JPH02234677A - D-aminoacylase acting on acidic d-amino acid and production thereof - Google Patents
D-aminoacylase acting on acidic d-amino acid and production thereofInfo
- Publication number
- JPH02234677A JPH02234677A JP1052830A JP5283089A JPH02234677A JP H02234677 A JPH02234677 A JP H02234677A JP 1052830 A JP1052830 A JP 1052830A JP 5283089 A JP5283089 A JP 5283089A JP H02234677 A JPH02234677 A JP H02234677A
- Authority
- JP
- Japan
- Prior art keywords
- aminoacylase
- acetyl
- glutamic acid
- activity
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、アルカリゲネス属に属する細菌をＮアセチル
ーＤ−グルタミン酸を含有する培地で培養して産出され
るＤ−アミノアシラーゼ及びその製造方法に関するもの
である。Detailed Description of the Invention [Field of Industrial Application] The present invention relates to D-aminoacylase produced by culturing bacteria belonging to the genus Alcaligenes in a medium containing N-acetyl-D-glutamic acid, and a method for producing the same. It is.
Ｄ−アミノ酸の製造方法としては、ＤＬ−アミノ酸をア
セチル化してＮ−アセチルーＤＬ−アミノ酸としたのち
、これに微生物の生産するＤ−アミノアシラーゼを反応
させ酵素的加水分解反応によりＤ−アミノ酸を生成させ
て、これを常法により分離する方法が知られている。ま
たＤ−アミノアシラーゼに関してはシュードモナス属に
属するファカルタテイブメタノール資化性細菌類が生産
するＤ−アミノアシラーゼ（特開昭５５− ４２５３４
号、特公昭６０−３１４７７号）、ストレブトミセス属
放線菌の生産するＤ−アミノ了シラーゼ（特開昭５３５
９０９２号、特公昭５ ３−３ ６ ０ ３ ５号）が
知られている。また最近アルカリゲネス属に属するアル
カリゲネス・デニトリフイカンス・サブスピーシーズ・
キシロースオキシダンスＭＩ−４の生産するＤ−アミノ
アシラーゼが報告されている（特開昭６ ４，−５ ４
８ ８号）。The method for producing D-amino acids is to acetylate DL-amino acids to N-acetyl-DL-amino acids, and then react this with D-aminoacylase produced by microorganisms to produce D-amino acids through an enzymatic hydrolysis reaction. There is a known method of separating this by a conventional method. Regarding D-aminoacylase, D-aminoacylase produced by facultative methanol-assimilating bacteria belonging to the genus Pseudomonas (Japanese Unexamined Patent Publication No. 55-42534
(Japanese Patent Publication No. 60-31477), and D-aminoylase produced by Strebtomyces actinomycetes (Japanese Patent Publication No. 535-1989).
No. 9092 and Japanese Patent Publication No. 53-36035) are known. Recently, Alcaligenes denitrificans subsp., which belongs to the genus Alcaligenes,
D-aminoacylase produced by xylose oxidans MI-4 has been reported (Japanese Unexamined Patent Publication No. 1986-44, -5-4).
8 No. 8).
一般にＤ−アミノアシラーゼは基質特異性が高く、Ｎ−
アセチルーＤ−アミノ酸の種類によってアシラーゼ活性
が種々異なるが、これ等公知のＤアミノアシラーゼはＮ
−アセチルーＤ−メチオニン、Ｎ−アセチルーＤ−アラ
ニン、Ｎ−アセチルーＤ−フエニルアラニン、Ｎ−アセ
チルーＤ一バリン等の中性アミノ酸に対しては活性を示
すが、Ｎ−アセチルーＤ−グルタミン酸の如き酸性アミ
ノ酸に対しては殆んど活性を示さないものである。In general, D-aminoacylases have high substrate specificity and N-
Acylase activity varies depending on the type of acetyl-D-amino acid, but these known D-aminoacylases are
-It shows activity against neutral amino acids such as acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, and N-acetyl-D-valine, but it shows activity against neutral amino acids such as N-acetyl-D-glutamic acid. It shows almost no activity against acidic amino acids.
Ｄ−グルタミン酸は、生理活性ペプチドの構成アミノ酸
、合成抗生物質の側鎖成分、脳機能改善剤の合成原料等
の用途を有する重要なＤ−アミノ酸であるが、公知のＤ
−アミノアシラーゼを用いて製造することが出来なかっ
た。D-glutamic acid is an important D-amino acid that has uses such as a constituent amino acid of physiologically active peptides, a side chain component of synthetic antibiotics, and a synthetic raw material for brain function improving agents.
- It could not be produced using aminoacylase.
斯かる実状のおいて、本発明者は鋭意研究の結果、Ｎ−
アセチルーＤ−グルタミン酸に特異的に作用してＤ−グ
ルタミン酸を生成するＤ−アミノアシラーゼを発見し、
このＤ−アミノアシラーゼの理化学的性質および酵素化
学的性質を明らかにして本発明を完成した。Under such circumstances, the present inventor has conducted extensive research and found that N-
Discovered D-aminoacylase, which acts specifically on acetyl-D-glutamic acid to produce D-glutamic acid,
The present invention was completed by clarifying the physicochemical and enzymatic properties of this D-aminoacylase.
すなわち、本発明は、Ｎ−アセチルーＤ−グルタミン酸
に対し特異的に作用する新規なＤ−アミノアシラーゼ及
びその製造法を提供するものである。That is, the present invention provides a novel D-aminoacylase that specifically acts on N-acetyl-D-glutamic acid and a method for producing the same.
本発明者はＮ−アセチルーＤ−アラニンを炭素源、窒素
源とする培地で、土壌中の微生物を検索し、Ｎ−アセチ
ルーＤ−グルタミン酸を誘導剤として添加した培地で培
養した結果、Ｎ−アセチル−Ｄ−グルタミン酸に特異的
に作用するＤ−アミノアシラーゼ生産菌を単離すること
に成功した。The present inventor searched for microorganisms in soil using a medium containing N-acetyl-D-alanine as a carbon source and nitrogen source, and as a result of culturing them in a medium containing N-acetyl-D-glutamic acid as an inducer, N-acetyl -We succeeded in isolating a D-aminoacylase-producing bacterium that specifically acts on D-glutamic acid.
本菌株の菌学的性質は次のとおりである。The mycological properties of this strain are as follows.
形態
■．細胞の形：かん状
２．細胞の多形性：なし
３，運動性の有無：あり
４．胞子の有無：なし
５．グラム染色性：陰性
６．抗酸性：なし
生育状況
１．ブイヨン寒天平板培養：
円形、僅かに盛上り、白色、平滑、光たくあり、半透明
生理学的性質
１．硝酸塩の還元：＋
２．亜硝酸塩の還元：＋
３．インドールの生）Ｉｉ．：
４．クエン酸の利用：＋
５．ゼラチンの加水分解：
６．ウレアーゼ：
７．オキシターゼ：十
８，カタラーゼ：＋
９．０−Ｆテスト
弱酸化性
グルコース：
キシロース：酸生成
１０．了ルギニンの分解：
１１ 生育温度．４１℃では生育するが、４５℃では
生育しない
１２ エスタリンの分解
１３．β−ガラクトシダーゼ：
１４ チ１−クロームオキシダーゼ：」一炭素源の資
化性
１，グルコース：十
２．アラビノース：
３，マンノース：十
４．マンニトール；
５．Ｎ−アヤチルクルコザミン：
６ マルトース：
７．グルコン酸 」一
８．カフ゜ロン酸一
９，アジピン酸：十
１０．リンゴ酸：＋
１１．フエニル酢酸一十
１２．キシロース：」ー
糖からの酸及びガスの生成：ブドウ糖より酸を生成しな
い
以」二の菌学的諸性質をバージーズ・マニュアル・オブ
・システマテック・バクテリオロジ（Ｂｅｒ巳ｅｙ
ｓ Ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ
Ｂａｃｔｅｒｉｏｌｏｇｙ）第１巻にて検索すると
、
本菌株はダラム陰性かん菌で、運動性あり、好気性、カ
タラーゼ陽性、オキシターゼ陽性、ペプトン含有培地で
ブドウ糖より酸を生成しない等の性質よりアルカリゲネ
ス属に属する細菌である。Form ■． Cell shape: cane-shaped 2. Cell pleomorphism: No 3, Motility: Yes 4. Presence or absence of spores: None5. Gram staining: negative6. Anti-acidity: None Growth status 1. Bouillon agar plate culture: round, slightly raised, white, smooth, shiny, translucent Physiological properties 1. Nitrate reduction: +2. Nitrite reduction: +3. Raw indole) Ii. : 4. Use of citric acid: +5. Hydrolysis of gelatin: 6. Urease: 7. Oxidase: 18, Catalase: + 9.0-F test Weakly oxidizing glucose: Xylose: Acid production 10. Decomposition of Ryoruginine: 11 Growth temperature. Grows at 41°C but not at 45°C 12 Decomposition of Estarin 13. β-galactosidase: 14 Thi1-chrome oxidase: Assimilation of carbon source 1, Glucose: 12. Arabinose: 3, Mannose: 14. Mannitol; 5. N-ayathylcurcozamine: 6 Maltose: 7. Gluconic acid”-18. Cafuronic acid: 19, adipic acid: 110. Malic acid: + 11. Phenyl acetic acid 112. Xylose: ``Generation of acid and gas from sugar: does not produce acid from glucose'' 2 Mycological properties are described in Bersey's Manual of Systematic Bacteriology.
s Manual of Systematic
According to a search in Volume 1 of Bacteriology, this strain belongs to the genus Alcaligenes because it is a Durham-negative rod, is motile, aerobic, positive for catalase, positive for oxidase, and does not produce acid rather than glucose in a peptone-containing medium. It's a bacteria.
また硝酸塩の還元反応、亜硝酸塩の還元反応、Ｄグルコ
ース、Ｄ−キシロース、Ｄ−マンノース、Ｄ−グルコン
酸、了ジピン酸の資化性を有し、ＯＦ培地でＤ−キシロ
ースから酸を生成することから、本菌株はアルカリゲネ
ス・キシロースオキシダンス・ザブスピーシーズ・キシ
ロースオキシダンス （八ｌｃａｌｉｇｅｎｅｓ Ｘｙ
ｌｏｓｏｘｙｄａｎｓ ＳｕｂｓｐＸｙｌｏｓｏｘｙｄ
ａｎｓ）に属すると考えられ、アルカリゲネス・キシロ
ース才キシダンス・サブスピーシーズ・キシロースオキ
シダンスＡ−６と命名し（以下単に八−６株と呼ぶ）、
工業技術院微生物工業技術研究所に微工研閑寄第１０５
／Ｉ９号（ＦεＲＭＰ−１．０５４９）として寄託した
。It also has the ability to reduce nitrate, reduce nitrite, assimilate D-glucose, D-xylose, D-mannose, D-gluconic acid, and dipic acid, and generate acid from D-xylose in OF medium. Therefore, this bacterial strain is Alcaligenes xyloseoxydans,
losoxydans SubspXylosoxyd
ans), and was named Alcaligenes xylose-oxydans subspecies xylose-oxydans A-6 (hereinafter simply referred to as strain 8-6).
Microbiological Research Center No. 105 at the Institute of Microbial Technology, Agency of Industrial Science and Technology
/I9 (FεRMP-1.0549).
本発明のＤ−アミノアシラーゼは、アルカリゲネス属に
属するＤ−アミノアシラーゼ生産菌をＮアセチルーＤ−
クルタミン酸を含む培地中で培養し、培養物よりＤ一了
ミノアシラーゼを採取することにより製造される。The D-aminoacylase of the present invention is a D-aminoacylase producing bacterium belonging to the genus Alcaligenes.
It is produced by culturing in a medium containing curtamic acid and collecting D-minoacylase from the culture.
Ａ−６株の培養には炭累源としてクエン酸、クルコン酸
が、窒素源としてペプトン、酵母エキス、ｒ−−グルタ
ミン酸が良好であるが、炭、窒素源としてベプ１−ンを
用いると最も高い活性が得られる。For culturing A-6 strain, citric acid and ccurconic acid are good as charcoal sources, and peptone, yeast extract, and r-glutamic acid are good as nitrogen sources, but when using vep1-one as charcoal and nitrogen sources, The highest activity is obtained.
誘導剤としてはＮ−アセチルーＤ−グルタミン酸の代り
に安価なＸ一了セチル−０Ｌ−グルタミン酸を用いるこ
とができ、これの培地中の濃度は約０． ２％が好まし
い。また培地ρ１１は７付近が好ましい。約１日間の培
養で菌体中にＤ一了ミノ了シラーセが生産される。As an inducer, an inexpensive X-cetyl-0L-glutamic acid can be used instead of N-acetyl-D-glutamic acid, and its concentration in the medium is about 0. 2% is preferred. Further, the culture medium ρ11 is preferably around 7. After culturing for about one day, D-Ichiryominoryo Shirase is produced in the bacterial cells.
生産されたＤ−アミノアシラーゼは、培養物を遠心分朗
１して菌体を染め、超音波破砕により無細胞抽出液とし
て得ることが出来るので、これより硫安分画、ＤＢＡＢ
−セルロース力ラムクロマトクラフィー、セファデック
ス０１５０カラムクロマトグラフィー等の公知手段を適
宜組合せてＤ−アミノアシラーゼを単離精製することが
できる。The produced D-aminoacylase can be obtained as a cell-free extract by centrifuging the culture, staining the bacterial cells, and crushing them with ultrasonic waves.
- D-aminoacylase can be isolated and purified by appropriately combining known means such as cellulose column chromatography and Sephadex 0150 column chromatography.
このようにして得られる本発明のＤ−アミノアシラーゼ
の理化学的性質は次のとおりである。The physicochemical properties of the D-aminoacylase of the present invention thus obtained are as follows.
■ 作用及び特異性
Ｎ−アセチルーＤ−グルタミン酸に作用してＤ−グルタ
ミン酸および酢酸を生成し、Ｎ−アセチルーＤ−アスパ
ラギン酸、Ｎ−アセチルーＤ−アラニン、Ｎ−アセチル
ーＤ一ロイシン、Ｎ−アセチルーＤ−メチオニン、Ｎ−
アセチルＤ−フェニル了ラニン、Ｎ−アセチルーＤ−ト
リプトファン、Ｎ−アセチルーＤ−バリンに対しては活
性を示さない。■ Action and specificity Acts on N-acetyl-D-glutamic acid to produce D-glutamic acid and acetic acid, and produces N-acetyl-D-aspartic acid, N-acetyl-D-alanine, N-acetyl-D-leucine, and N-acetyl-D-leucine. -Methionine, N-
It does not show any activity against acetyl D-phenyltransamine, N-acetyl-D-tryptophan, and N-acetyl-D-valine.
■ 至適ｐ１１： ｐＨ６．８〜７．２（リン酸カリウム緩衝液）である。■ Optimal p11: pH 6.8-7.2 (potassium phosphate buffer).
■ 最適温度： 活性発現の最適温度は４５〜５０ｔである。■ Optimal temperature: The optimum temperature for expression of activity is 45-50t.
■ 熱安定性：
６０℃１０分間の加熱処理後、約４０％の活性が残存し
ている。■ Thermal stability: Approximately 40% activity remains after heat treatment at 60°C for 10 minutes.
■ 金属イオンの作用：
金属イオンにより活性化されず、ｐｅ２＋ ［：，２
＋１１ｇ”， Ｃｏ”， Ｎｉ２＋により活性阻害され
る。■ Effect of metal ions: Not activated by metal ions, pe2+ [:,2
The activity is inhibited by +11g'', Co'', and Ni2+.
■ 分子量： 約４２，０００ （ゲル濾過法による）。■ Molecular weight: Approximately 42,000 (by gel filtration method).
本発明のＤ−アミノアシラーゼはＮ−アセチルＤＬ−グ
ルタミン酸よりＤ−グルタミン酸を製造する方法に利用
できる。The D-aminoacylase of the present invention can be used in a method for producing D-glutamic acid from N-acetyl DL-glutamic acid.
次に実施例を挙げて説明する。 Next, an example will be given and explained.
実施例１
（ｉ＞Ｄ−アミノアシラーゼの産生
Ｎ−アセチルーＤＬ−グルタミン酸０．１％、グルコー
ス１％、ペプトン１％、ＫＨ．ＰＯ． ０．１％、Ｋ２
ＨＰ０． ０．１％、ＭｇＳ０ ４・７Ｈ２００．０１
％、酵母エキス０．０１％を含むｐＨ１．０の培地１０
０ｎｕ中でＡ−６株を２２時間培養し、その無細胞抽出
液中のＤ−アミノアシラーゼ活性を測定した。Example 1 (i>Production of D-aminoacylase N-acetyl-DL-glutamic acid 0.1%, glucose 1%, peptone 1%, KH.PO. 0.1%, K2
HP0. 0.1%, MgS0 4.7H200.01
%, pH 1.0 medium containing 0.01% yeast extract 10
The A-6 strain was cultured in 0nu for 22 hours, and the D-aminoacylase activity in the cell-free extract was measured.
活性の測定はリン酸カリウム緩衝液ｐＨ７．０５ Ｑ
）ｔｍｏｌｌ， Ｎ−アセチルアミノ酸５，ｌｊｍｏｊ
！及びＤ−アミノアシラーゼを含む０．５ｍｌの抽出液
を３０分間反応させた後、５０％トリクロロ酢酸０．０
５＋＋＋Ｉｌを加えて反応を停止させ、残存するトリク
ロ口酢酸をエーテルで抽出除去したのちｌ−ＩＰＬＣで
Ｄ−アミノ酸或いはＬアミノ酸を定量した。Activity was measured using potassium phosphate buffer pH 7.05 Q
)tmoll, N-acetylamino acid 5, ljmoj
! After reacting 0.5 ml of the extract containing D-aminoacylase for 30 minutes, 50% trichloroacetic acid 0.0
After the reaction was stopped by adding 5+++ Il and the remaining trichloroacetic acid was extracted and removed with ether, D-amino acid or L-amino acid was quantified by l-IPLC.
（ｉｉ）Ｄ−アミノアシラーゼ活性とＬ−アミノアシラ
ーゼ活性の分離
上記無細胞抽出液をＤＢＡＢセルロースに吸着させ、食
塩水のグラジエント溶離を行い、Ｌアミノアシラーゼ活
性部分とＤ−アミノアシラーゼ活性部分を分離した。そ
の溶出パターンは第１図のとおりである。(ii) Separation of D-aminoacylase activity and L-aminoacylase activity The above cell-free extract was adsorbed onto DBAB cellulose, and gradient elution with saline was performed to separate the L-aminoacylase active part and the D-aminoacylase active part. did. The elution pattern is shown in FIG.
（ｉｉｉ）Ｄ−アミノアシラーゼ活性及びＬ−アミノア
シラーゼ活性に対する誘導剤の効果
Ｉ２
Ａ，−６株の培養培地に添加する誘導剤Ｎ−アセチルー
ＤＬ−グルタミン酸の濃度を変えて培養し、その無細胞
抽出液中のＤ−アミノアシラーゼ活性とＬ−アミノアシ
ラーゼ活性を分離定量し、第２図に示す結果を得た。即
ちＮ−アセチルーＤＬ−グルタミン酸の添加によりＤ−
アミノアシラーゼ活性の産生は顕著に上昇し、その最適
添加濃度は０． ２％であった。これに対しＬ−アミノ
アシラーゼ活性は全く変化を受けず、且つその活性は極
めて僅かなものであった。(iii) Effect of inducer on D-aminoacylase activity and L-aminoacylase activity I2 A, -6 strain was cultured with varying concentrations of the inducer N-acetyl-DL-glutamic acid added to the culture medium, and the cell-free D-aminoacylase activity and L-aminoacylase activity in the extract were separated and quantified, and the results shown in FIG. 2 were obtained. That is, by adding N-acetyl-DL-glutamic acid, D-
The production of aminoacylase activity increases significantly, and its optimal addition concentration is 0. It was 2%. On the other hand, the L-aminoacylase activity did not change at all, and the activity was extremely small.
実施例２
Ａ−６株の培養における培地成分の検討（炭素源の検討
）：
実施例に記載した培養条件において、窒素源として硫安
１％を用い、炭素源として種々の化合物を実験した。培
養菌体の無細胞抽出液中のＤ−アミノアシラーゼ活性を
Ｎ−アセチルーＤ−グルタミン酸を用いて測定し、表−
１の結果を得た。酵素単位および比活性は実施例１の活
性測定法により下式に従って算出した。Example 2 Examination of medium components in culture of A-6 strain (examination of carbon source): Under the culture conditions described in the example, 1% ammonium sulfate was used as the nitrogen source, and various compounds were experimented as the carbon source. The D-aminoacylase activity in the cell-free extract of cultured bacteria was measured using N-acetyl-D-glutamic acid, and the results are shown in Table-
1 result was obtained. Enzyme units and specific activity were calculated using the activity measurement method of Example 1 according to the following formula.
表−１
Δ−６株の培養における培地成分の検討（窒素源の検討
）：
実施例１に記載した培養条件において、炭素源としてグ
ルコース１％を用い、窒素源として種々の化合物を実験
し、実施例２と同様にＤ一丁ミノアシラーゼ生成量を測
定し、表−２の結果を得た。Table 1 Examination of medium components in culture of Δ-6 strain (examination of nitrogen source): Under the culture conditions described in Example 1, using 1% glucose as a carbon source and various compounds as a nitrogen source, The amount of D-1-chominoacylase produced was measured in the same manner as in Example 2, and the results shown in Table 2 were obtained.
表−２
以下余白
実施例４
Ａ−６株培養液よりＤ−アミノアシラーゼの分離精製：
Ａ−６株をペブトンを炭素、窒素源、０、２％Ｎ−アセ
ヂルーＤＬ−グルタミン酸を誘導剤として実施例１に従
って３０℃で１８時間振盪培養し、その培養液４ｐを遠
心分離し菌体を分離する。得られた閑体２１．６ｇ（湿
重量）を１０ｍＭＩＪン酸カリウム緩衝液ｐｌ１７．０
に懸濁し、超音波により菌体を破砕した。この様にして
得た無細胞抽出液を硫安分画（２０〜６０％）し、１０
ｍＭリン酸カリウム緩衡液ｐｌ＋７．０で透析したのち
、この緩衝液で平衡化したＤＢ八Ｂ−セルロースに吸着
させた。カラムを０． １ Ｍ − ＮａＣｊ７で洗浄
後、０． １ ５ Ｍ − ＮａＣ．ｉ！でＤ−アミノ
アシラーゼ活性を溶離した。この活性画分を濃縮後セフ
ァデックスＧ−１５０カラムクロマトグラフィーによっ
て精製して精製Ｄ一了ミノアシラーゼを得た。各精製段
階の収率および活性の」二昇は表−３に示すとおりであ
る。Table 2 Below is the blank space Example 4 Isolation and purification of D-aminoacylase from A-6 strain culture solution: A-6 strain was carried out using pebtone as a carbon and nitrogen source, and 0.2% N-acetyl-DL-glutamic acid as an inducer. According to Example 1, culture was carried out with shaking at 30°C for 18 hours, and the culture solution 4p was centrifuged to separate the bacterial cells. 21.6g (wet weight) of the obtained blank was added to 10mM J potassium phosphate buffer pl17.0
and the bacterial cells were disrupted by ultrasonication. The cell-free extract thus obtained was subjected to ammonium sulfate fractionation (20-60%) and
After dialyzing with mM potassium phosphate buffer pl+7.0, it was adsorbed onto DB8B-cellulose equilibrated with this buffer. Column 0. After washing with 1 M-NaCj7, 0. 15M-NaC. i! D-aminoacylase activity was eluted with. This active fraction was concentrated and purified by Sephadex G-150 column chromatography to obtain purified D-minoacylase. The yield and activity of each purification step are shown in Table 3.
以下余白
実施例５
Ｄ−アミノアシラーゼの基質特異性：
実施例４により得た精製酵素を用いて各種Ｎアセチルー
Ｄ−アミノ酸に対するＤ−アミノアシラーゼ活性を実施
例１に従って測定した。活性はＮ−アセチルーＤ−グル
タミン酸に対するＤ−アミノアシラーゼ活性を１００と
して計算し、表４に示す結果を得た。Example 5 Substrate specificity of D-aminoacylase: D-aminoacylase activity toward various N-acetyl-D-amino acids was measured according to Example 1 using the purified enzyme obtained in Example 4. The activity was calculated by setting the D-aminoacylase activity against N-acetyl-D-glutamic acid as 100, and the results shown in Table 4 were obtained.
表−４
実施例６
Ｄ−アミノアシラーゼ活性に及ぼす金属イオンの影曹：
実施例４に従って精製したＤ−アミノアシラーゼを用い
て、Ｄ−アミノアシラーゼ活性に及ぼす金属イオンの影
蕾を実験した。活性の測定は実施例１に記載する方法に
従って行ない、金属イオン１ ｍＭを添加して、無添加
の場合を１００とした相対活性により金属イオンの影曹
を求め、表−５に示す結果を得た。Table 4 Example 6 Effect of metal ions on D-aminoacylase activity: Using D-aminoacylase purified according to Example 4, an experiment was conducted to examine the effect of metal ions on D-aminoacylase activity. The activity was measured according to the method described in Example 1. 1mM of metal ions was added, and the relative activity of the metal ions was determined based on the relative activity with the case of no addition being set as 100. The results shown in Table 5 were obtained. Ta.
以下余白
表−５
実施例７
Ｄ−アミノアシラーゼ活性に及ぼすｒｏｌｌの影響：実
施例４に従って精製したＤ−アミノアシラーゼを用いて
、実施例１に記載する活性測定に用いるリン酸緩衝液の
ｐＨを変えて、Ｎ−アセチルーＤ一グルタミン酸に対す
るＤ−アミノアシラーゼ活性を測定した。結果は第３図
のとおりである。Below is a margin table-5 Example 7 Effect of roll on D-aminoacylase activity: Using D-aminoacylase purified according to Example 4, the pH of the phosphate buffer used in the activity measurement described in Example 1 was adjusted. Alternatively, D-aminoacylase activity against N-acetyl-D-glutamic acid was measured. The results are shown in Figure 3.
実施例８
Ｄ−アミノアシラーゼ活性の熱安定性：実施例４に従っ
て精製したＤ−アミノアシラーゼをリン酸カリウム緩衝
液ｐＨ７．０に溶解して３０〜７０℃で１０分間加熱処
理したのち残存するＤアミノアシラーゼ活性を実施例１
に従って測定した。その結果は第４図のとおりである。Example 8 Thermostability of D-aminoacylase activity: D-aminoacylase purified according to Example 4 was dissolved in potassium phosphate buffer pH 7.0 and heated at 30 to 70°C for 10 minutes. Example 1 Aminoacylase activity
Measured according to The results are shown in Figure 4.
第１図はＡ−６株の培養物の無細胞抽出液をＤＢＡＢセ
ノレロース力ラムク口マトグラフィ−１こイ寸シたとき
の溶出パターンを示す図である。第２図はＤ一及びＬ−
アミノアシラーゼ活性とＮ−アセチルーＤＬ−グルタミ
ン酸添加濃度との関係を示す図である。第３図はＤ−ア
ミノアシラーゼ活性と培地ｐａｌとの関係を示す図であ
る。第４図はＤ−アミノアシラーゼ活性の熱安定性を示
す図である。
以 上FIG. 1 is a diagram showing the elution pattern when a cell-free extract of a culture of strain A-6 was subjected to DBAB Senorellostomography-1. Figure 2 shows D1 and L-
FIG. 3 is a diagram showing the relationship between aminoacylase activity and the concentration of N-acetyl-DL-glutamic acid added. FIG. 3 is a diagram showing the relationship between D-aminoacylase activity and medium pal. FIG. 4 is a diagram showing the thermostability of D-aminoacylase activity. that's all
Claims (3)
２＾＋、Ｈｇ＾２＾＋、Ｃｏ＾２＾＋、Ｎｉ＾２＾＋に
より活性阻害される。 ［６］分子量： 約４２，０００（ゲル濾過法による）。(1) D-aminoacylase with the following physical and chemical properties [1] Action and specificity Acts on N-acetyl-D-glutamic acid to produce D-glutamic acid and acetic acid, N-acetyl-D-aspartic acid, For N-acetyl-D-alanine, N-acetyl-D-leucine, N-acetyl-D-methionine, N-acetyl-D-phenylalanine, N-acetyl-D-tryptophan, N-acetyl-D-valine shows no activity. [2] Optimum pH: pH 6.8 to 7.2 (potassium phosphate buffer). [3] Optimal temperature: The optimal temperature for activity expression is 45 to 50°C. [4] Thermal stability: Approximately 40% activity remains after heat treatment at 60°C for 10 minutes. [5] Effect of metal ions: Not activated by metal ions, Fe^2^+, Cu^
The activity is inhibited by 2^+, Hg^2^+, Co^2^+, and Ni^2^+. [6] Molecular weight: Approximately 42,000 (according to gel filtration method).
生産菌をＮ−アセチル−Ｄ−グルタミン酸を含む培地中
で培養し、培養物よりＤ−アミノアシラーゼを採取する
ことを特徴とするＤ−アミノアシラーゼの製造方法。(2) Production of D-aminoacylase, which comprises culturing D-aminoacylase-producing bacteria belonging to the genus Alcaligenes in a medium containing N-acetyl-D-glutamic acid, and collecting D-aminoacylase from the culture. Method.
生産菌がアルカリゲネス・キシロースオキシタンス・サ
ブスピーシーズ・キシロースオキシタンス（Ａｌｃａｌ
ｉｇｅｎｅｓ ＸｙｌｏｓｏｘｙｄａｎｓＳｕｂｓｐ．
Ｘｙｌｏｓｏｘｙｄａｎｓ）Ａ−６である請求項２記載
のＤ−アミノアシラーゼの製造方法。(3) The D-aminoacylase-producing bacteria belonging to the genus Alcaligenes is Alcaligenes xylose oxytans subspecies
igenes Xylosoxydans Subsp.
The method for producing D-aminoacylase according to claim 2, wherein the D-aminoacylase is D-aminoacylase A-6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1052830A JP2869793B2 (en) | 1989-03-07 | 1989-03-07 | D-aminoacylase acting on acidic D-amino acid and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1052830A JP2869793B2 (en) | 1989-03-07 | 1989-03-07 | D-aminoacylase acting on acidic D-amino acid and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02234677A true JPH02234677A (en) | 1990-09-17 |
| JP2869793B2 JP2869793B2 (en) | 1999-03-10 |
Family
ID=12925762
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1052830A Expired - Fee Related JP2869793B2 (en) | 1989-03-07 | 1989-03-07 | D-aminoacylase acting on acidic D-amino acid and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2869793B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994001577A1 (en) * | 1992-07-03 | 1994-01-20 | Research Association For Biotechnology Of Agricultural Chemicals | Process for producing optically active d-amino acid |
| WO2000023598A1 (en) * | 1998-10-20 | 2000-04-27 | Chirotech Technology Limited | Aminoacylase and its use in the production of d-aminoacids |
| JP2008061642A (en) * | 2006-08-10 | 2008-03-21 | Toyobo Co Ltd | Method for producing D-amino acid |
-
1989
- 1989-03-07 JP JP1052830A patent/JP2869793B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994001577A1 (en) * | 1992-07-03 | 1994-01-20 | Research Association For Biotechnology Of Agricultural Chemicals | Process for producing optically active d-amino acid |
| WO2000023598A1 (en) * | 1998-10-20 | 2000-04-27 | Chirotech Technology Limited | Aminoacylase and its use in the production of d-aminoacids |
| JP2008061642A (en) * | 2006-08-10 | 2008-03-21 | Toyobo Co Ltd | Method for producing D-amino acid |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2869793B2 (en) | 1999-03-10 |
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