ｌｅｔｔ、）ＶＯＬ、１９
． Ｐ １６６−１６８．（１９７１） ； ’ｉムケ
、７−ＪＬｔ。Mycoplasma arsulitidis
sma arthritidis) [Kakimoto,
Kakimoto, T, etal FEBS Lett, VOL, 19
．． P 166-168. (1971);'i Muke, 7-JLt.
ティー、 （Ｓｈｉｍｋｅ、Ｒ，Ｔ、） メソッド
オン エンザイモロジ−（Ｍｅｔｈ、Ｅｎｚｙ＋ｎｏ１
．） ＶＯＬ、１７Ａ、Ｐ ３１０−３１３、 （１９
７０） ｉワイクマン、ジュー。エル、アンドファルニ
ー、デイ−、イー、 （Ｗｅｉｃｋｍａｎ、Ｊ、Ｌ、
＆ Ｆａｈｒｎｅｙ、 Ｄ、Ｅ、） ジャーナル オ
ン バイオロジカル ケミストリー（Ｊ、Ｂｉｏｌ、Ｃ
ｈｅａ＋、） ＶＯＬ、２５２．Ｐ２６１５２６２０、
（１９７７） ）。Tee, (Shimke, R, T,) Method
On Enzymology (Meth, Enzy+no1
．． ) VOL, 17A, P 310-313, (19
70) iWeikman, Zhu. L, Andfarny, D, E, (Weickman, J, L,
& Fahrney, D, E,) Journal on Biological Chemistry (J, Biol, C
hea+,) VOL, 252. P26152620,
(1977)).
また、Ｌ−アルギニンを分解する酵素であるＬ−アルギ
ナーゼが抗ｍｉ作用を有していることが知られているこ
とから、アルギニン・デイミナーゼへの同様な作用への
期待はされていた〔特開昭４８−５８１８７号〕。Furthermore, since L-arginase, an enzyme that decomposes L-arginine, is known to have anti-mitrointestinal effects, there were expectations for a similar effect on arginine deiminase [JP-A No. 1973-58187].
［発明が解決しようとする問題点］
しかしながら、これら従来のアルギニン・デイミナーゼ
生産菌による生産にはいくつかの問題点があった。すな
わち、これらのアルギニン・デイミナーゼ生産蘭は、ア
ルギニン・デイミナーゼ生産性が低いこと；マイコプラ
ズマに属するマイコプラズマ・ホミニス（Ｍｙｃｏｐｌ
ａｓｍａ ｈｏｍｉｎｉｓ）、マイコプラズマ・アルス
リディス（Ｍｙｃｏｐｌａｓｍａ ａｒｔｈｒｉｔｉｄ
ｉｓ）から採取する場合は培養管理が煩雑であり、該酵
素の大量生産における酵素源として用いるには難点があ
ること；精製純化するのに極めて困難である等の問題が
あった。[Problems to be Solved by the Invention] However, there are several problems with production using these conventional arginine deiminase producing bacteria. In other words, these arginine deiminase-producing orchids have low arginine deiminase productivity; Mycoplasma hominis (Mycopl.
asma hominis), Mycoplasma arthritis
When harvesting from is), culture management is complicated, and there are problems such as difficulty in using it as an enzyme source in mass production of the enzyme; and extremely difficult purification.
また、マイコプラズマ由来のアルギニン・デイミナーゼ
が、！！ＩＩＩＸ細胞増殖阻害を示す明確な報告もない
。Also, arginine deiminase derived from mycoplasma! ! There are also no clear reports showing inhibition of IIIX cell growth.
本発明者らは、動物１１１５＆細胞の中から、培養液が
顕著に増殖阻害作用を示す細胞株を見出し、その株につ
いて、種々研究を行った結果、その株に寄生したマイコ
プラズマ属に属する菌株（ＴＧＩＦ）が、産生ずる蛋白
質が、腫瘍細胞増殖阻害を示すことを見出した。また研
究者らは、該蛋白質を、前記マイコプラズマに属する菌
株（ＴＧＴＦ）を培養し、単離精製することにより均質
に得、この蛋白質が新規なアルギニン・デイミナーゼで
あることも見出し本発明を完成した。The present inventors found a cell line among animal 1115 & cells whose culture solution exhibits a remarkable growth-inhibitory effect, and conducted various studies on that strain. It was discovered that the protein produced by TGIF) inhibits tumor cell growth. The researchers also found that the protein was obtained homogeneously by culturing the aforementioned Mycoplasma strain (TGTF), and isolated and purified it, and discovered that this protein was a novel arginine deiminase, and completed the present invention. .
すなわち、本発明は下記の理化学的性状を有するアルギ
ニン・デイミナーゼであり、
（ａ） 作用；Ｌ−アルギニンと水からシトルリンと
アンモニアを生成する酵素反応を触媒する（ｂ） 基
質特異性；Ｌ−アルギニンに基質特異性を有する
（ｃ）至適ｐＨ；７．０〜７．５
（ｄ）ｐＨ安定性；５．０以上
（ｅ）分子量 ；４７０００±５０００ （ＳＤＳＰＡ
ＧＥ法による）
（ｆ） 等電点 ；７．４±０．５
マイコプラズマ・オラーレに属するアルギニン・デイミ
ナーゼ生産蘭を培地に培養し、培養物より咳アルギニン
・デイミナーゼを採取することを特徴とするアルギニン
・デイミナーゼの製造法であり、更にマイコプラズマ属
に属する微生物由来のアルギニン・デイミナーゼ活性を
有する腫瘍細胞増殖阻害活性物質を提供するものである
。That is, the present invention is an arginine deiminase having the following physical and chemical properties: (a) Action: catalyzes an enzymatic reaction that produces citrulline and ammonia from L-arginine and water (b) Substrate specificity: L-arginine (c) Optimum pH; 7.0 to 7.5 (d) pH stability; 5.0 or more (e) Molecular weight; 47,000 ± 5,000 (SDSPA
(by GE method) (f) Isoelectric point; 7.4±0.5 Arginine deiminase produced by culturing an arginine deiminase-producing orchid belonging to Mycoplasma orale in a medium and collecting cough arginine deiminase from the culture. - A method for producing deiminase, and further provides a tumor cell growth inhibiting substance having arginine deiminase activity derived from a microorganism belonging to the genus Mycoplasma.
本発明の新規なアルギニン・デイミナーゼ生産菌の分類
学的性状は以下の通りである。The taxonomic properties of the novel arginine deiminase producing bacterium of the present invention are as follows.
■、培地における生育状態
マイコプラズマ・オラーレ（ＴＧＩＦ）の？ａ体培地〔
培地組成；培地１２中にＰ Ｐ、 Ｌ ＯプロスＷ１０
（ＤＩＦＣＯ社製）１５ｇ、Ｌ−アルギニン塩酸塩５ｇ
、 ウマ血清２００ ｄ、酵母エキス２５ｇ、酢酸タ
リウム０．２５ ｇ 、 ペニシリンＧカリウム１０万
単位、フエーノールレンド５ｍｇを含有する）に於ける
増殖は、種菌としてｌＸ１０５ＣＦＵ／成〔ＣＦＵは集
落形成単位（ｃｏｌｏｎｙ Ｆｏｍｉｎｇ Ｕｎｉｔ）
の略記号である〕接種し、３７°Ｃで、静置培養した結
果、誘導期に続いて対数増殖期に入り、培養開始４〜５
日後に菌数が最大になる定常期に至る。定常期の期間は
短く、培養によって異なり、且つほぼ数時間で、減数期
に入る生育形態をとる。■What is the growth status of Mycoplasma orale (TGIF) in the culture medium? a-body culture medium [
Medium composition: PP, LO Pros W10 in medium 12
(manufactured by DIFCO) 15g, L-arginine hydrochloride 5g
, containing 200 d of horse serum, 25 g of yeast extract, 0.25 g of thallium acetate, 100,000 units of potassium penicillin G, and 5 mg of phenollend). colony forming unit)
As a result of inoculation and static culture at 37°C, the logarithmic growth phase follows the lag phase, and 4 to 5 minutes after the start of culture.
After a few days, the stationary phase is reached where the number of bacteria reaches its maximum. The period of the stationary phase is short and varies depending on the culture, and the growth form enters the mitotic phase in approximately a few hours.
■、生理的諸条件
生育し得るｐＨ；６〜８
至適ｐＨ；７．４〜７．６
生育し得る温度；３０〜３７°Ｃ
至適生育温度；３７°Ｃ
■、顕微鏡下における形態的特徴
寒天培地〔培地組成；培地ｌｌ中に、バクトアガール（
ＤＩＦＣＯ社製ＮＯｇ、ＰＰＬＯブロスＷ１０ （、％
’ （Ｄ Ｉ ＦＣＯ社製） １４．５．ｇ、 Ｌ−
フルギニン塩酸塩５ｇ、Ｌ−グルタミン３ｇ、ＤＮＡ（
粗製） ２０ｍｇ、 Ｎ Ａ Ｄ １５０ｍｇ、グルコ
ース１０ｇ、イーグルビタミン液（Ｘ１００）１０成、
ウマ血清２００ ｄ酵母エキス２５ｇを含有する〕に於
ける集落の大きさは、１０〜５００ａｍであり、中心部
が濃く、周辺が薄く、いわゆる目玉焼状を呈する。染色
はＤｉｅｎｅｓ （ディエネス）液〔メチレンブルー２
．５ｇ； アズールＩＩ １．２５ｇ；マルトース１
０．０ｇ：Ｎ ａ 、ＣＯｓ ０．２５ｇ；蒸留水 １
００ｄ）を−用いて行った。染色液を寒天面に流し、１
〜２分後に余分の液を捨て、顕微鏡観測した。集落の中
心部は濃青色に周辺部は淡青色に染まって見える。１０
０倍の顕微鏡倍率下では、周辺部は顆粒状に見える。■, Physiological conditions pH for growth; 6-8 Optimal pH; 7.4-7.6 Temperature for growth; 30-37°C Optimal growth temperature; 37°C ■, Morphological conditions under the microscope Characteristics Agar medium [Medium composition; Bacto agar (
DIFCO NOg, PPLO broth W10 (,%
' (manufactured by DI FCO) 14.5. g, L-
Fulginine hydrochloride 5g, L-glutamine 3g, DNA (
crude) 20mg, NAD 150mg, glucose 10g, Eagle vitamin liquid (X100) 10 parts,
The size of the colony in [Horse serum 200 d containing 25 g of yeast extract] is 10 to 500 am, and has a so-called fried egg shape, with the center being thicker and the periphery being thinner. Staining was done with Dienes solution [Methylene Blue 2
．． 5g; Azure II 1.25g; Maltose 1
0.0g: Na, COs 0.25g; distilled water 1
00d) was used. Pour the staining solution onto the agar surface and
After ~2 minutes, excess liquid was discarded and microscopic observation was performed. The center of the village appears dark blue and the periphery appears pale blue. 10
Under 0x microscopic magnification, the periphery appears granular.
■、抗体による発育阻害
各マイコプラズマ種の抗血清を用い、ワラス（Ｗａｌｌ
ａｃｅ Ａ、ＣＩｙｄｅｌＪＲ）著の文献（ジャーナル
オン イムノロジー（Ｊ、 Ｉｍｍｕｎｏ＋、） Ｖ
ＯＬ、９２．Ｐ、９５８９６５、 （１９６４） ）に
従い同定の結果〔東京大学医学部付属動物実験施設にて
〕、マイコプラズマ・オラーレと同定された。■ Antibody-induced growth inhibition Using antiserum of each mycoplasma species, Wallas (Wall)
ace A, CIydelJR) (Journal on Immunology (J, Immuno+,) V
OL, 92. P., 958965, (1964)) [at the Animal Experiment Facility attached to the Faculty of Medicine, University of Tokyo], it was identified as Mycoplasma orale.
■、グルコース、アルギニン、尿素の分解試験被検菌を
１０％（Ｖ／Ｖ）ウマ血清添加ハートインツユジョンブ
ロス（Ｉｌｅａｒｔ １ｎｆｕｓｉｏｎ ｂｒｏｔｈ）
ＣＤ ｉ ｆｃｏ社製）で、２４時間培養し、その１
成をそれぞれグルコース、アルギニン、尿素を含む被検
培地および対照培地に接種した。培養試験管を密栓して
、３７°Ｃで１週間培養した。被検物質を含まない対照
と０．５以上のｐＨの下降または上昇のみられたものを
陽性として判定した結果以下の通りであった。■Glucose, arginine, urea decomposition test Heart infusion broth containing 10% (V/V) horse serum supplemented with test bacteria (Ileart 1nfusion broth)
CD (manufactured by fco) for 24 hours, part 1
were inoculated into test and control media containing glucose, arginine, and urea, respectively. The culture test tube was tightly stoppered and cultured at 37°C for one week. The results were determined as positive if the pH decreased or increased by 0.5 or more compared to the control that did not contain the test substance.
グルコース分解活性 ； 陰性
アルギニン分解活性 ： 陽性
尿素分解活性 ； 陰性
以上の結果から、細胞壁の完全欠如、直径０．１〜０．
２μｍの基本小体を有し、集落形態として、寒天培地に
くい込んで増殖し、目玉焼状集落を形成する特徴を有し
、発育に血清を必要とすることからマイコプラズマ属に
属する。そこで、抗体による増殖■害試験により、本菌
株（ＴＧＩＦ）は、マイコプラズマ・オラーレ（ＴＧＩ
Ｆ）（Ｍｙｃｏｐｌａｓｍａ ｏｒａｌｅ （ＴＧＩ
Ｆ））と同定命名した。Glucose decomposition activity; Negative arginine decomposition activity; Positive urea decomposition activity; Negative or higher results indicate complete absence of cell wall, diameter 0.1-0.
It has an elementary body of 2 μm, and its colony form is characterized by embedding it in agar medium and multiplying, forming fried egg-shaped colonies, and because it requires serum for growth, it belongs to the genus Mycoplasma. Therefore, through growth and damage tests using antibodies, this strain (TGIF) was found to be Mycoplasma orale (TGIF).
F) (Mycoplasma orale (TGI)
F)).
なお、本菌株は、工業技術院微生物工業技術研究所にマ
イコプラズマ・オラーレ（ＴＧＩＦ）（Ｍｙｃｏｐｌａ
ｓｍａ ｏｒａｌｅ （ＴＧＩＦ）］微工研菌条寄第
１２７０号（ＦＥＲＭ 、ＢＰ−／２７０） とし
て寄託されている。This strain was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology as Mycoplasma orale (TGIF).
sma orale (TGIF)] FERM, BP-/270.
本発明のアルギニン・デイミナーゼを生産するに当たっ
ては、このアルギニン・デイミナーゼ生産菌を酵素など
を生産する通常の方法で培養する。In producing the arginine deiminase of the present invention, this arginine deiminase producing bacterium is cultured by a conventional method for producing enzymes and the like.
培養の方法は、液体培養でも固体培養でもよいが、工業
的にはアルギニン・デイミナーゼ生産菌をその生産用培
地に接種し、静置培養を行うのが有利である。アルギニ
ン・デイミナーゼを培養するための培地組成は、微生物
、特にマイコプラズマを培養するのに通常用いられるも
のが広く用いられる。窒素源として利用可能な窒素化合
物であればよく、例えば酵母エキス、ラクトアルブミン
氷解物、尿素、ペプトン、肉エキスなどが使用される。The culture may be carried out by liquid culture or solid culture, but from an industrial perspective, it is advantageous to inoculate arginine deiminase-producing bacteria into a production medium and perform static culture. As the medium composition for culturing arginine deiminase, those commonly used for culturing microorganisms, particularly mycoplasma, are widely used. Any nitrogen compound that can be used as a nitrogen source may be used, such as yeast extract, lactalbumin thawed product, urea, peptone, meat extract, etc.
炭素源としては資化可能な炭素化合物であればよく、例
えばブドウ糖などが使用される。その他マイコプラズマ
の培養に必須である血清またはステロール類を適宜添加
すればよく、また種々の無機塩が必要に応じて使用され
る。培養温度は菌が発育し、アルギニン・デイミナーゼ
を生産する範囲内で適宜変更し得るが、３０〜３７°Ｃ
１好ましくは３５〜３７°Ｃである。培養時間は、条件
によって多少異なるが、通常４〜８日、好ましくは５〜
６である。しかしながら、本菌株の増殖が最高力価に達
する時期を見計らって適当な時期に培養を終了するのは
当然のことである。The carbon source may be any carbon compound that can be assimilated, such as glucose. In addition, serum or sterols essential for culturing mycoplasma may be added as appropriate, and various inorganic salts may be used as necessary. The culture temperature can be changed as appropriate within the range that allows the bacteria to grow and produce arginine deiminase, but it is 30 to 37°C.
1 Preferably 35 to 37°C. The culture time varies depending on the conditions, but is usually 4 to 8 days, preferably 5 to 8 days.
It is 6. However, it is a matter of course that the culture should be terminated at an appropriate time, taking into account the time when the growth of this strain reaches its maximum titer.
培養終了後、該培養物より本酵素を採取するには通常の
酵素採取手段を用いることができる。なお、本酵素は、
菌体内酵素であるので、超音波処理、凍結菌体衝撃破砕
法（低温細胞破砕法）、各種薬剤による方法等菌体内よ
り酵素を採取する通常の手段を用いて、粗酵素液を得る
。このようにして得られた粗酵素液は、さらに公知の蛋
白質、酵素などの単離、精製手段を用いて精製し、精製
されたアルギニン・デイミナーゼが得られる。例えば、
粗製のアルギニン・デイミナーゼ含有液を、トリス−塩
酸緩衝液等の緩衝液で緩衝化し、これをカルボキシ−セ
ルロース、カルボキシメチルデキストランゲル、スルホ
プロピル−デキストランゲルなどのイオン交換樹脂やデ
キストランゲルやポリアクリルアマイドなどのゲル濾過
剤によるクロマトグラフィーを適宜組み合わせて精製し
、ついで、必要に応じて濃縮し緩衝液に溶解した溶液と
してし、精製アルギニン・デイミナーゼ溶液を得ること
ができる。After the culture is completed, the present enzyme can be collected from the culture using a conventional enzyme collection method. In addition, this enzyme is
Since the enzyme is an intracellular enzyme, a crude enzyme solution is obtained using conventional means for collecting the enzyme from within the microbial cell, such as ultrasonication, frozen cell shock disruption (low-temperature cell disruption), and methods using various drugs. The crude enzyme solution thus obtained is further purified using known means for isolating and purifying proteins, enzymes, etc., to obtain purified arginine deiminase. for example,
The crude arginine deiminase-containing solution is buffered with a buffer such as Tris-HCl buffer, and then mixed with an ion exchange resin such as carboxy-cellulose, carboxymethyl dextran gel, sulfopropyl-dextran gel, dextran gel, or polyacrylamide. A purified arginine deiminase solution can be obtained by performing purification by appropriately combining chromatography using a gel filtration agent such as arginine deiminase, etc., and then concentrating as necessary to prepare a solution dissolved in a buffer solution.
このようにして得られたアルギニン・デイミナーゼの理
化学的性質は以下に述べる通りである。The physicochemical properties of the arginine deiminase thus obtained are as described below.
（１）アルギニン・デイミナーゼ活性測定法基質溶液の
調整；
２００ｍＭ）リス−塩酸緩衝？＆ （Ｐ Ｈ７，２）
２００μ２に５０ｎＭＤＴＴ （デイチオスレイトール
）８０μｊ２，１０Ｏｎ＋Ｍアルギニン・塩酸付加物４
０μｌを溶解して、基質溶液を調整する。(1) Arginine deiminase activity measurement method Preparation of substrate solution; 200mM) Lis-HCl buffer? & (PH7,2)
200μ2, 50nMDTT (Dethiothreitol) 80μj2, 10On+M arginine/hydrochloric acid adduct 4
Prepare the substrate solution by dissolving 0 μl.
酵素溶液の調整：
後述の実施例により得られた本発明アルギニン・デイミ
ナーゼ溶液を１０ｍＭ）’Ｊス塩酸緩衝液（ｐＨ７，０
） で０Ｄｚｓｏ＝０．００５になる様ニ希釈して酵素
溶液とした。Preparation of enzyme solution: The arginine deiminase solution of the present invention obtained in the example described later was added to 10 mM)'JS hydrochloric acid buffer (pH 7.0).
) to obtain an enzyme solution.
活性測定：
前記の基質溶液３２０μｌを小試験管に取り、３７°Ｃ
１水浴中に２分間静置した後、酵素溶ｉ８０μ！添加し
、反応を開始する。正確に３０分間３７°Ｃで反応した
後、５Ｍ過塩素酸１００μ２を添加し反応を停止する。Activity measurement: Take 320 μl of the above substrate solution into a small test tube and heat at 37°C.
1 After leaving it in a water bath for 2 minutes, apply Enzyme Solu i80μ! and start the reaction. After reacting for exactly 30 minutes at 37°C, 100 μ2 of 5M perchloric acid is added to stop the reaction.
その後、反応液に、酸化還元緩衝液（９，０ｇ ＮＨ４
Ｆｆｌ（ＳＯ４）ｚ −１２Ｈｚ０，１１．０ｇ （Ｎ
Ｈ４） Ｊｅ（Ｓｏｎ） ｚ ・６ＨｚＯをＩＮ硫酸溶
液に溶解し１００成とした溶液］ １２５μｌを加え、
その後酸混合液（蒸留水２００ ｄ、 Ｈ１ＰＯ４（ｄ
＝１．７４）’ ３００ ｄ。After that, a redox buffer (9.0 g NH4
Ffl(SO4)z -12Hz0,11.0g (N
H4) Je(Son) z ・6HzO dissolved in IN sulfuric acid solution to make 100% solution] Add 125μl,
Then add an acid mixture (distilled water 200 d, H1PO4 (d
=1.74)' 300 d.
ＨｆｆｉＰＯ４（ｄ＝１．８４） １００ ｄ） ６
２５μｌを加え、ジアセチルモノキシム溶液〔ジアセチ
ルモノキシム０．７５ｇを蒸留水に溶解し１００−とし
た溶液〕２５０μｌを加え、遮光下、２０分間沸騰水中
で加熱し、その後冷却し、４９０ ｎｍの吸光度を測定
する。シトルリン生成量を算定するための標準線は、１
Ｍ過塩素酸で調整した種々の濃度（０，０１〜０．０５
ｐ ｍｏｌｅ／ ｍｌ ）のＬ−シトルリン溶液５０
０μｊ２を用い、上記酸化還元緩衝液１２５μｉ添加以
降の操作行ってシトルリン濃度に対する４９０ｎｍの吸
光度の関係をプロットして作成し、直線領域を標準検量
線として使用した。HffiPO4 (d=1.84) 100 d) 6
Add 25 μl of diacetyl monoxime solution [a solution of 0.75 g of diacetyl monoxime dissolved in distilled water to give a solution of 100%], and heat in boiling water for 20 minutes in the dark, then cool and measure the absorbance at 490 nm. Measure. The standard line for calculating the amount of citrulline produced is 1
Various concentrations (0.01 to 0.05) adjusted with M perchloric acid
p mole/ml) L-citrulline solution 50
Using 0 μj2, the operations after adding 125 μi of the redox buffer were performed to plot the relationship between absorbance at 490 nm and citrulline concentration, and the linear region was used as a standard calibration curve.
（２）基質特異性：
遊離アミノ酸に対する反応の特異性を調べるために、ア
ミノ酸混合液（Ｉｌの１０ｍＭ ）リス塩酸緩衝液ｐ
Ｈ７、０） Ｌ−ｙルギニン２００ｍｇ 、Ｌ−
アスパラギン・１１□０５６．８ｍｇ、Ｌ−ｙスバラギ
ン酸２０ｍｇ、Ｌ−シスチン５抛ｇ、Ｌ−クルタミン酸
２０ｍｇ、Ｌ−クルクミン３００ｍｇ 、グツ９フ１
０ドロキシプロリン２０ｍｇルーイ加インン５０ｍｇ，
Ｌ−ｏイラン５０ｍｇ，Ｌ−リジン５１１ｃ Ｑ ４０
ｍｇ，Ｌ−メチオニン１５ｍｇ，Ｌ−フェニルアラニン
１５ｍｇ．Ｌ−プロリン２０ｍｇ，Ｌ−セリン３０ｍｇ
．Ｌ−スレオニン２０ｍｇ．Ｌ−チ■シシ２０Ｉｌ＋ｇ
，Ｌ−バリン２０ｍｇ，Ｌ−トリプトフ７：７ ｍ
ｇを含有する） ８００ｔｔｌ に２００μｌの本発
明アルギニン・デイミナーゼ溶液（ＯＤ２，。、＝０．
０１６ ）を添加し、３７°Ｃ１１８時間インキエヘー
トした後、アミノ酸濃度の変化をアミノ酸自動分析装置
（日立 Ｌ−８５００型）を用いて解析した。対照とし
て、本発明アルギニン・デイミナーゼ溶液の代わりに１
０ｍＭ トリス塩酸緩衝液を添加して同じ操作を行った
。その結果第１表に示す如く本発明アルギニン・デイミ
ナーゼは、Ｌ−アルギニンに特異的に作用してシトルリ
ンを生成する反応を触媒する酵素であることがわかった
。(2) Substrate specificity: In order to examine the specificity of the reaction to free amino acids, an amino acid mixture (10 mM of Il) was prepared in Lis-HCl buffer p
H7,0) L-y Luginine 200mg, L-
Asparagine 11□056.8mg, L-ysubaragic acid 20mg, L-cystine 5g, L-curtamic acid 20mg, L-curcumin 300mg, Gutu 9fu 1
0 Droxyproline 20mg Louie 50mg,
L-o ylang 50mg, L-lysine 511c Q 40
mg, L-methionine 15mg, L-phenylalanine 15mg. L-proline 20mg, L-serine 30mg
．． L-Threonine 20mg. L-chi■shishi20Il+g
, L-valine 20 mg, L-tryptoph 7:7 m
200 μl of the arginine deiminase solution of the present invention (OD2, ., = 0.g) in 800 ttl.
After adding 016) and incubating at 37°C for 118 hours, changes in amino acid concentration were analyzed using an automatic amino acid analyzer (Hitachi Model L-8500). As a control, instead of the arginine deiminase solution of the present invention,
The same operation was performed with the addition of 0 mM Tris-HCl buffer. As a result, as shown in Table 1, the arginine deiminase of the present invention was found to be an enzyme that specifically acts on L-arginine to catalyze the reaction that produces citrulline.
第１表 （３）酵素作用： 次の反応を触媒する。Table 1 (3) Enzyme action: Catalyze the following reaction.
Ｌ−アルギニン ＋ Ｈ，Ｏ→ シトルリン 十 ＮＨ。L-arginine + H, O → Citrulline 10 NH.
（４）ｐＨ安定性：
１０μ２の本発明酵素溶液（ＯＤｚａ。＋１１１ ＝Ｏ
＋８）を４０μ２の試験ＴＩＨ緩衝液と混合し、３７°
Ｃ２３時間インキエベートした後、９５０μ２のＰＢＳ
を加えて、２０倍に希釈し、残存する酵素活性を前記酵
素活性測定法に従って計測した。その結果、ｐＨ５，０
〜１０．０まで安定であった。(4) pH stability: 10 μ2 of the present enzyme solution (ODza. +111 = O
+8) with 40μ2 of test TIH buffer and incubate at 37°
After incubation for 23 hours, 950μ2 PBS
was added, diluted 20 times, and the remaining enzyme activity was measured according to the enzyme activity measurement method described above. As a result, pH5.0
It was stable up to 10.0.
尚、試験ｐＨ緩衝液は、ｐＨ３，０〜６．０までは０．
１Ｍクエン酸−クエン酸ナトリウム緩衝液、ｐＨ７，０
〜９．０まではＯ，ＩＭＦリス塩酸緩衝液、ｐＨ１０，
０はＯ，１Ｍグリシン−ＮａＯＨ緩衝液を用いた。In addition, the test pH buffer solution has a pH of 0.0 to 6.0.
1M citric acid-sodium citrate buffer, pH 7.0
~9.0, O, IMF Liss-HCl buffer, pH 10,
0, 1M glycine-NaOH buffer was used.
（５）至適ｐＨ；
種りのｐＨ値の緩衝液を用いて、前述の酵素活性測定法
に従い、測定した結果、ｐＨ７，０〜７５の範囲に至適
ｐＨを有していた。(5) Optimum pH; As a result of measurement using a buffer solution with a suitable pH value and according to the enzyme activity measurement method described above, it was found that the optimal pH was in the range of pH 7.0 to 75.
（６）分子量：
４７０００±５０００ （ＳＤＳ−ＰＡＧＥ法）５５０
００±５０００ （ゲル濾過法）（７）等電点：
アンフオライン ピーエージ−プレート（Ａ Ｍ Ｐ
ｌｌ０ＬＩＮＥ ＰＡＧＰＬＡＴＥ ； Ｌ Ｋ Ｂ社
製）を用いる等電点電気泳動法により測定した結果、ｐ
Ｈ７，４±０゜５に等電点を有する。(6) Molecular weight: 47000±5000 (SDS-PAGE method) 550
00±5000 (Gel filtration method) (7) Isoelectric point: Ampholine PAGE plate (AMP
As a result of measurement by isoelectric focusing using LL0LINE PAGPLATE (manufactured by LKB), p
It has an isoelectric point at H7,4±0°5.
（８）Ｎ末端アミノ酸配列解析
後述の実施例に基づいて得られた本発明酵素（総ＯＤ、
、。１１．＝０．０１）を用いて液相プロティン シー
ケンサ−（ベックマン社製： ＢＥＣＫＭＡＮ Ｓｙｓ
ｔｅｍ ８９０Ｍ Ｅ ）によりＮ末端側からのアミノ
酸配列を解析した結果、まず、以下の如く５番目までの
配列が明らかとなり、
Ｘ−３ｅ ｒ−Ｖａ １−Ｐｈｅ−３ｅ ｒ−Ａｓｐ−
（式中、Ｘは水素原子又はＭｅｔを示す）更に、総ＯＤ
ｇｅｏ−＝０． ０５のサンプルで解析の結果、以下の
如く３００番目での配列が明らかとなった。(8) N-terminal amino acid sequence analysis of the enzyme of the present invention (total OD,
,. 11. = 0.01) using a liquid phase protein sequencer (manufactured by Beckman: BECKMAN Sys).
As a result of analyzing the amino acid sequence from the N-terminal side using X-3e r-Va 1-Phe-3e r-Asp-
(In the formula, X represents a hydrogen atom or Met) Furthermore, the total OD
geo-=0. As a result of analysis of sample No. 05, the sequence at position 300 was revealed as shown below.
Ｘ−Ｓｅｒ−Ｖａ １−Ｐｈｅ−ＳｅｒＡｓｐ−Ｌｙｓ
−Ｐｈｅ−Ａｓｎ−Ｇｌｙ−Ｉ Ｉ ｅ−Ｈｉ ５−Ｖ
ａ Ｉ−Ｔｙ ｒ−３ｅ ｒＧｌｕ−１１ｅ−Ｇｌｙ−
Ａｓｐ−Ｌｅｕ−Ｇｌｕ−Ｓｅｒ−Ｖａｌ−Ｌｅｕ−Ｖ
ａ ＩＨｉｓ−Ｇｌｕ−Ｐｒｏ−Ｇｌｙ−Ｌｙｓ１ｕ−
（式中、Ｘは水素原子又はＭｅｔを示す）（９）アミノ
酸組成分析
精製した本発明酵素標品（ＯＤｚｅ。、、＝０．０８）
１ｍｌに１２Ｎ塩酸を等量刑え、】０５°Ｃ，２４時間
加水分解した後、アミノ酸自動分析装置（日立Ｌ−８５
００型）を用いて解析した。本発明酵素の分子量はＳＤ
Ｓ電気泳動から約４７０００であり、アミノ酸の平均分
子量を１１０とすると構成アミノ酸は約４２７残基とな
ることから、分析結果を４２７残基当たりのアミノ酸残
基数で表し、その結果を第２表に示す。X-Ser-Va 1-Phe-SerAsp-Lys
-Phe-Asn-Gly-I Ie-Hi 5-V
a I-Tyr-3e rGlu-11e-Gly-
Asp-Leu-Glu-Ser-Val-Leu-V
a IHis-Glu-Pro-Gly-Lyslu- (In the formula, X represents a hydrogen atom or Met) (9) Amino acid composition analysis Purified enzyme preparation of the present invention (ODze., = 0.08)
Add an equal amount of 12N hydrochloric acid to 1ml, and hydrolyze at 05°C for 24 hours.
00 type). The molecular weight of the enzyme of the present invention is SD
It is approximately 47,000 from S electrophoresis, and if the average molecular weight of amino acids is 110, the constituent amino acids are approximately 427 residues.The analysis results are expressed as the number of amino acid residues per 427 residues, and the results are shown in Table 2. Shown below.
第２表
以上の諸性質を公知のアルギニン・デイミナーゼと比較
すると、いずれの酵素とも異なることが判る。Comparing the properties shown in Table 2 and above with those of known arginine deiminase, it is found that it is different from any other enzyme.
また、本発明酵素は腫瘍細胞にたいする強い増殖阻害活
性を示すことから抗癌剤としての用途が考えられる。以
下に本発明酵素の腫瘍細胞に対する理化学的諸性状を示
す。Furthermore, since the enzyme of the present invention exhibits strong growth-inhibiting activity against tumor cells, it can be used as an anticancer agent. The physicochemical properties of the enzyme of the present invention against tumor cells are shown below.
（ａｌ 細胞増殖抑制活性の測定法
測定に用いる細胞：本発明酵素に対し阻害感受性の高い
細胞としてＣＣＲＦ−ＣＥＭ細胞（ヒトリンパ球系白血
病細胞；大日本製薬味から購入）を選定し、検定に使用
した。(al) Method for measuring cell proliferation inhibitory activity Cells used for measurement: CCRF-CEM cells (human lymphoid leukemia cells; purchased from Dainippon Pharmaceutical Aji) were selected as cells with high inhibition sensitivity to the enzyme of the present invention and used for the assay. did.
培養用培地：１０％生胎児血清（Ｆｅ２）添加ＲＰ Ｍ
ｌ−１６４０培地（１００ｍｇ／ ｌのペニシリンＧ
及び硫酸ジヒドロストレプトマイシンを含む）。Culture medium: RP M supplemented with 10% live fetal serum (Fe2)
l-1640 medium (100 mg/l penicillin G
and dihydrostreptomycin sulfate).
測定方法：上記培養用培地で培養された対数増殖期にあ
るＣＣＲＦ−ＣＥＭ細胞を集め、新鮮な培養用培地に懸
濁し、細胞濃度を１．ｌｌＸｌ０’個／ ｍ ｌに調整
する。その後、得られた細胞含有溶液を２４穴マルチプ
レート（ＭｔｌＬＴＩＤＩＳＩＩ ｉ Ｎ Ｕ ＮＣ
社製）に９００ｕＮ／穴（ｗｅｌｌ）づつ均質な細胞懸
濁液を播種する。フィルター（ＭＩＬＬＥＸ ＧＶフィ
ルター；ミリポア社製）で濾過滅菌した試験溶液を１０
０μＩ！、／匈ｅ１１添加し、ゆるく攪拌する。Measurement method: CCRF-CEM cells in the logarithmic growth phase cultured in the above culture medium were collected, suspended in fresh culture medium, and the cell concentration was adjusted to 1. Adjust to llXl0' pieces/ml. Thereafter, the obtained cell-containing solution was placed in a 24-well multiplate (MtlLTIDISII i N U NC
A homogeneous cell suspension is inoculated at 900 uN/well into a tube (manufactured by Seibu University). The test solution was sterilized by filtration using a filter (MILLEX GV filter; manufactured by Millipore).
0μI! Add 11 liters of water and stir gently.
コントロールとしてＰＢＳ （リン酸緩衝液生理食塩水
）１００μｆｆｉ／ｗｅｌｌ添加する。細胞懸濁液を播
種した２４穴マルチプレートそれぞれを、５％ＣＣＬ、
９５％空気、１００％湿度、３７°Ｃのインキュベータ
ーで４日間培養した後、各−ｅ１１内の細胞数をコール
タ−カウンター（米国；コールタ−社製）を用いて計測
する。As a control, 100 μffi/well of PBS (phosphate buffered saline) is added. Each of the 24-well multi-plates seeded with cell suspension was treated with 5% CCL,
After culturing in an incubator of 95% air, 100% humidity, and 37°C for 4 days, the number of cells in each -e11 is counted using a Coulter Counter (manufactured by Coulter Inc., USA).
以下の式に従い増殖抑制率（％）を算出する。Calculate the growth inhibition rate (%) according to the following formula.
（％） Ｃ−Ｂ
Ａ；試験液細胞数
Ｂ；播種細胞数
Ｃ；コントロール溶液の細胞数
この系で、ＣＣＲＦ−ＣＥＭ細胞の増殖を５０％阻害す
る活性を１型位（ｕ）とした。(%) CB A; Number of cells in test solution B; Number of cells seeded C; Number of cells in control solution In this system, the activity of inhibiting the proliferation of CCRF-CEM cells by 50% was defined as type 1 (u).
（ト））細胞増殖抑制に対するｐ Ｈ安定性１０ｔｔｌ
！の本発明酵素溶？（１（１００００ｕ／ｍｌ）を４０
μｌの試験ｐＨ緩衝液と混合し、３７°Ｃ１３時間イン
キエベートした後、９５０μｉのＰＢＳを加えて、２０
倍に希釈し、残存する細胞増殖抑制活性を前記細胞増殖
抑制活性の測定法に示した方法に従って計測した。その
結果、ｐＨ５，０〜１０．０まで安定であった。(g)) pH stability for cell growth inhibition 10ttl
! The enzyme solution of the present invention? (1 (10000u/ml) at 40
After mixing with μl of test pH buffer and incubating for 13 hours at 37°C, 950 μl of PBS was added and 20
The solution was diluted twice and the remaining cell proliferation inhibitory activity was measured according to the method described above in the method for measuring cell proliferation inhibitory activity. As a result, it was stable from pH 5.0 to 10.0.
尚、試験ｐＨ緩衝液は、ｐＨ３，０〜６．０までは０．
１Ｍクエン酸−クエン酸ナトリウム緩衝液、ｐＨ７，０
〜９．０まではＯ，ＬＭ）リス塩酸緩衝液、ｐＨ１０，
０は０．１Ｍグリシン−ＮａＯＨａｌｔ衝液を用いた。In addition, the test pH buffer solution has a pH of 0.0 to 6.0.
1M citric acid-sodium citrate buffer, pH 7.0
~9.0 O, LM) Liss-HCl buffer, pH 10,
0, a 0.1M glycine-NaOHalt solution was used.
（ｃ）細胞増殖抑制に対する熱安定性
１０１！の本発明酵素溶液（１００００ｕ／ｍｅ）を９
９０μｌの０．０５Ｍトリス塩酸緩衝液（ｐＨ７，０）
に加え、３７゛Ｃで２４時間、６０°Ｃで１時間、１０
０ ’Ｃで５分間処理した後水冷し、残存する細胞増殖
抑制活性を測定することにより評価した。以下の第３表
にその結果を示す。(c) Thermostability against cell growth inhibition 101! of the present invention enzyme solution (10000 u/me)
90μl 0.05M Tris-HCl buffer (pH 7.0)
in addition to 24 hours at 37°C and 1 hour at 60°C
After treatment at 0'C for 5 minutes, the mixture was cooled with water and evaluated by measuring the remaining cell proliferation inhibitory activity. The results are shown in Table 3 below.
第３表
（ｄ）細胞増殖抑制作用の各種酵素に対する安定性各種
の酵素を用い、それぞれの至適ｐＨで３７°Ｃ１６時間
反応した後、残存する細胞増殖抑制活性を測定すること
により評価した。以下にその結果を第４表に示す。Table 3 (d) Stability of cell proliferation inhibitory activity against various enzymes.Evaluation was made by measuring the remaining cell proliferation inhibitory activity after reacting with various enzymes at their respective optimum pHs at 37°C for 16 hours. The results are shown in Table 4 below.
第４表
（ｅ）細胞増殖抑制作用の各種の有機酸および有機溶媒
に対する安定性
本発明酵素溶液（２０００ｕ／ｍｊりに各種の有機酸お
よび有機溶媒を各添加濃度（Ｖ／Ｖ％）で添加し、２５
°Ｃ１１時間反応した。その後反応液をＰＢＳを用いて
希釈し、本発明酵素濃度を１００ｕ／−になる様に調整
した後、残存する細胞増殖抑制活性を測定することによ
り評価した。以下にその結果を第５表に示す。Table 4 (e) Stability of cell proliferation inhibitory effect against various organic acids and organic solvents Enzyme solution of the present invention (adding various organic acids and organic solvents at various concentrations (V/V%) to 2000 u/mj) 25
The reaction was carried out at °C for 11 hours. Thereafter, the reaction solution was diluted with PBS and the concentration of the enzyme of the present invention was adjusted to 100 u/-, and then the remaining cell proliferation inhibitory activity was measured and evaluated. The results are shown in Table 5 below.
第５表
（ｆ）各種腫瘍細胞に対する増殖抑制作用各種培養系の
腫瘍細胞に対する本発明酵素の増殖抑制作用について、
ヒトリンパ球系白血病由来細胞株（ｃＣＲＦ−ＣＥＭ、
ＣＣＲＦ−Ｈ３Ｂ２゜ＣＣＲＦ−３Ｂ、ＲＰＭＩ−８２
２６）、ヒト骨髄系白血病由来細胞株（Ｋ−５６２，Ｈ
ＥＬ９２・１・７）、ヒト腎癌由来細胞株（ＴＲ（、−
２９Ｒ）、ヒト肺癌由来細胞株（ＴＬＣ−７ＮＣ３）マ
ウス乳癌由来細胞株（ｃ−１２７Ｉ）を用い、これらの
細胞株の増殖に及ぼす影響を調べた。Table 5 (f) Growth inhibitory effect on various tumor cells Regarding the growth inhibitory effect of the enzyme of the present invention on tumor cells in various culture systems,
Human lymphoid leukemia-derived cell line (cCRF-CEM,
CCRF-H3B2゜CCRF-3B, RPMI-82
26), human myeloid leukemia-derived cell line (K-562, H
EL92・1・7), human renal cancer-derived cell line (TR(,-
29R), a human lung cancer-derived cell line (TLC-7NC3), and a mouse breast cancer-derived cell line (c-127I), the effects on the proliferation of these cell lines were investigated.
培養に用いた培地は、Ｃ−１２７１細胞株には、Ｅ−Ｍ
ＥＭ培地に１０％牛脂児血清を添加した培地を使用し、
それ以外の細胞株は、ＲＰＭＩ−１６４０培地に１０％
牛脂児血清を添加した培地を使用した。それぞれの細胞
株を各培地で培養し、対数増殖期にあるこれらの腫瘍細
胞株を集め、新鮮な培養用培地に懸濁し、細胞濃度をｌ
Ｘｌ０’個／ ｍ ｌになる様に調整した。２４穴マル
チプレートに均一に懸濁した細胞浮遊液を１ｍＩ！、／
ｗｅｌｌに播種した後、ＰＢＳに溶解した本発明酵素の
種々の濃度の希釈液１００ ｕ ｌ ／ｗｅｌｌ添加し
、５％ＣＯ２９５％空気、１００％湿度、３７℃のイン
キュベーターで４日間培養した。対照として本発明酵素
の希釈液の代わりにＰＢＳを添加して培養した。The medium used for culture was E-M for C-1271 cell line.
Using EM medium supplemented with 10% tallow serum,
For other cell lines, add 10% to RPMI-1640 medium.
A medium supplemented with beef tallow serum was used. Each cell line was cultured in each medium, and these tumor cell lines in the logarithmic growth phase were collected, suspended in fresh culture medium, and the cell concentration was adjusted to l.
Adjustment was made so that the number was Xl0' pieces/ml. 1 mI of cell suspension uniformly suspended in a 24-well multi-plate! ,/
After seeding in a well, 100 ul/well of diluted solutions of the enzyme of the present invention dissolved in PBS at various concentrations were added, and cultured for 4 days in an incubator at 37°C in 5% CO2, 95% air, and 100% humidity. As a control, culture was performed by adding PBS instead of the diluted solution of the enzyme of the present invention.
培養後の細胞数の測定は、浮遊系の細胞（白血病由来細
胞）は、ｌ５ＯＴＯＮｎ＠ （米国；コールタ−社製）
で２０倍に希釈し、付着性細胞は、培地を吸引除去した
後０．１％トリプシン溶液１ｍｌで、培養基質から細胞
を剥離し、ｌ５ＯＴＯＮＨ■で２０倍に希釈して測定用
サンプルとし、コールタ−カウンター（米国；コールタ
−社製）を用いて計測した。更に以下の式に従い増殖抑
制率（％）を算出する。To measure the number of cells after culture, suspending cells (leukemia-derived cells) should be measured using l5OTONn@ (USA; manufactured by Coulter).
To remove adherent cells, remove the culture medium by suction, detach the cells from the culture substrate with 1 ml of 0.1% trypsin solution, dilute 20 times with l5OTONH to prepare a sample for measurement, and use a coulter. - Measured using a counter (manufactured by Coulter, USA). Furthermore, the growth inhibition rate (%) is calculated according to the following formula.
Ａ；試験液細胞数
Ｂ；播種細胞数
Ｃ；対照溶液の細胞数
その結果、試験に供したすべての腫瘍細胞株で増殖抑制
作用が認められ、５０％の増殖阻害をしめす本発明酵素
濃度は第６表に示す如く、２８０ｎｍに於けるＯＤ値が
１０−’〜１０−６の領域にあり、極めて微量で作用す
ることが明らかとなった。A: Number of cells in the test solution B; Number of cells seeded C: Number of cells in the control solution As a result, a growth inhibitory effect was observed on all tumor cell lines subjected to the test, and the concentration of the enzyme of the present invention that showed 50% growth inhibition was As shown in Table 6, the OD value at 280 nm was in the range of 10-' to 10-6, and it became clear that it was effective in extremely small amounts.
第６表
（ｇ） −１１正常細胞に対する本発明酵素の影響マウ
ス３Ｔ３細胞は長期***増殖可能であるが、造腫瘍能を
有さないことから、正常細胞に近い細胞として、細胞変
異及び細胞毒性の研究に広く用いられており、今回一般
正常細胞に対する本発明酵素の影響の指標として用いた
。マウス３Ｔ３細胞をｌＯ％牛脂児血清を含むイーグル
ＭＥＭ培地に懸濁し、５Ｘ１０’個／ ｍ ｌになるよ
うに調整し、φ３５ＩＩＩ１１の組織培養用プラスチッ
クデイシュに２ｍｆｆ１づつ播種した。更にＰＢＳに溶
解した０１）ｚａ。い、値４．７Ｘ１０−’の本発明酵
素溶液を終濃度がＯＤ ｔ ＠。、値２．３５Ｘ１０−
’（１０，２ｕ ／ ｍｌ ）になるように添加し、５
％Ｃｏｔ、９５％空気、１００％湿度、３７’Ｃのイン
キュベーターで培養した。対照として本発明酵素溶液の
代わりにＰＢＳを添加して培養した。２日間及び４日間
培養した後、０．１％トリプシン溶液を用いて細胞を剥
離し、再度培養用培地に懸濁させて単細胞浮遊液とした
。細胞浮遊／（！１容にＰＢＳに溶解した０、１％トリ
バンプルー溶液９容を加えた後、ノイバウエル氏血球計
測盤を用いて細胞数を計測した。トリパンブルーにより
青色に染まる細胞は死細胞、染まらない細胞は生細胞で
あるので、その数を計測した結果、第７表に示す如く、
本発明酵素添加培養と無添加培養の場合に於いて、細胞
生存率に有意な差は認められず、本発明酵素が、正常細
胞に対して直接的な毒性を有さないことを示した。Table 6 (g) -11 Effect of the enzyme of the present invention on normal cells Although mouse 3T3 cells are capable of long-term division and proliferation, they do not have tumorigenic ability, and therefore, as cells close to normal cells, they are considered to be susceptible to cell mutations and cytotoxicity. This enzyme has been widely used in research on general normal cells, and was used as an indicator of the effect of the enzyme of the present invention on general normal cells. Mouse 3T3 cells were suspended in Eagle's MEM medium containing 10% tallow serum, adjusted to 5 x 10' cells/ml, and seeded at 2mff1 into φ35III11 tissue culture plastic dishes. Furthermore, 01)za dissolved in PBS. The enzyme solution of the present invention having a value of 4.7×10−′ has a final concentration of OD t @. , value 2.35X10-
'(10,2u/ml),
% Cot, 95% air, 100% humidity, and cultured in an incubator at 37'C. As a control, culture was performed by adding PBS instead of the enzyme solution of the present invention. After culturing for 2 and 4 days, the cells were detached using a 0.1% trypsin solution and resuspended in a culture medium to obtain a single cell suspension. After adding 9 volumes of 0.1% trivan blue solution dissolved in PBS to 1 volume of cell suspension/(!), the number of cells was counted using a Neubauer hemocytometer. Cells that stain blue with trypan blue are dead cells. , Since unstained cells are living cells, as a result of counting their number, as shown in Table 7,
No significant difference in cell survival rate was observed between cultures with and without the enzyme of the present invention, indicating that the enzyme of the present invention has no direct toxicity to normal cells.
第７表
また本発明酵素を有効成分とする腫瘍細胞増殖阻害剤に
おいて、例えば、注射用蒸留水または緩衝液Ｏ１１〜５
ｍｆ当りこの本発明酵素１×１０３〜ｌＸ１０’ｕを溶
解して調整し、また必要に応じて、安定化剤例えばヒト
血清アルブミン、乳糖グルコース、サッカロース、デキ
ストリン マンニット、イノジット サイクロデキスト
リン等の季唐類、グルタミン、グルタミン酸、アスパラ
ギンアスパラギン酸、グリシン、アラニン等のアミノ酸
類を０．１％以上、好ましくは０．２〜１０％濃度とし
て添加調整し、凍結乾燥または真空乾燥等の通常の乾燥
手段により調整でき、用時溶解用注射剤として製剤化す
れよい。また上記の安定化剤や、またはプロピレングリ
コール、グリセリンエチレングリコール ポリエチレン
グリコールソルビトール等の多価アルコール類の５〜３
０％注射用蒸留水または緩衝液の溶液として、この０゜
５〜５ｍｌ当りこの本発明酵素１×１０３〜１×１０５
ｕを添加して注射剤として調整してもよい。Table 7 Also, in the tumor cell growth inhibitor containing the enzyme of the present invention as an active ingredient, for example, distilled water for injection or buffer solution O11-5
The enzyme of the present invention is prepared by dissolving 1x103 to 1x10'u of the enzyme of the present invention per mf, and if necessary, stabilizers such as human serum albumin, lactose glucose, saccharose, dextrin mannitol, inojito cyclodextrin, etc. are added. Amino acids such as glutamine, glutamic acid, asparagine, aspartic acid, glycine, and alanine are added and adjusted to a concentration of 0.1% or more, preferably 0.2 to 10%, and then dried by normal drying means such as freeze drying or vacuum drying. It can be prepared as an injection to be dissolved before use. In addition, the above stabilizers or polyhydric alcohols such as propylene glycol, glycerin ethylene glycol, polyethylene glycol sorbitol, etc.
As a solution of 0% distilled water for injection or buffer solution, 1 x 103 to 1 x 105 of the enzyme of the present invention per 0.5 to 5 ml.
It may be prepared as an injection by adding u.
さらにこの本発明酵素を用いるイン・ビトロ用の試薬調
製としては、例えば、注射用蒸留水、緩衝液または細胞
培養用培地０． ５〜５ｍｌ当り本発明酵素を１×ｌＯ
″〜ｌＸ１０Ｓｕを溶解して使用するのが簡便である。Further, in vitro reagent preparation using the enzyme of the present invention includes, for example, distilled water for injection, buffer solution, or cell culture medium 0.00. 1 x lO of the enzyme of the present invention per 5-5 ml
It is convenient to dissolve and use 1X10Su.
これらの製剤は、適宜塩化ナトリウムやグルコースの等
張化剤を添加して浸透性を調製し、静脈内注射または腫
瘍患部近辺に直接注入することが簡便である。These preparations can be conveniently adjusted for permeability by appropriately adding tonicity agents such as sodium chloride or glucose, and then injected intravenously or directly into the vicinity of the tumor-affected area.
次いで本発明の実施例を挙げるが、本発明はこれらによ
って何ら限定されるものではない。Next, examples of the present invention will be given, but the present invention is not limited thereto.
実施例 Ｉ
ＰＰＬＯプロス（Ｄ’ＩＦＣＯ社製）７容、ウマ血清２
容、酵母エキス１容、ペニシリンＧ１０００ｕ／ｒｄ、
酢酸タリウム５００μｇ／ｄを加えた液体培地５００ｄ
に、Ｉ Ｘ １０ｂＣＦＵ／ｍｌのマイコプラズマ・オ
ラーレ（Ｔ Ｇ Ｉ Ｆ）を１０ｄ接種し、３７’Ｃ，
５日間静置培養した。墳養液を高速冷却遠心分離機（日
立５ＣＲ−２０ＢＡ型）を用い、４°Ｃ，１１０００ｒ
、ｐ、ｍ 、３０分間遠心し、ペレット状に集菌した。Example I PPLO Pross (manufactured by D'IFCO) 7 volumes, horse serum 2
volume, yeast extract 1 volume, penicillin G1000u/rd,
500 d of liquid medium containing 500 μg/d of thallium acetate
was inoculated with Mycoplasma orale (TGIF) at I x 10 bCFU/ml for 10 days, and then incubated at 37'C.
It was statically cultured for 5 days. The culture solution was heated at 4°C and 11,000 r using a high-speed refrigerated centrifuge (Hitachi 5CR-20BA model).
, p, m, centrifuged for 30 minutes to collect bacteria into a pellet.
得られたマイコプラズマ・オラーレＴＧＩＦの菌体１ｇ
（温重量）を３０蔵のＰＢＳに懸濁した後、水冷上超音
波処理（１２０秒、４〜５回）により菌体を破砕した。1 g of the obtained Mycoplasma orale TGIF cells
(warm weight) was suspended in 30 volumes of PBS, and then the bacterial cells were disrupted by ultrasonication over water cooling (120 seconds, 4 to 5 times).
破砕液を４ ’Ｃ，１８０００ｒ、ｐ、ｍで４０分間遠
心分離し、上清を分離した。沈澱物は、再度３０−のＰ
ＢＳに懸濁した後、上記と同一の操作を行い、上清液を
分離し、先に得た上清と混合して透析用セルロースチュ
ーブ（米国、■ｌ５ＫＡＳＥ社製）に入れ、１０ｍＭト
リス塩酸緩衝液（ｐＨ７，０）３１に対し４°Ｃでの透
析を２４時間づつ２回くり返した。The disrupted solution was centrifuged at 4'C, 18000 r, p, m for 40 minutes, and the supernatant was separated. The precipitate is again 30-P
After suspending in BS, perform the same operation as above to separate the supernatant, mix it with the previously obtained supernatant, put it in a cellulose tube for dialysis (manufactured by KASE, USA), and add 10mM Tris-HCl. Dialysis at 4°C against buffer solution (pH 7,0) 31 was repeated twice for 24 hours each.
得られた菌体抽出液を、予め同一緩衝液〔１０１トリス
塩酸緩衝液（ｐＨ７，０））で平衡化したＤＥＡＥ−３
ｅｐｈａｒｏｓｅカラム（φ１６Ｘ２００ｍｍ）に吸着
させ、カラム容量の３倍の同一緩衝液で洗浄した後、同
一緩衝液に溶解したＮａＣＩ！によるＯから２００ｍＭ
の直線的濃度勾配で溶出を行った。溶出液は６絨ごとの
分画に集め、各分画の細胞増殖抑制活性を前記の活性測
定法に示した方法により調べた結果、ＮａＣｊ”１度２
０ｍＭから１００１までの分画に活性の溶出が認められ
た。The obtained bacterial cell extract was pre-equilibrated with DEAE-3 in the same buffer [101 Tris-HCl buffer (pH 7,0)].
NaCI! was adsorbed onto an epharose column (φ16 x 200 mm), washed with the same buffer of 3 times the column volume, and then dissolved in the same buffer! 200mM from O by
Elution was performed with a linear concentration gradient of The eluate was collected into 6-cell fractions, and the cell proliferation inhibitory activity of each fraction was examined using the method described in the activity measurement method described above.
Elution of activity was observed in the fractions from 0mM to 1001.
ＤＥＡＲ−３ｅｐｈａｒｏｓｅのイオン交換クロマトグ
ラフィーで精製された細胞増殖抑制活性画分を集め、限
外′ａ過膜（ＹＭ−１０ｉ米国アミコン社製）を用いて
９．５ｍＡに濃縮した後、予め５０ｍＭのＮａＣ１を含
む１０ｍ１リス塩酸緩衝液（ｐＨ７，０）で平衡化した
ゲル濾過（Ｕ１ｔｒｏｇｅｌＡｃＡ４４ ；スウェー
デン、ＬＫＢ社製）のカラム（φ２６Ｘ９３０ｍｌｌ）
にチャージし、同一緩衝液を用いて？容出した。The cell proliferation-inhibiting active fraction purified by ion exchange chromatography on DEAR-3 epharose was collected, concentrated to 9.5 mA using an ultra'a membrane (YM-10i, manufactured by Amicon, USA), and then pre-injected with 50 mM A gel filtration (U1trogelAcA44; manufactured by LKB, Sweden) column (φ26 x 930 ml) equilibrated with 10 ml of Lis-HCl buffer (pH 7,0) containing NaCl.
and using the same buffer? I let it out.
溶出液は５Ｉｎｌの分画に集め、各分画の細胞増殖抑制
活性を前記の活性測定法に示した方法により調べた結果
、溶出液量の２４０ ｍｌから３１０成の範囲に細胞増
殖抑制活性の溶出が認められた。The eluate was collected into 5 Inl fractions, and the cell proliferation inhibitory activity of each fraction was examined using the method described in the activity measurement method described above. Elution was observed.
ゲル濾過によって精製された細胞増殖抑制活性画分を集
めることにより６１成の溶液が得られた。A 61-component solution was obtained by collecting the cell proliferation-inhibiting activity fractions purified by gel filtration.
得られた溶液を限外濾過膜（ＹＭ−１０；米国アミコン
社製）を用いて５ｄに濃縮した後、透析用セルロースチ
ューブ（米国、ＶＩＳＫＡＳＥ社製）を用いて１０ｍＭ
）リス塩酸緩衝ｉ！（ｐＨ７，０）２！に対し、４°Ｃ
で２回（４８時間）透析した。The obtained solution was concentrated to 5d using an ultrafiltration membrane (YM-10; manufactured by Amicon, USA), and then concentrated to 10mM using a cellulose tube for dialysis (manufactured by VISKASE, USA).
) Liss hydrochloric acid buffer i! (pH7,0)2! against 4°C
Dialysis was performed twice (48 hours).
透析後の溶液の一部を予め１０ｍＭ）リス塩酸緩衝液（
ｐＨ７，０）に平衡化したイオン交換クロマトグラフィ
ー（ＭｏｎｏＱ １ｌＲ５１５カラム；スウェーデン、
ファルマシア社製）に吸着させた後、同一緩衝液に溶解
したＮａＣｅによるＯから２００ｍＭの直線的濃度勾配
で溶出した。流速１ｄ／分で２０ｄの溶媒を使用した。A portion of the solution after dialysis was preliminarily diluted with 10mM) Lis-HCl buffer (10mM).
Ion exchange chromatography (MonoQ 1lR515 column; Sweden, equilibrated to pH 7,0)
After adsorption to 200 mM linear concentration gradient from O to 200 mM using NaCe dissolved in the same buffer. 20 d of solvent was used at a flow rate of 1 d/min.
溶出液の蛋白濃度変化を２８０ｎｍの紫外線吸収で観測
しながら、蛋白質溶出ピーク毎に分画し、各分画の細胞
増殖抑制活性を前記の活性測定法に示した方法により調
べた結果、ＮａＣｌ ４度が５５ｍＭから６５ｍＭに）
容量する蛋白ピークに存在することが明らかとなった。While observing changes in the protein concentration of the eluate using ultraviolet absorption at 280 nm, each protein elution peak was fractionated, and the cell proliferation inhibitory activity of each fraction was examined using the method shown in the activity measurement method above. As a result, NaCl 4 (from 55mM to 65mM)
It was found that this protein exists in the protein peaks that contain the same amount of protein.
前記のイオン交換クロマトグラフィー（ＭｏｎｏＱ）Ｉ
Ｒ５１５カラム；スウェーデン、ファルマシア社製）に
よって精製された活性蛋白ピーク画分をさらに精製する
ために、得られた活性蛋白ピーク画分を集め、この溶液
を透析用セルロースチューブ（米国、ＶＩＳＫＡＳＥ社
製）を用いて１０ｍＭトリス塩酸緩衝液（ｐＨ７，０）
３１に対し、４°Ｃで２４時間透析した後、透析後の溶
液の一部を予め１０１トリス塩酸緩衝液（ｐＨ７，０）
に平衡化したイオン交換クロマトグラフィー（Ｍｏｎｏ
Ｓ ｌｌＲ５１５カラム；スウェーデン、ファルマシア
社製）に吸着させた。吸着した蛋白質の溶出は、同一緩
衝液に溶解したＮａＣＱによって行い、流速１ｍ／分で
２０分後からＮａＣｌの濃度を０から１００ｍＭまでの
直線的濃度勾配で溶出させた。溶出液の蛋白濃度変化を
２８０ ｎｍの紫外線吸収で観測しながら、蛋白質溶出
ピーク毎に分画し、各分画の細胞増殖抑制活性を前記の
活性測定法に示した方法により調べた結果、ＮａＣｌ濃
度が６０ｍＭ付近にある単一ピークに局在して溶出する
ことがわかり、その分画を採取した。Ion exchange chromatography (MonoQ) I
In order to further purify the active protein peak fraction purified using R515 column (manufactured by Pharmacia, Sweden), the obtained active protein peak fractions were collected and the solution was passed through a dialysis cellulose tube (manufactured by VISKASE, USA). using 10mM Tris-HCl buffer (pH 7,0)
After dialysis against 31 for 24 hours at 4°C, a portion of the dialyzed solution was preliminarily added to 101 Tris-HCl buffer (pH 7,0).
Ion exchange chromatography (Mono
It was adsorbed onto a SIIR515 column (manufactured by Pharmacia, Sweden). The adsorbed protein was eluted with NaCQ dissolved in the same buffer solution, and elution was performed with a linear concentration gradient of NaCl from 0 to 100 mM starting after 20 minutes at a flow rate of 1 m/min. While observing changes in the protein concentration of the eluate using ultraviolet absorption at 280 nm, each protein elution peak was fractionated, and the cell proliferation inhibitory activity of each fraction was examined using the method shown in the activity measurement method above. It was found that the elution was localized to a single peak at a concentration of around 60 mM, and that fraction was collected.
イオン交換クロマトグラフィー（ＭｏｎｏＱ ＨＲ５１
５カラム；スウェーデン、ファルマシア社製）およびイ
オン交換クロマトグラフィー（ＭｏｎｏＳ １ｌＲ５１
５カラム：スウェーデン、ファルマシア社製）を用いる
精製操作を繰り返し全試料の処理を行った結果、本発明
酵素標品として約３■を得た。Ion exchange chromatography (MonoQ HR51
5 column; manufactured by Pharmacia, Sweden) and ion exchange chromatography (MonoS 11R51
As a result of repeating the purification operation using 5 columns (manufactured by Pharmacia, Sweden) and treating all samples, approximately 3 μl of the enzyme preparation of the present invention was obtained.
試験例（純度検定）
（１）逆相高速液体クロマトグラフィーによる検定本発
明酵素の０．１％トリフルオロ酢酸溶液（ｌＯＯμｇ／
−）５００μ２をシリカゲルを担体とした逆相クロマト
グラフィー用カラム（ＯＤＳ）にかけて高速液体クロマ
トグラフィーをおこなった。溶出は流速１ｍ／分で０．
１％トリフルオロ酢酸を溶媒とし、アセトニトリル（ｃ
Ｈ，ＣＮ）の濃度を０〜６０％まで１％／分の速度の直
線的濃度勾配で溶出させた。その結果４３〜４５％のア
セトニトリル濃度に単一のピークとして溶出されること
から、均一な物質であることがわった。Test Example (Purity Assay) (1) Assay by reverse phase high performance liquid chromatography 0.1% trifluoroacetic acid solution of the enzyme of the present invention (lOOμg/
-) 500μ2 was applied to a column for reverse phase chromatography (ODS) using silica gel as a carrier, and high performance liquid chromatography was performed. Elution was performed at a flow rate of 1 m/min with a flow rate of 0.
Using 1% trifluoroacetic acid as a solvent, acetonitrile (c
The concentration of H, CN) was eluted from 0 to 60% with a linear concentration gradient at a rate of 1%/min. As a result, it was found that it was a homogeneous substance because it was eluted as a single peak at an acetonitrile concentration of 43 to 45%.
（２）ポリアクリルアミドゲル電気泳動による検定本発
明酵素の１２％ポリアクリルアミドスラブゲルを用いた
ＳＤＳ電気泳動を常法〔電気泳動単金属、電気泳動実験
法 ２０８頁、（１９７６年）文光堂〕に従って行った
。泳動に供した本発明酵素標品は１％ＳＤＳ （ラウリ
ル硫酸ナトリウム）存在下で０１１Ｍ２−メルカプトエ
タノール添加及び無添加で、３７°Ｃｌ２Ｏ時間インキ
ュベートして変性させることにより調整した。泳動終了
後、日本バイオランドラボラトリーズ社製の銀染色キッ
トを用い、これに添付されている処方に従って泳動蛋白
染色を行った。その結果本発明酵素標品は、還元剤の存
在下でも非存在下でも単一なバンドになることから、均
一な蛋白質であることが明らかになった。また同時に泳
動した分子量マーカーとの相対泳動度から本発明酵素の
分子量は約４７０００であることがわかった。(2) Assay by polyacrylamide gel electrophoresis The enzyme of the present invention was subjected to SDS electrophoresis using a 12% polyacrylamide slab gel according to the conventional method [Electrophoresis Single Metals, Electrophoresis Experimental Methods, p. 208, (1976) Bunkodo]. went. The enzyme preparations of the present invention used for electrophoresis were prepared by denaturation by incubation at 37 DEG Cl2O in the presence of 1% SDS (sodium lauryl sulfate) with and without the addition of 011M2-mercaptoethanol. After completion of electrophoresis, electrophoretic protein staining was performed using a silver staining kit manufactured by Nippon Bioland Laboratories according to the instructions attached to the kit. As a result, the enzyme preparation of the present invention was found to be a homogeneous protein, as it formed a single band both in the presence and absence of a reducing agent. Furthermore, the molecular weight of the enzyme of the present invention was found to be about 47,000 from the relative migration with the molecular weight marker that was electrophoresed at the same time.
本発明は新規なアルギニン・デイミナーゼを提供するも
ので、また本アルギニン・デイミナーゼは、腫瘍細胞増
殖阻害作用を示し、抗腫瘍剤としての用途が期待される
ものである。The present invention provides a novel arginine deiminase, which exhibits an inhibitory effect on tumor cell growth and is expected to be used as an antitumor agent.
Claims (8)
ン・デイミナーゼ。 （ａ）作用；Ｌ−アルギニンと水からシトルリンとアン
モニアを生成する酵素反応を触媒する（ｂ）基質特異性
；Ｌ−アルギニンに基質特異性を有する （ｃ）至適ｐＨ；７．０〜７．５ （ｄ）ｐＨ安定性；５．０以上 （ｅ）分子量；４７０００±５０００（ＳＤＳ−ＰＡＧ
Ｅ法による） （ｆ）等電点；７．４±０．５(1) Arginine deiminase having at least the following physical and chemical properties. (a) Action: Catalyzes the enzymatic reaction that produces citrulline and ammonia from L-arginine and water (b) Substrate specificity: Has substrate specificity for L-arginine (c) Optimum pH: 7.0-7 .5 (d) pH stability; 5.0 or more (e) Molecular weight; 47,000 ± 5,000 (SDS-PAG
(by E method) (f) Isoelectric point; 7.4±0.5
ある特許請求の範囲第１項記載のアルギニン・デイミナ
ーゼ。(2) The arginine deiminase according to claim 1, which is an enzyme derived from a strain belonging to the genus Mycoplasma.
、Ｘは水素原子又はＭｅｔを示す） のアミノ酸配列を有するポリペプチドを構成成分として
なる特許請求の範囲第１項記載のアルギニン・デイミナ
ーゼ。(3) Claims consisting of a polypeptide having an amino acid sequence of at least X-Ser-Val-Phe-Ser-Asp- (wherein, X represents a hydrogen atom or Met) from the N-terminal side. Arginine deiminase according to item 1.
デイミナーゼ生産菌を培地に培養し、培養物より該アル
ギニン・デイミナーゼを採取することを特徴とするアル
ギニン・デイミナーゼの製造法。(4) Arginine belonging to Mycoplasma orale
A method for producing arginine deiminase, which comprises culturing deiminase-producing bacteria in a medium and collecting the arginine deiminase from the culture.
項記載のアルギニン・デイミナーゼである特許請求の範
囲第４項記載の製造法。(5) Arginine deiminase is the first claim.
5. The method for producing arginine deiminase according to claim 4.
デイミナーゼ生産菌が、マイコプラズマ・オラーレ（Ｔ
ＧＩＦ）（ＦＥＲＭＢＰ−１９７０）である特許請求の
範囲第４項記載の製造法。(6) Arginine belonging to Mycoplasma orale
The deiminase-producing bacterium is Mycoplasma orale (T
GIF) (FERMBP-1970).
・デイミナーゼ活性を有する腫瘍細胞増殖阻害活性物質
。(7) A tumor cell growth inhibiting substance having arginine deiminase activity derived from a strain belonging to the genus Mycoplasma.
項記載のアルギニン・デイミナーゼである特許請求の範
囲第７項記載の腫瘍細胞増殖阻害活性物質。(8) The tumor cell growth inhibiting active substance is claimed in claim 1.
The tumor cell growth inhibiting active substance according to claim 7, which is arginine deiminase according to claim 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63184556A JPH0235081A (en) | 1988-07-26 | 1988-07-26 | Arginine deiminase and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63184556A JPH0235081A (en) | 1988-07-26 | 1988-07-26 | Arginine deiminase and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0235081A true JPH0235081A (en) | 1990-02-05 |
| JPH0331436B2 JPH0331436B2 (en) | 1991-05-07 |
Family
ID=16155274
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63184556A Granted JPH0235081A (en) | 1988-07-26 | 1988-07-26 | Arginine deiminase and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0235081A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998024473A1 (en) * | 1996-12-03 | 1998-06-11 | Kyowa Hakko Kogyo Co., Ltd. | Tissue fibrosis inhibitor |
| US9125876B2 (en) | 2005-04-25 | 2015-09-08 | Erytech Pharma | Erythrocytes containing arginine deiminase |
| US9364504B2 (en) | 2007-08-08 | 2016-06-14 | Erytech Pharma | Compostition and therapeutic anti-tumour vaccine |
| EP3449935A1 (en) | 2017-08-31 | 2019-03-06 | Erytech Pharma | Arginine deiminase encapsulated inside erythrocytes and their use in treating cancer and arginase-1 deficiency |
| WO2019042628A1 (en) | 2017-08-31 | 2019-03-07 | Erytech Pharma | Arginine deiminase encapsulated inside erythrocytes and their use in treating cancer and arginase-1 deficiency |
-
1988
- 1988-07-26 JP JP63184556A patent/JPH0235081A/en active Granted
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998024473A1 (en) * | 1996-12-03 | 1998-06-11 | Kyowa Hakko Kogyo Co., Ltd. | Tissue fibrosis inhibitor |
| US9125876B2 (en) | 2005-04-25 | 2015-09-08 | Erytech Pharma | Erythrocytes containing arginine deiminase |
| US9364504B2 (en) | 2007-08-08 | 2016-06-14 | Erytech Pharma | Compostition and therapeutic anti-tumour vaccine |
| US9950049B2 (en) | 2007-08-08 | 2018-04-24 | Erytech Pharma | Composition and therapeutic anti-tumour vaccine |
| US10780151B2 (en) | 2007-08-08 | 2020-09-22 | Erytech Pharma | Composition and therapeutic anti-tumour vaccine |
| EP3449935A1 (en) | 2017-08-31 | 2019-03-06 | Erytech Pharma | Arginine deiminase encapsulated inside erythrocytes and their use in treating cancer and arginase-1 deficiency |
| WO2019042628A1 (en) | 2017-08-31 | 2019-03-07 | Erytech Pharma | Arginine deiminase encapsulated inside erythrocytes and their use in treating cancer and arginase-1 deficiency |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0331436B2 (en) | 1991-05-07 |
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