JP4253195B2 - Modified ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase - Google Patents

Description

【００５６】
なお、ブレブンディモナス・ディミニュタ（Brevundimonas diminuta）MR-E001株は、独立行政法人産業技術総合研究所 特許生物寄託センター（茨城県つくば市東１丁目１番地１ 中央第６）にFERM P-19203として2003年2月5日付けで寄託されている。
【００５７】
【実施例】
以下、本発明を実施例によりさらに具体的に説明する。しかしながら、実施例は本発明の具体的な認識を得る一助とみなすべきものであり、本発明の範囲を何等限定するものではない。
【００５８】
[実施例1] MR-E001株由来野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子の取得
(1) MR-E001株染色体DNAの調製
MR-E001株を 100mlのEDDS培地 (0.2%エチレンジアミン-N,N'-ジコハク酸、0.2%グルコース、0.1%バクトイーストエキス、 0.05%ポリペプトン、0.1%硫酸マグネシウム・7水塩、25%(v/v)燐酸緩衝液(1M 、pH 7.0) 、0.5%(v/v) 金属塩混合物溶液（硫酸ナトリウム 56g、塩化マグネシウム・6水塩 8g 、塩化カルシウム0.8g 、硫酸マンガン・4水塩 0.6g 、塩化第二鉄・6水塩 0.12g、硫酸亜鉛 0.06g/100ml）中、30℃にて4日間振盪培養した後に集菌し、この菌体を Saline-EDTA溶液(0.1M EDTA、15M NaCl、pH 8.0) 4ml に懸濁し、リゾチーム 8mgを加えて37℃で1時間振盪した後凍結した。次に、10mlの Tris-SDS 液(1%SDS、0.1M NaCl 、0.1M Tris 、pH 9.0) を穏やかに振盪しながら加え、さらにプロテイナーゼK（メルク社製）（終濃度 1mg） を加え37℃で1時間振盪した。次に、等量のTE飽和フェノール(TE:10mM Tris 、1mM EDTA、pH 8.0)を加えて撹拌後遠心し、上層を採り2倍量のエタノールを加えた後ガラス棒でDNAを巻きとり、90% 、80% 、70% のエタノールで順次フェノールを取り除いた。次に、DNAを 3mlのTE緩衝液に溶解させ、リボヌクレアーゼ A溶液(100℃、15分間の熱処理済み)を 10mg/mlの終濃度となるように加え37℃で30分間振盪した。さらにプロテイナーゼ Kを加え37℃で30分間振盪した後、等量のTE飽和フェノールを加え遠心し、上層と下層に分離させ上層を採取した（以後この操作をフェノール抽出と呼ぶ）。フェノール抽出を2回繰り返した後、同量のクロロホルム (4%イソアミルアルコール含有)を加え同様の抽出操作を繰り返した（以後この操作をクロロホルム抽出と呼ぶ）。その後上層に2倍量のエタノールを加えガラス棒でDNAを巻き取り回収し、染色体DNA標品を得た。
【００５９】
(2) プローブの調製
本出願人らは先に、ブレブンディモナス（Brevundimonas）sp. TN-3株よりエチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子の単離に成功し、そのアミノ酸配列および遺伝子配列を初めて明らかにしている（特開平10-210984号公報記載）。該公報において用いられたデジェネレイトプライマー、すなわち、配列番号3および配列番号4に示されるような配列を有する合成DNA（それぞれ、プライマー#1 および #2とする）を用い、工程(1)で得られたMR-E001株の染色体DNAを鋳型としてPCRを行った。
プライマー#1：ATGACICCIC AYAAYCCIGA YGC（配列番号３）
プライマー#2：CCDATYTGCAT YTTICCIGC RACIGAICCD ATYTC（配列番号４）
【００６０】
すなわち、MR-E001株染色体DNA 1μl 、10倍濃度の反応緩衝液 10μl 、10mM dNTP 4μl 、プライマー#1および#2各々1μl(100pmol 濃度相当)、ExTaq （宝酒造社製）1μl を加えて100 μl とした。この溶液について、95℃、30秒間（デナチュレーション過程）、55℃、30秒間（アニーリング過程）、72℃、2分間（エクステンション過程）のインキュベーションを30サイクル行った。反応終了後、フェノール抽出およびクロロホルム抽出を行い、エタノール沈殿により増幅されたDNAを回収した。これを1.0%アガロースゲル電気泳動で分離後、MR-E001株のエチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子の一部をコードすると考えられる約300bpのDNA断片を得た。こうして得られたDNA断片をDIG DNA Labeling kit（ロシュ・ダイアグノスティックス社製）を用いて標識し、プローブとした。
【００６１】
(3) DNAライブラリーの作製
MR-E001株染色体DNA 10μl に10倍濃度制限酵素反応用緩衝液5μl 、滅菌水33μl 、制限酵素 KpnI 2μl を加え、37℃にて16時間反応させた後エタノール沈殿によりDNAを回収した。アガロースゲル電気泳動を行い、6.5Kb から 5.5KbのDNA断片をゲルから切り出し、DNA PREP（ダイアトロン社製）を用いて回収した。このDNA断片をDNA Ligation Kit Ver.1（宝酒造社製）を用いて大腸菌ベクター pUC18のKpnI部位に挿入し、組換えDNAライブラリーを作製した。ライゲーションに用いた pUC18断片は次のように作製した。pUC18 保存液 2μl に対し、10倍濃度制限酵素用緩衝液5μl 、滅菌水40μl 、制限酵素 KpnI 3μl を加え、37℃で2時間反応後、フェノール抽出およびクロロホルム抽出を行い、エタノール沈殿させた後乾燥して50μl の滅菌水に溶解させた。さらにアルカリフォスファターゼ（宝酒造社製）1μl 、10倍濃度緩衝液10μl 、滅菌水39μl を加え65℃で反応後、フェノール抽出およびクロロホルム抽出を行い、エタノール沈殿後乾燥して滅菌水に溶解させた。
【００６２】
(4) 大腸菌形質転換体の作製および組換えDNAの選別
大腸菌 JM109株をLB培地(1% バクトトリプトン、0.5%バクトイーストエキス、0.5% NaCl) 1mlに接種し37℃、5時間好気的に前培養し、この培養物 0.4mlを SOB培地 40ml(2%バクトトリプトン、0.5%バクトイーストエキス、10mM NaCl 、2.5mM KCl 、1mM MgSO₄ 、1mM MgCl₂ ) に加え、18℃で20時間培養した。この培養物を遠心分離により集菌した後、冷TF溶液(20mM PIPES-KOH(pH 6.0) 、200mM KCl 、10mM CaCl₂ 、40mM MnCl₂ ) を13ml加え、0℃で10分間放置した。その後、再度遠心し、上澄を除いた後、沈殿した大腸菌を冷TF溶液 3.2mlに懸濁し、0.22mlのジメチルスルフォキシドを加え0℃で10分間放置した。こうして作製したコンピテントセル 200μl に工程(3)で作製した組換えプラスミドDNAを含有する溶液（DNAライブラリー）を10μl 加え、0℃で30分放置後、42℃で30秒間ヒートショックを与え、0℃で2分間冷却後、SOC 培地(20mM グルコース、2%バクトトリプトン、0.5%バクトイーストエキス、10mM NaCl 、2.5mM KCl 、1mM MgSO₄ 、1mM MgCl₂ )1ml を添加して37℃にて1時間振盪培養した。これを 200μl ずつLBAmp寒天培地（アンピシリン 100mg/L 、1.5％寒天を含有するLB培地）にまき、37℃で培養した。寒天培地上に生育した形質転換体コロニーについて、コロニーハイブリダイゼーション法によりエチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子を保有すると考えられる形質転換体を選別した。すなわち、寒天培地上に生育した形質転換体をナイロンメンブレン（バイオダイン A：日本ポール社製）上に移し、菌体を溶かしてDNAを固定した後、これを工程(2)で作製したプローブ（約300bp）で処理し、DIG Luminescent Detection Kit （ロシュ・ダイアグノスティックス社製）を用い、目的の組換えDNAを持つコロニーを選択した。
【００６３】
(5) 組換えプラスミドの調製
工程(4) で選択した形質転換体を 100mlのLBAmp培地（アンピシリン 100mg/Lを含有するLB培地）にて37℃で一晩培養し、集菌後、Flexi Prep（アマシャムバイオサイエンス社製）を用いてプラスミドDNAを回収した。得られた組換えプラスミドDNAを pEDS9001と名付けた。
【００６４】
(6) 制限酵素地図の作製およびエチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子領域の特定
工程(5) で得られたプラスミド pEDS9001を数種の制限酵素を用いて切断し制限酵素地図を作製した（図1）。さらに、通常行われるようにサブクローニングを行った。すなわち、pEDS9001 を制限酵素 BamHIで切断した後アガロースゲル電気泳動を行い、約5.3KbのDNA断片をゲルから切り出し、DNA PREP（ダイアトロン社製）を用いて回収した。DNA Ligation Kit Ver.1（宝酒造社製）を用いて自己連結反応を行った後、大腸菌 JM109株を形質転換することにより、エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子を含むと考えられる約2.6Kb の断片が挿入されたプラスミド(pEDS9003)（図2）を得た。
【００６５】
(7) 塩基配列の決定
工程(6) で特定された領域周辺の塩基配列を蛍光シーケンサ ALFII（アマシャムバイオサイエンス社製）を用いて決定した。その結果、配列番号１に示されるアミノ酸配列から成るオープンリーディングフレームをコードする塩基配列（配列番号２）が見い出された。
【００６６】
[実施例2] MR-E001株由来野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子を導入した形質転換体の活性評価
実施例1の工程(6)で得られたエチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子を有する組換えプラスミドpEDS9003 2μl に対し、10倍濃度制限酵素用緩衝液2μl 、滅菌水15μl 、制限酵素 KpnI 1μl を添加し、37℃にて2時間反応させた。エタノール沈殿によりプラスミドDNAを回収し、乾燥後17μl の滅菌水、 2μl の10倍濃度制限酵素緩衝液、制限酵素 BamHI 1μlを添加して37℃にて2時間反応させた。この反応液からアガロースゲル電気泳動により約2.6Kb の断片を調製し、大腸菌ベクター pUC119 に挿入した。作製したライゲーション溶液を用いて大腸菌 JM109を形質転換して目的のプラスミドを得た。ここで作製したプラスミドを pEDS9020 （図3）と名付け、また形質転換体を JM109/ pEDS9020と名付けた。JM109/ pEDS9020 およびコントロールとしてJM109/ pEDS020（特開平10-210984号公報記載）を、それぞれLBAmp培地1mlに接種して37℃にて8時間振盪培養後、1mM isopropyl-β-thiogalactosideを含有する40mlのLBAmp培地で37℃、30時間培養した。得られた培養物を10mMリン酸ナトリウム緩衝液(pH 8.0)で洗浄後、2ml の同緩衝液に懸濁した。得られた菌体懸濁液の一部を 342mMフマル酸と 171mMエチレンジアミンを含むpH 8.0の水溶液50ml に懸濁し30℃で反応させた。時間間隔を空けて反応混合液の一部を（0.1ml）を取り出し、0.42NのNaOH水溶液0.9ml中に加え反応を停止した。菌体を遠心分離により除いた後、HPLCを用いて生成したS,S-エチレンジアミン-N,N'-ジコハク酸を分析した（WAKOSIL5C8 （和光純薬社製）〔溶出液；10mM水酸化テトラ-n-ブチルアンモニウムと0.4mMCuSO₄ を含む50mMリン酸;pH 2〕）。上記測定条件下において1分間に1μmolのS,S-エチレンジアミン-N,N'-ジコハク酸を生成させる酵素量を酵素単位（U）として、JM109/ pEDS9020 およびJM109/ pEDS020の菌体あたり（OD630あたり）の活性を求めたところ、それぞれ1.22 mU/ODml、0.89mU/ODmlであり、MR-E001株由来エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼが高い該酵素活性を有することが確認された。なお、pEDS9020は、独立行政法人産業技術総合研究所 特許生物寄託センター（茨城県つくば市東１丁目１番地１ 中央第６）にFERM P-19202として、2003年2月5日付で寄託されている。
【００６７】
[実施例3] 改変型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子の取得
(1)野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子への変異導入
実施例2で得られたプラスミドpEDS9020をもとに、野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子へのランダム変異導入を行った。変異導入は、PCRにおけるヌクレオチドの誤取り込みによる塩基置換を利用した。エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子開始コドン領域を含むオリゴヌクレオチドED-01（配列番号5）と、該遺伝子終止コドンより約50bp下流領域を含むED-02（配列番号6）を変異誘発PCRのプライマーとし、以下のような組成のPCR反応液100μlを調製した。
プライマー：
ED-01：CGCCATGGCC CCGCATAACC CAGATGCCAC C（配列番号５）
（下線部分は制限酵素NcoI切断認識部位）
ED-02：AAACAAGCTT CGTCATGGCT ATCCCCTC（配列番号６）
（下線部分は制限酵素HindIII切断認識部位）
反応液組成：
鋳型DNA（上記工程で調製したpEDS9020） 1μl
10× PCR Buffer（GIBCO社製） 10μl
50mM MgCl₂（GIBCO社製） 3μl
プライマーED-01 1μl
プライマーED-02 1μl
2.5mM dNTP 各々2μl
10mM dITP 2μl
10mM dBraUTP 2μl
滅菌水 71μl
Taq DNAポリメラーゼ（GIBCO社製） 1μl
【００６８】
上記反応液に対し、94℃、30秒（デナチュレーション過程）、68℃、180秒（アニーリング過程・エクステンション過程）のインキュベーションを30サイクル行った。上記PCR終了後、反応液10μlを0.7％アガロースゲルにより電気泳動を行って、約1.5kbの増幅断片の検出を行った。また、耐熱性評価のコントロールとして用いるため、通常のPCRにて野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子を増幅した。すなわち、以下のような組成のPCR反応液100μlを調製した。
鋳型DNA（pEDS9020） 1μl
10× Pyrobest Buffer（宝酒造社製） 10μl
プライマーED-01 1μl
プライマーED-02 1μl
5mM dNTP 各々2μl
滅菌水 78μl
Pyrobest^TM DNAポリメラーゼ（宝酒造社製） 1μl
【００６９】
上記反応液に対し、94℃、30秒（デナチュレーション過程）、68℃、180秒（アニーリング過程・エクステンション過程）のインキュベーションを30サイクル行った。上記PCR終了後、反応液10μlを0.7％アガロースゲルにより電気泳動を行って、約1.5kbの増幅断片の検出を行った。
【００７０】
プライマーED-01およびED-02中には、制限酵素NcoI切断認識部位および制限酵素HindIII切断認識部位がそれぞれ導入されており（プライマーED-01及びED-02の塩基配列の下線部分）、増幅DNA産物を両制限酵素で切断することにより、後述する発現ベクターpFY529VのNcoI部位−HindIII部位間に容易に挿入することが可能となる。
【００７１】
(2)発現ベクターの構築
後述のスクリーニング工程において、熱処理後のエチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ残存活性を効率よく検出するため、コピー数と発現効率の高い大腸菌発現ベクターpFY529Vを作製した（図4）。trcプロモーターを有する発現ベクターpKK233-2（アマシャム社製）5μlに制限酵素NaeI 1μl 、ScaI 1μl、10倍濃度制限酵素反応用緩衝液1μl、滅菌水2μlを加え、37℃にて12時間切断反応を行った。切断後、0.7％アガロースゲルにより電気泳動を行い、該プラスミドの複製開始点を含まないNaeI-ScaI断片（1.2kb）を切り出し、DNA PREP（ダイアトロン社製）を用いて、該DNA断片を含むTE溶液(10mM Tris、1mM EDTA、pH8.0)3μlを回収した。この操作と並行して、高コピー数ベクターであるpUC18 2μlに制限酵素PvuII 1μl 、ScaI 1μl、10倍濃度制限酵素反応用緩衝液1μl、滅菌水5μlを加え、37℃にて12時間切断反応を行った。切断後、0.7％アガロースゲルにより電気泳動を行い、該プラスミドの複製開始点を含むPvuII-ScaI断片（1.6kb）を切り出し、DNA PREP（ダイアトロン社製）を用いて、該DNA断片を含むTE 1μlを回収した。こうして得られた両DNA断片をDNA Ligation Kit Ver.1（宝酒造社製）により連結させた。pKK233-2由来NaeI-ScaI断片溶液3μl、pUC18由来PvuII-ScaI断片溶液1μl、キット中のA液16μl、キット中のＢ液4μlを混合し、16℃にて16時間連結反応を行った。連結反応後の反応液を用い、実施例1 (4)記載の方法により大腸菌JM109を形質転換した。得られた形質転換体コロニーより約10クローンをLBAmp培地1.5mlに接種し、37℃で12時間振盪培養した。培養後、この培養物を遠心分離により集菌した後、Flexi Prep（アマシャムバイオサイエンス社製）を用いることにより、プラスミドDNAを抽出した。得られたプラスミドDNAを制限酵素ScaIで切断後、0.7％アガロースゲルにより電気泳動を行い、pKK233-2由来NaeI-ScaI断片（1.2kb）とpUC18由来PvuII-ScaI断片（1.6kb）が正しく連結されているクローンを選んでpFY529Vと命名し、変異ライブラリーの発現ベクターとして用いた。
【００７２】
（3）変異ライブラリーの作製
工程(1)のPCRで得られた変異導入エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子を含む反応液を、常法に従ってエタノール沈殿により精製を行い、沈殿物を70μlの滅菌水に再懸濁した。10倍濃度制限酵素反応用緩衝液10μl、制限酵素NcoI 10μl、HindIII 10μlを加え、37℃にて12時間切断反応を行った。切断反応後、フェノール抽出およびクロロホルム抽出を行った後、エタノール沈殿を行い、沈殿物を100μlの滅菌水に再懸濁して変異DNA断片溶液とした。この操作と並行して、工程(2)で作製した発現ベクターpFY529V 3μlに、滅菌水67μl、10倍濃度制限酵素反応用緩衝液10μl、制限酵素NcoI 10μl、HindIII 10μlを加え、37℃にて12時間切断反応を行った。切断後、フェノール抽出およびクロロホルム抽出を行い、エタノール沈殿により精製した後、沈殿物を10μlの滅菌水に再懸濁して切断済みpFY529V溶液とした。変異DNA断片と発現ベクターpFY529Vの連結は、DNA Ligation Kit Ver.1（宝酒造社製）を用いて行った。上記の変異DNA断片溶液3μl、切断済みpFY529V溶液1μl、キット中のA液16μl、キット中のＢ液4μlを混合し、16℃にて16時間連結反応を行った。連結反応後の反応液を用いて、実施例1（工程(4)）記載の方法により大腸菌JM109を形質転換し、種々の変異導入EDDSase遺伝子を保有する形質転換体を得た。また、以上と同様の操作を、工程(1)で増幅した野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子を挿入断片として行った。得られた形質転換体コロニーよりプラスミドを抽出し、蛍光シークエンサーALF II（アマシャムバイオサイエンス社製）を用いて塩基配列を確認した結果、実施例1（工程(7)）で決定した野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼの塩基配列（配列番号２）と同一であった｛但し、プライマーED-01のNcoI部位導入による、4番目の塩基変化（A→G）は除く｝。このプラスミドをpEDTrc9003と命名し、該プラスミドを含む大腸菌、すなわちJM109/ pEDTrc9003を、後述する(4)の耐熱性向上エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼのスクリーニング工程におけるコントロールとして用いた。なお、pEDTrc9003は、独立行政法人産業技術総合研究所 特許生物寄託センター（茨城県つくば市東１丁目１番地１ 中央第６）にFERM P-19201として2003年2月5日付けで寄託されている。
【００７３】
(4)耐熱性向上エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼのスクリーニング
工程(3)で得られた変異エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子を含むJM109形質転換体およびコントロールとするJM109/ pEDTrc9003を、48穴マルチディッシュに1.5mlずつ分注したLBAmp培地にそれぞれ接種し、37℃にて12時間液体培養した。得られた培養物を、50℃の温度下、30分間の熱処理に供した後、実施例2記載の方法により、残存エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ活性の測定を行った。工程(3)で得られた形質転換体約10000株についてスクリーニングを行った結果、野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼを保有するJM109/ pEDTrc9003がもはや完全にその活性を消失していたのに対し、該酵素活性が残存している株が4株得られた。
【００７４】
(5)変異の同定
工程(4)で得られた4株の耐熱性向上候補株に含有される変異導入エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子について、どの部位にどのような変異が導入されたのかを確認すべく以下のように解析を行った。4株の耐熱性向上候補株に含まれる組換えプラスミドDNAを、Flexi Prep（アマシャムバイオサイエンス社製）を用いて精製し、得られた組換えプラスミドDNAをそれぞれpEDTrcI-2、pEDTrcI-23、pEDTrcJ-05、pEDTrcK-01と命名した。また、それぞれのプラスミドに含まれる変異酵素自体をI-2、I-23、J-05、K-01と命名した。これらの組換えプラスミドDNAに含まれる変異エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子の塩基配列を、蛍光シークエンサーALF II（アマシャムバイオサイエンス社製）を用いて決定した。決定した各変異エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子の塩基配列と野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼの塩基配列（配列番号２）とを比較解析した結果、pEDTrcI-2では166番目のイソロイシン残基（ATC）がスレオニン（ACC）に、pEDTrcI-23では120番目のリジン残基（AAA）がグルタミン酸（GAA）に、pEDTrcJ-05では166番目のイソロイシン残基（ATC）がセリン（AGC）に、pEDTrcK-01では365番目のアラニン残基（GCC）がバリン（GTC）にそれぞれ置換されている単変異体であることが明らかになった[（）内は、各々のアミノ酸をコードする塩基配列の変化を示す]。
【００７５】
(6)単変異体の耐熱性評価
工程(5)で同定された4種類の単変異体エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ（I-2、I-23、J-05、K-01）の耐熱性をより詳細に調べた。単変異体エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼを保有する大腸菌4株（JM109/pEDTrcI-2、JM109/pEDTrcI-23、JM109/pEDTrcJ-05、JM109/pEDTrcK-01）、および野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼを保有する大腸菌JM109/pEDTrc9003を、LBAmp培地1.5mlに接種して30℃にて8時間振盪培養した後、それぞれの培養液を500ml容の三角フラスコに調製したLBAmp培地40mlに400μlずつ接種し、37℃にて12時間振盪培養した。得られた培養物より1.5mlを採取し、遠心分離により集菌した後、50mMホウ酸緩衝液（pH9.0）で洗浄した後、1.5mlの同緩衝液に懸濁することにより菌体懸濁液を調製した。この菌体懸濁液に対して超音波破砕を行い、粗酵素抽出液を得た。得られた粗酵素抽出液を40℃、45℃、50℃、55℃、60℃、65℃の各温度下、それぞれ30分間熱処理した後、直ちに4℃に冷却した。熱処理後、実施例2記載の方法により、エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ活性の測定を行い、熱処理を行わずに4℃で保冷したものをそれぞれの相対活性値のコントロール（100%）として、残存相対活性を求めた。
【００７６】
結果を図5に示す。図５において、縦軸は残存エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ活性を、横軸は処理温度を示す。野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼは50℃で完全に該酵素活性が消失していたのに対し、単変異体I-2、I-23、J-05、K-01はそれぞれ50℃で75%、50℃で85%、60℃で12%、55℃で12％の残存相対活性を保持しており、野生型と比較してこれらの単変異体の耐熱性が向上していることが確認された。
【００７７】
[実施例4] 多重変異体の作製と耐熱性評価
(1) 多重変異体の作製
実施例3で耐熱性の向上が確認された4種類の単変異体におけるアミノ酸残基の置換を組み合わせることにより、多重変異体を作製した。単変異体I-2、I-23、J-05、K-01の変異個所は、それぞれ166番目のイソロイシン残基、120番目のリジン残基、166番目のイソロイシン残基、366位のアラニン残基であり、エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子DNA断片を制限酵素EcoT22I、AccI、ClaIで切断することにより、これらの変異個所を各々一つずつ含むDNA断片が得られる（図6）。切断した単変異体のDNA断片を、異なる単変異体由来の対応するDNA断片と置き換えて再連結することにより、二重変異あるいは三重変異を持つキメラ酵素遺伝子を作製した（図6）。例えば、二重変異体Ch-4の作製は、先ず、単変異体I-23をコードする遺伝子を含むpEDTrcI-23を制限酵素NcoIおよびAccIで切断し、切断の結果生じた短いほうのDNA断片（約0.5kb）をアガロースゲル電気泳動により切り出した。同時に、単変異体J-05をコードする遺伝子を含むpEDTrcJ-05を制限酵素NcoIおよびAccIで切断し、切断の結果生じた長いほうのDNA断片（約3.8kb、ベクター含む）をアガロースゲル電気泳動により切り出した。両DNA断片を、DNA PREP（ダイアトロン社製）を用いて回収し、DNA Ligation Kit Ver.1（宝酒造社製）を用いて連結した後、常法に従って大腸菌JM109を形質転換した。得られた形質転換体よりプラスミドを抽出し、制限酵素NcoIおよびAccIで切断した後、アガロースゲル電気泳動により正しく連結されていることを確認し、このプラスミドDNAをpEDTrcCh-4、キメラ酵素をCh-4と命名した。同様に、単変異体I-23由来NcoI-AccI断片（約0.5kb）と、単変異体I-2由来NcoI-AccI断片（約3.8kb、ベクター含む）を連結して得られるプラスミドDNAをpEDTrcCh-6、キメラ酵素をCh-6と命名した。同様な手法により、単変異体J-05由来NcoI-ClaI断片（約1kb）と、単変異体K-01由来NcoI-ClaI断片（約3.3kb、ベクター含む）を連結して得られるプラスミドDNAをpEDTrcCh-1、キメラ酵素をCh-1とし、単変異体I-2由来NcoI-ClaI断片（約1kb）と、単変異体K-01由来NcoI-ClaI断片（約3.3kb、ベクター含む）を連結して得られるプラスミドDNAをpEDTrcCh-2、キメラ酵素をCh-2とした。また、三重変異体についても、単変異体I-23由来NcoI-AccI断片（約0.5kb）と単変異体J-05由来AccI-ClaI断片（約0.5kb）および単変異体K-01由来NcoI-ClaI断片（約3.3kb、ベクター含む）を連結して得られるプラスミドDNAをpEDTrcCh-8、キメラ酵素をCh-8とし、単変異体I-23由来NcoI-AccI断片（約0.5kb）と単変異体I-2由来AccI-ClaI断片（約0.5kb）および単変異体K-01由来NcoI-ClaI断片（約3.3kb、ベクター含む）を連結して得られるプラスミドDNAをpEDTrcCh-10、キメラ酵素をCh-10とした。かくして得られたキメラ酵素の構造を図6に示す。
【００７８】
(2) 多重変異体の耐熱性評価
工程(1)で作製した6種類の多重変異体エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ（Ch-4、Ch-6、Ch-1、Ch-2、Ch-8、Ch-10）の耐熱性を調べた。多重変異体エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼを保有する大腸菌6株（JM109/ pEDTrcCh-4、JM109 /pEDTrcCh-6、JM109/pEDTrcCh-1、JM109/pEDTrcCh-2、JM109 /pEDTrcCh-8、JM109/ pEDTrcCh-10）、および野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼを保有する大腸菌JM109/pEDTrc9003を、LBAmp培地1.5mlに接種して30℃にて8時間振盪培養した後、それぞれの培養液を500ml容の三角フラスコに調製したLBAmp培地40mlに400μlずつ接種し、37℃にて12時間振盪培養した。得られた培養物より1.5mlを採取し、遠心分離により集菌した後、50mMホウ酸緩衝液（pH9.0）で洗浄した後、1.5mlの同緩衝液に懸濁することにより菌体懸濁液を調製した。この菌体懸濁液に対して超音波破砕を行い、粗酵素抽出液を得た。得られた粗酵素抽出液を40℃、45℃、50℃、55℃、60℃、65℃の各温度下、それぞれ30分間熱処理した後、直ちに4℃に冷却した。熱処理後、実施例2記載の方法により、エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ活性の測定を行い、熱処理を行わずに4℃で保冷したものをそれぞれの相対活性値のコントロールとして、残存相対活性を求めた。結果を図7に示す。図７において、縦軸は残存エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ活性を、横軸は処理温度を示す。野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼは50℃で完全に該酵素活性が消失していたのに対し、多重変異体Ch-4、Ch-6、Ch-1、Ch-2、Ch-8、Ch-10はそれぞれ60℃で72%、55℃で89%、60℃で56%、55℃で25％、60℃で90%、55℃で97％の残存相対活性を保持しており、野生型および単変異体と比較してこれら多重変異体の耐熱性が向上していることが確認された。
【００７９】
【発明の効果】
本発明により、ブレブンディモナス・ディミニュタ（Brevundimonas diminuta）MR-E001株由来野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼの塩基配列およびアミノ酸配列が提供される。また、野生型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼより誘導された改変型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼの塩基配列およびアミノ酸配列が提供される。さらには、野生型および改変型エチレンジアミン-N,N'-ジコハク酸：エチレンジアミンリアーゼ遺伝子を含む組換えDNA、該組換えDNAを含む形質転換体または形質導入体、該形質転換体または形質導入体を用いたジアミノアルキレン-N,N'-ジコハク酸の製造法が提供される。本発明により、効率よくジアミノアルキレン-N,N'-ジコハク酸を製造することが可能となる。
【００８０】
【配列表】

【００８１】
【配列表フリーテキスト】
配列番号１：XaaはMet又は欠失を表す
配列番号３：合成DNA
配列番号４：合成DNA
配列番号５：合成DNA
配列番号６：合成DNA
【図面の簡単な説明】
【図１】プラスミドpEDS9001の制限酵素地図。
【図２】プラスミドpEDS9003の制限酵素地図。
【図３】プラスミドpEDS9020の制限酵素地図。
【図４】発現ベクターpFY529Vの構築図。
【図５】単変異体の耐熱性評価を示す図。
【図６】単変異体および多重変異体の構造を示す模式図。□はT497C（Ile166Thr）の変異を、△はA358G（Lys120Glu）の変異を、○はT497G（Ile166Ser）の変異を、◇はC1094T（Ala365Val）の変異をそれぞれ示す。
【図７】多重変異体の耐熱性評価を示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel protein having ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity and a gene DNA encoding the same. Furthermore, a modified ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase that can be induced as a mutant of the enzyme, a genetic DNA encoding the modified ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase, and the modified ethylenediamine -N, N'-disuccinic acid: a recombinant DNA containing a gene DNA encoding ethylenediamine lyase, a trait including a recombinant DNA containing a genetic DNA encoding the modified ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase The present invention relates to a transformant or transductant, and a method for producing diaminoalkylene-N, N′-disuccinic acid using the transformant or transductant.
[0002]
[Prior art]
While diaminoalkylene-N, N'-disuccinic acid is important as an intermediate for the synthesis of medicines and agrochemicals, it has the unique property of capturing heavy metals, so it may be susceptible to biodegradation after being released to nature. The optically active form of the compound possessed is expected to be used for chelating agents and detergent builders.
[0003]
The present applicants have previously described a novel production method for efficiently synthesizing optically active S, S-diaminoalkylene-N, N'-disuccinic acid from fumaric acid or maleic acid and various amines utilizing the catalytic action of microorganisms. (Japanese Patent Laid-Open Nos. 9-140390, 9-289895, and 10-52292). Furthermore, by isolating and identifying the ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase gene and using genetic recombination techniques, we succeeded in improving the catalytic activity of the cell and improving the cell productivity. (Described in JP-A-10-210984).
[0004]
By the way, it is widely known that fumarase generally exists in microbial cells. Fumarase is an enzyme that produces malic acid by adding water to fumaric acid. Therefore, when producing diaminoalkylene-N, N'-disuccinic acid using a microorganism having ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase activity, in order to avoid the formation of by-products such as malic acid It is necessary to inactivate fumarase in the microbial cells. As means for solving this problem, the present applicants have previously found that intracellular fumarase activity can be reduced by treating microbial cells in an aqueous alkaline solution (described in JP-A-11-196882).
[0005]
[Problems to be solved by the invention]
In the fumarase deactivation treatment by the above method, the deactivation rate of the fumarase depends on the treatment temperature, so that the higher the temperature, the shorter the deactivation. Further, although the stability of fumarase varies depending on the host microorganism, even when a microorganism in which fumarase is hardly inactivated is used, inactivation treatment at a higher temperature is desired. Accordingly, an object of the present invention is to provide ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase having improved heat resistance.
[0006]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have newly discovered the breven dimonas diminuta ( Brevundimonas diminuta ) By replacing at least one amino acid residue with a residue selected from the group of natural amino acids in the amino acid sequence of ethylenediamine-N, N′-disuccinate: ethylenediamine lyase derived from MR-E001 strain, The inventors have found that the heat resistance of the enzyme is improved, and have completed the present invention. That is, this application provides the following invention.
[0007]
(1) A protein having the amino acid sequence set forth in SEQ ID NO: 1 and having ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity.
(2) The amino acid sequence described in SEQ ID NO: 1 consists of an amino acid sequence in which one or more amino acid residues are deleted, substituted or added, and has ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase activity protein.
(3) In the amino acid sequence described in SEQ ID NO: 1, at least one amino acid residue of the 120th lysine residue, the 166th isoleucine residue and the 365th alanine residue was substituted with another amino acid residue A protein having an amino acid sequence and having ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity.
(4) A protein having an amino acid sequence in which at least the 120th lysine residue is substituted with glutamic acid in the amino acid sequence described in SEQ ID NO: 1, and having ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity.
(5) A protein having an amino acid sequence in which at least the 166th isoleucine residue is substituted with serine in the amino acid sequence described in SEQ ID NO: 1 and having ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity.
(6) A protein having an amino acid sequence in which at least the 166th isoleucine residue is substituted with threonine in the amino acid sequence described in SEQ ID NO: 1 and having ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity.
(7) A protein having an amino acid sequence in which at least the 365th alanine residue is substituted with valine in the amino acid sequence described in SEQ ID NO: 1 and having ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity.
(8) The amino acid sequence described in SEQ ID NO: 1 comprises an amino acid sequence in which at least the 120th lysine residue is substituted with glutamic acid and the 166th isoleucine residue is substituted with serine, and ethylenediamine-N, N′- Disuccinic acid: A protein having ethylenediamine lyase activity.
(9) The amino acid sequence described in SEQ ID NO: 1 comprises an amino acid sequence in which at least the 120th lysine residue is substituted with glutamic acid and the 166th isoleucine residue is substituted with threonine, and ethylenediamine-N, N′- Disuccinic acid: A protein having ethylenediamine lyase activity.
(10) The amino acid sequence described in SEQ ID NO: 1 consists of an amino acid sequence in which at least the 166th isoleucine residue is substituted with serine and the 365th alanine residue is substituted with valine, and ethylenediamine-N, N'- Disuccinic acid: A protein having ethylenediamine lyase activity.
[0008]
(11) The amino acid sequence described in SEQ ID NO: 1 comprises an amino acid sequence in which at least the 166th isoleucine residue is substituted with threonine and the 365th alanine residue is substituted with valine, and ethylenediamine-N, N'- Disuccinic acid: A protein having ethylenediamine lyase activity.
(12) consisting of an amino acid sequence in which at least the 120th lysine residue is substituted with glutamic acid, the 166th isoleucine residue is substituted with serine, and the 365th alanine residue is substituted with valine. And ethylenediamine-N, N′-disuccinic acid: a protein having ethylenediamine lyase activity.
(13) The amino acid sequence of SEQ ID NO: 1, comprising at least the 120th lysine residue substituted with glutamic acid, the 166th isoleucine residue replaced with threonine, and the 365th alanine residue replaced with valine. And ethylenediamine-N, N′-disuccinic acid: a protein having ethylenediamine lyase activity.
(14) A genetic DNA encoding the protein described in (1).
(15) The following gene DNA (a) or (b):
(A) A gene DNA comprising the base sequence described in SEQ ID NO: 2.
(B) Hybridizes under stringent conditions with a DNA comprising a sequence complementary to the DNA comprising the base sequence described in SEQ ID NO: 2 or a partial sequence thereof, and ethylenediamine-N, N′-disuccinic acid: Genetic DNA encoding a protein having ethylenediamine lyase activity
(16) A genetic DNA encoding the protein described in (2).
(17) The gene DNA according to (16), comprising a base sequence in which one or more bases are deleted, substituted or added in the base sequence described in SEQ ID NO: 2.
(18) A genetic DNA encoding the protein described in (3).
(19) Consists of a base sequence in which at least one base is replaced with another different base from the 358th to 360th, 496th to 498th and 1093th to 1095th bases in the base sequence described in SEQ ID NO: 2. 19. The genetic DNA according to claim 18.
(20) A gene DNA encoding the protein described in (4).
[0009]
(21) The gene DNA according to (20), comprising a base sequence in which the 358th to 360th base AAA is replaced with GAA or GAG in the base sequence described in SEQ ID NO: 2.
(22) The gene DNA according to (20), comprising a base sequence in which the 358th base adenine is substituted with guanine in the base sequence described in SEQ ID NO: 2.
(23) A genetic DNA encoding the protein described in (5).
(24) The nucleotide sequence of SEQ ID NO: 2 consists of a nucleotide sequence in which the 496th to 498th nucleotide ATC is replaced by AGC, AGT or TCN (N represents A, G, C or T) (23 ) The gene DNA described.
(25) The gene DNA according to (23), comprising a base sequence in which the 497th base thymine is replaced with guanine in the base sequence described in SEQ ID NO: 2.
(26) A genetic DNA encoding the protein described in (6).
(27) The gene according to (26), comprising a nucleotide sequence in which the 496th to 498th nucleotide ATC is replaced with ACN (N represents A, G, C or T) in the nucleotide sequence described in SEQ ID NO: 2 DNA.
(28) The gene DNA according to (26), comprising a base sequence in which the 497th base thymine is replaced with cytosine in the base sequence described in SEQ ID NO: 2.
(29) A genetic DNA encoding the protein described in (7).
(30) The gene according to (29), comprising a nucleotide sequence in which nucleotides 1093 to 1095 in the nucleotide sequence of SEQ ID NO: 2 are replaced with GTN (N represents A, G, C or T) DNA.
[0010]
(31) The gene DNA according to (29), comprising a base sequence in which the 1094th base cytosine is replaced with thymine in the base sequence described in SEQ ID NO: 2.
(32) A gene DNA encoding the protein described in (8).
(33) In the base sequence described in SEQ ID NO: 2, base AAA from 358 to 360 is GAA or GAG, base ATC from 496 to 498 is AGC, AGT or TCN (N is A, G, C or T) The gene DNA according to (32), which comprises a base sequence each substituted by
(34) The gene DNA according to (32), comprising a base sequence in which the 358th base adenine is replaced with guanine and the 497th base thymine is replaced with guanine in the base sequence described in SEQ ID NO: 2.
(35) A gene DNA encoding the protein described in (9).
(36) In the base sequence described in SEQ ID NO: 2, the 358th to 360th base AAA is GAA or GAG, and the 496th to 498th base ATC is ACN (N represents A, G, C or T.) The gene DNA according to (35), comprising a base sequence each substituted on
(37) The gene DNA according to (35), comprising a base sequence in which the 358th base adenine is replaced with guanine and the 497th base thymine is replaced with cytosine in the base sequence described in SEQ ID NO: 2.
(38) A gene DNA encoding the protein described in (10).
(39) In the base sequence described in SEQ ID NO: 2, the 496th to 498th bases ATC are AGC, AGT or TCN (N represents A, G, C or T), and the 1093rd to 1095th bases GCC. The gene DNA according to (38), comprising a nucleotide sequence each substituted with GTN (N represents A, G, C or T).
(40) The gene DNA according to (38), comprising a base sequence in which the 497th base thymine is replaced with guanine and the 1094th base cytosine is replaced with thymine in the base sequence described in SEQ ID NO: 2.
[0011]
(41) A gene DNA encoding the protein described in (11).
(42) In the base sequence described in SEQ ID NO: 2, the 496th to 498th base ATC is ACN (N represents A, G, C or T), and the 1093rd to 1095th base GCC is GTN (N Represents A, G, C, or T.) The gene DNA according to (41), which comprises a nucleotide sequence each substituted by
(43) The gene DNA according to (41), comprising a base sequence in which the 497th base thymine is replaced with cytosine and the 1094th base cytosine is replaced with thymine in the base sequence described in SEQ ID NO: 2.
(44) A gene DNA encoding the protein described in (12).
(45) In the base sequence described in SEQ ID NO: 2, the 358th to 360th base AAA is GAA or GAG, and the 496th to 498th base ATC is AGC, AGT or TCN (N is A, G, C or T). Wherein the 1093rd to 1095th base GCC is replaced by GTN (N represents A, G, C or T), respectively.
(46) The nucleotide sequence described in SEQ ID NO: 2, comprising a nucleotide sequence in which the 358th base adenine is replaced with guanine, the 497th base thymine is replaced with guanine, and the 1094th base cytosine is replaced with thymine. Genetic DNA.
(47) A genetic DNA encoding the protein described in (13).
(48) In the base sequence described in SEQ ID NO: 2, the 358th to 360th base AAA is GAA or GAG, and the 496th to 498th base ATC is ACN (N represents A, G, C, or T). Furthermore, the gene DNA according to (47), comprising a base sequence in which the 1093rd to 1095th base GCC are each replaced by GTN (N represents A, G, C or T).
(49) The base sequence described in SEQ ID NO: 2 consisting of a base sequence in which the 358th base adenine is replaced by guanine, the 497th base thymine is replaced by cytosine, and the 1094th base cytosine is replaced by thymine, respectively (47) Genetic DNA.
(50) A recombinant DNA obtained by inserting the gene DNA described in (14) to (49) into a vector DNA.
[0012]
(51) A transformant or transductant comprising the recombinant DNA according to (50).
(52) Ethylenediamine-N, N ′ characterized by culturing the transformant or transductant according to (51) and collecting ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase from the resulting culture. -Disuccinic acid: A method for producing ethylenediamine lyase.
(53) Fumaric acid and diamine are reacted in the presence of the transformant or transductant according to (51), and diaminoalkylene-N, N′-disuccinic acid is collected from the resulting reaction product. And a process for producing diaminoalkylene-N, N′-disuccinic acid.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail. In the present invention, ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase (also referred to as “EDDSase”) is an enzyme capable of reversibly generating ethylenediamine-N, N′-disuccinic acid from fumaric acid and ethylenediamine. As shown, ethylenediamine-N-monosuccinic acid may be produced depending on the reaction conditions. In addition, the enzyme is reactive with diamines other than ethylenediamine and produces the corresponding diaminoalkylene-N, N′-disuccinic acid. In addition, diaminoalkylene-N, N′-disuccinic acid to be produced is optically active in many cases, but there is also an enzyme that produces a racemate. A group of enzymes exhibiting such reactivity has been found in bacteria belonging to a plurality of genera isolated and identified from the natural world by the present applicants. These bacteria are disclosed in the above-mentioned JP-A-9-140390. Nos. 9-289895 and 10-52292. Furthermore, the present applicants isolated the ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase gene from the Brevundimonas sp. TN-3 strain described in JP-A-10-52292, and the amino acid sequence thereof. In addition, the gene sequence has been clarified for the first time, and a transformant having the possibility of mass expression of the gene product has been successfully produced (described in JP-A-10-210984).
[0014]
On the other hand, due to recent advances in recombinant DNA technology, mutations in which one or more of the constituent amino acids of the enzyme are deleted, added, deleted, inserted, or replaced with other amino acids without substantially changing the action of the enzyme It is possible to make a body. These mutants are more resistant to organic solvents, depending on where the amino acid residues are substituted, deleted, added, deleted, or inserted, and depending on the type of amino acid that is substituted. It is known that performances such as heat resistance, acid resistance, alkali resistance, substrate specificity, and substrate affinity are significantly improved. These improvements in performance may lead to a significant reduction in production costs in industrial production using enzyme reactions through stabilization of the enzyme as a catalyst, simplification of the reaction process, improvement in reaction yield, and the like. Therefore, creation of useful improved enzymes having various performances improved in many enzymes.
[0015]
In the present specification, “wild type” means that an amino acid sequence constituting an enzyme retained in a microorganism isolated from the natural world and a base sequence of a gene encoding the enzyme are intentionally or unintentionally. Deletion, deletion, insertion, or substitution with other amino acids or bases.
[0016]
Applicants screened for microorganisms possessing the enzyme activity in search of further useful ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase. As a result, a breven dimonas diminuta MR-E001 strain (hereinafter sometimes referred to as MR-E001 strain) having high enzyme activity was isolated, and ethylenediamine-N, N′-disuccinic acid from MR-E001 strain: The ethylenediamine lyase gene was obtained. Furthermore, the inventors have found that the heat resistance of the enzyme is improved by substituting at least one amino acid residue in the amino acid sequence of the enzyme with a residue selected from the group of natural amino acids, and the present invention is completed. It came.
[0017]
The modified ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase of the present invention can be obtained, for example, as follows. First, wild type ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase gene is obtained from MR-E001 strain. Any known means may be used for obtaining the gene. For example, PCR (Polymerase Chain Reaction) is performed using chromosomal DNA prepared from the MR-E001 strain as a template to obtain a DNA fragment containing a part of the gene. As a primer used for PCR, generally, a degenerate primer designed based on amino acid sequence information obtained by conducting amino acid analysis after isolating and purifying the enzyme can be used. In addition, when “the sequence of the target gene is expected to be homologous to the sequence of the gene derived from another species already known” or “from the species of another species already known In the case of “for the purpose of obtaining a gene having homology with the sequence of the gene”, it is also possible to design and use a degenerate primer from the amino acid sequence information encoded by the gene derived from the known other species It is. Using the primers thus designed, PCR is performed using the chromosomal DNA of the MR-E001 strain as a template, and the obtained amplified DNA product is used as a probe for colony hybridization performed later.
[0018]
Subsequently, a DNA library is prepared. The MR-E001 strain chromosome prepared according to a known method, for example, the method of Saito and Miura et al. (Biochem. Biophys. Acta, 72, 619 (1963)) is cleaved or partially cleaved with an appropriate restriction enzyme. A DNA library of the chromosome is obtained by ligating to a vector DNA treated with a restriction enzyme capable of generating a cleaved end that can be ligated to the end, and producing a transformant or transductant of an appropriate microbial host. . The microorganism used as a host for the transformant or the transductant is not particularly limited. For example, when E. coli is used, E. coli K12 strain, JM109 strain, XL1-Blue strain and the like can be mentioned. Examples of plasmid DNA used for the preparation of transformants include pBR322, pUC18, and pBluescript II SK (+) having a region capable of autonomous replication in E. coli when E. coli is used as a host. In addition, the present invention is not limited to the plasmid vector DNA, and a transductant may be prepared using other phage vector DNA.
[0019]
Here, in the description of the process relating to genetic manipulation, the microorganism containing the recombinant DNA to be produced is called a transformant when using plasmid vector DNA, and is called a transductant when using phage vector DNA. Both the above transformant and transductant are included in the present invention. Hereinafter, the case of a transformant using plasmid vector DNA will be described as an example.
[0020]
The thus obtained chromosomal DNA library is subjected to colony hybridization using the above amplified DNA product by PCR as a probe. Colony hybridization may be performed by a method that can be usually performed, for example, as follows. That is, the chromosomal DNA library transformant grown on an agar medium is copied onto a nylon membrane and then lysed to immobilize DNA. The amplified DNA product obtained by PCR is labeled with, for example, a DNA Labeling kit (manufactured by Roche Diagnostics) to form a probe, then hybridized to the membrane, and the DNA Luminescent Detection kit (Roche. Positive clones can be selected using a diagnostics). Plasmid DNA is prepared from the obtained positive clone according to a conventional method, and after subcloning as necessary, the base sequence of the inserted fragment is determined. The base sequence can be determined by any method, and can usually be determined by the dideoxy method (Methods in Enzymology, 101, 20-78, 1983) using a commercially available kit or the like. Thus, the MR-E001 strain-derived wild-type ethylenediamine-N, N′-disuccinate: ethylenediamine lyase gene can be obtained, and its amino acid sequence and base sequence can be determined.
[0021]
Subsequently, in the amino acid sequence of the obtained wild-type ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase, at least one is substituted with another natural amino acid residue. Modified ethylenediamine-N, N′-disuccinic acid: ethylenediamine Any method may be used to obtain lyase, and it can be carried out by a generally known method. In other words, wild-type ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase gene DNA, for example, a method of contacting and acting a drug that becomes a mutation source, such as hydroxylamine or nitrous acid, induces mutation by ultraviolet irradiation Methods, methods of introducing mutations randomly using PCR, methods of generating site-specific substitution using commercially available kits, selective cleavage of gene DNA, and then removal and addition of selected oligonucleotides And a connecting method.
[0022]
After obtaining ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase gene DNA having various mutations by any of the above treatments, transformants are prepared. As a plasmid vector that can be used for transformation, for example, when Escherichia coli is used as a host, the plasmid vector mentioned in the DNA library preparation step can be used. In the subsequent screening step, ethylenediamine- N, N'-disuccinic acid: In order to efficiently detect the residual activity of ethylenediamine lyase, an expression vector with high expression efficiency, such as an expression vector pKK233-2 (manufactured by Amersham) having a trc promoter, or pKK233 shown in a later example It is preferable to use the derivative pFY529V of -2.
[0023]
However, the vectors and hosts used in the present invention are not limited to the above plasmids and E. coli. For example, examples of the vector include plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA and the like.
[0024]
The ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase gene DNA of the present invention needs to be incorporated into a vector so that it can be expressed in a host organism into which the gene DNA is introduced. Therefore, in addition to the gene DNA of the present invention, a promoter, terminator, enhancer, splicing signal, poly A addition signal, selection marker, ribosome binding sequence (SD sequence) and the like can be linked to the vector of the present invention. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.
[0025]
The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the ethylenediamine-N, N′-disuccinate: ethylenediamine lyase gene can be expressed. Here, examples of the host include bacteria such as Escherichia coli and Bacillus subtilis. Yeast, animal cells, insect cells, plant cells, and the like can also be used.
[0026]
Examples of E. coli include Escherichia coli, and examples of Bacillus subtilis include Bacillus subtilis. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.
[0027]
When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like are used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.
[0028]
When animal cells are used as hosts, monkey cells COS-7, Vero, CHO cells, mouse L cells, rat GH3, human FL cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.
[0029]
When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used. As a method for introducing a recombinant vector into insect cells, for example, calcium phosphate method, lipofection method, electroporation method and the like are used.
[0030]
When plant cells are used as hosts, examples thereof include, but are not limited to, corn, rice, tobacco and the like. As a method for introducing a recombinant vector into a plant cell, for example, an Agrobacterium method, a particle gun method, a PEG method, an electroporation method, or the like is used.
[0031]
As described above, transformants carrying recombinant DNA containing various ethylenediamine-N, N′-disuccinate: ethylenediamine lyase genes can be obtained.
[0032]
When the host is Escherichia coli, the obtained transformant is cultured on an agar medium to form colonies, and then liquid culture is performed to produce ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase. The obtained culture was subjected to, for example, a heat treatment for 30 minutes at a temperature of 40 to 65 ° C., and then the residual ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity was measured, and the residual ethylenediamine-N, N '-Disuccinic acid: Select a transformant with high ethylenediamine lyase activity. Determination of the base sequence of the ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase gene inserted into the recombinant DNA of the excellent transformant thus obtained can be performed, for example, by the dideoxy method.
[0033]
SEQ ID NO: 2 illustrates the nucleotide sequence of the ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase gene DNA of the present invention, and SEQ ID NO: 1 illustrates the amino acid sequence encoded by the gene of the present invention.
[0034]
In addition, it is known that by combining multiple different single mutation substitutions that give heat resistance to the enzyme and making it a multiple mutant, it is possible to create a mutant enzyme with further improved heat resistance than the single mutant. ing. Any method can be used for preparing multiple mutants, for example, a method in which a site-specific substitution is generated using a synthetic single-stranded oligonucleotide, a DNA fragment containing a plurality of different single mutation sites is cleaved with a restriction enzyme. And the like.
[0035]
Accordingly, as long as ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase has the enzyme activity, deletion, substitution, addition, etc. are performed in plural, preferably one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1. Mutations may occur. For example, 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence shown in SEQ ID NO: 1 may be deleted, and 1 to 10, preferably 1 to 1, are added to the amino acid sequence shown in SEQ ID NO: 1. Five amino acids may be added, or 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence shown in SEQ ID NO: 1 may be substituted with other amino acids.
[0036]
In the present invention, in particular, the amino acid sequence shown in SEQ ID NO: 1 is preferably one in which at least one amino acid of the 120th Lys, the 166th Ile, and the 365th Ala is substituted with another amino acid. The above three amino acid substitutions can be arbitrarily combined. Preferred embodiments of substitution are shown below. In the following substitution mode, the number is the position number of the amino acid sequence shown in SEQ ID NO: 1, the alphabet to the left of the number is the amino acid before substitution (indicated by one letter), and the alphabet to the right of the number is the amino acid after substitution ( (Single character notation).
K120E
I166S
I166T
A365V
(K120E, I166S)
(K120E, I166T)
(I166S, A365V)
(I166T, A365V)
(K120E, I166S, A365V)
(K120E, I166T, A365V)
[0037]
In addition, base substitution for causing substitution of K120E is as follows.
The position of the base sequence shown in SEQ ID NO: 2 corresponding to the 120th Lys is from the 358th to the 360th, and the base sequence is “AAA”. On the other hand, since the codon of glutamic acid is GAA or GAG, in the present invention, the base can be substituted so that AAA becomes GAA or GAG. In particular, it is preferable to replace 358th A with G (AAA → GAA).
[0038]
In the same manner as described above, when causing substitution of I166S, the 496th to 498th base ATC in the base sequence shown in SEQ ID NO: 2 can be replaced with AGC, AGT, ACA, ACC, ACG or ACT. . In particular, it is preferable to replace the 497th T with G (ATC → AGC). When substitution of I166T is caused, the 496th to 498th base ATC in the base sequence shown in SEQ ID NO: 2 can be substituted with ACA, ACC, ACG or ACT. In particular, it is preferable to replace the 497th T with C (ATC → ACC).
[0039]
When causing A365V substitution, the 1093rd to 1095th base GCC in the base sequence shown in SEQ ID NO: 2 can be replaced with GTA, GTG, GTC or GTT. Substitution (GCC → GTC) is preferred.
[0040]
However, in the present invention, the amino acid after substitution is not limited to the above examples. Therefore, at least one base of the codon encoding the amino acid is replaced with another base so that it is at least one amino acid residue of the 120th, 166th, and 365th amino acids of the amino acid sequence shown in SEQ ID NO: 1. can do.
[0041]
Here, “ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity” means catalytic activity for reacting fumaric acid and diamine to produce diaminoalkylene-N, N′-disuccinic acid. .
[0042]
Further, it hybridizes under stringent conditions with a DNA consisting of a base sequence shown in SEQ ID NO: 2 or a partial sequence thereof, and a DNA complementary thereto, and ethylenediamine-N, N′-disuccinic acid: ethylenediamine A gene DNA encoding a protein having lyase activity is also included in the gene DNA of the present invention. Stringent conditions refer to conditions under which so-called specific hybrids are formed. For example, nucleic acids having high homology, that is, DNAs having a homology of 80% or more, preferably 90% or more, and DNAs encoding a protein having ethylenediamine-N, N'-disuccinate: ethylenediamine lyase activity Are hybridized and DNAs having lower homology do not hybridize with each other. More specifically, the sodium concentration is 300 to 2000 mM, preferably 600 to 900 mM, and the temperature is 40 to 75 ° C, preferably 55 to 65 ° C.
[0043]
Once the base sequence of the gene of the present invention has been determined, the DNA sequence is then synthesized by chemical synthesis, by PCR using the cloned DNA as a template, or by hybridization with a DNA fragment having the base sequence as a probe. The gene DNA of the invention can be obtained.
[0044]
In the present invention, heat resistance means a property capable of retaining enzyme activity even in a temperature range where wild-type ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase is deactivated, preferably 45 to 60 ° C., preferably Is 50-60 ° C. Within the range of 50 to 60 ° C., the modified ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase can maintain an enzyme activity of 60% compared to the enzyme activity at 50 ° C.
[0045]
Using the thus obtained transformant having a recombinant plasmid DNA containing an ethylenediamine-N, N'-disuccinate: ethylenediamine lyase gene into which single or multiple mutations are introduced, ethylenediamine-N, N ' -Disuccinic acid: ethylenediamine lyase can be produced. In addition, diaminoalkylene-N, N′-disuccinic acid can also be produced using the transformant (for example, using a transformant of E. coli).
[0046]
Ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase can be produced by culturing the transformant and collecting it from the culture. “Culture” means any of culture supernatant, cultured cells or cultured cells, or disrupted cells or cells. The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host.
[0047]
A medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms, and efficiently cultures the transformants. As long as the medium can be used, either a natural medium or a synthetic medium may be used. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like. Examples of the inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. The culture is usually performed under aerobic conditions such as shaking culture or aeration and agitation culture. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.
[0048]
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector having a promoter inducible with isopropyl-β-D-thiogalactoside (IPTG), IPTG or the like can be added to the medium. In addition, when culturing a microorganism transformed with an expression vector using a trp promoter inducible with indoleacetic acid (IAA), IAA or the like can be added to the medium.
[0049]
Examples of a medium for culturing a transformant obtained using animal cells as a host include generally used RPMI1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums. Culture is usually 5% CO ₂ Perform in the presence at 37 ° C. for 1-30 days. During culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary.
[0050]
When the protein of the present invention is produced in cells or cells after culturing, the target protein is collected by crushing the cells or cells by sonication, repeated freeze-thawing, homogenizer treatment, etc. . When the protein of the present invention is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, From the above, the protein of the present invention can be isolated and purified.
[0051]
When the transformant is a plant cell or a plant tissue, the culture can be performed by using a normal plant culture medium such as an MS basic medium or an LS basic medium. As a culture method, any of a normal solid culture method and a liquid culture method can be employed.
[0052]
In order to purify the protein of the present invention from the culture, cells are first destroyed by cell lysis treatment, ultrasonic disruption treatment, grinding treatment, etc. using enzymes such as cellulase and pectinase. Next, insoluble matters are removed by filtration or centrifugation to obtain a crude protein solution. In order to purify the protein of the present invention from the above crude solution, salting out, various chromatography (eg, gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc.), SDS polyacrylamide gel electrophoresis, etc. are used alone or as appropriate. Implement in combination.
[0053]
Diaminoalkylene-N, N′-disuccinic acid can be produced as follows. In order to culture the E. coli transformant, it may be cultured by a normal solid culture method, but it is preferable to employ a liquid culture method as much as possible. Examples of the culture medium used for cultivation include one or more nitrogen sources such as yeast extract, tryptone, polypeptone, corn steep liquor, soybean or wheat bran leachate, sodium chloride, monopotassium phosphate, dipotassium phosphate. In addition, one or more inorganic salts such as magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate are added, and if necessary, saccharide raw materials, vitamins and the like are appropriately added. It is appropriate to adjust the initial pH of the medium to 7-9. The culture is preferably performed at 25 to 42 ° C. for 6 to 24 hours by aeration and agitation deep culture, shaking culture, stationary culture, or the like.
[0054]
After completion of the culture, the obtained microbial cells are collected, washed with an appropriate buffer solution, for example, 50 mM borate buffer (pH 9.0), and suspended in the same buffer solution. A body suspension is prepared. For example, when this cell suspension is heat-treated at a temperature of 40 to 65 ° C. for 30 minutes to 72 hours, fumarase activity disappears and ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity is possessed. Bacteria can be prepared. The bacterial cells thus obtained are suspended and reacted in an aqueous solution containing fumaric acid or maleic acid and various amines, so as not to contain by-products such as malic acid, optically active S, S-diaminoalkylene-N, An N′-disuccinic acid aqueous solution can be produced.
In addition, Breven Dimonas Diminuta ( Brevundimonas diminuta ) Mycological properties of MR-E001 strain are as shown in the table below.
[0055]
[Table 1]

[0056]
The Brevundimonas diminuta MR-E001 strain was registered as FERM P-19203 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, 2003). Deposited as of February 5,
[0057]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the examples should be regarded as an aid for obtaining specific recognition of the present invention, and are not intended to limit the scope of the present invention.
[0058]
[Example 1] MR-E001 strain-derived wild-type ethylenediamine-N, N'-disuccinic acid: acquisition of ethylenediamine lyase gene
(1) Preparation of chromosomal DNA of MR-E001 strain
MR-E001 strain in 100 ml of EDDS medium (0.2% ethylenediamine-N, N'-disuccinic acid, 0.2% glucose, 0.1% bacto yeast extract, 0.05% polypeptone, 0.1% magnesium sulfate / 7-hydrate, 25% (v / v) Phosphate buffer (1M, pH 7.0), 0.5% (v / v) metal salt mixture solution (sodium sulfate 56g, magnesium chloride hexahydrate 8g, calcium chloride 0.8g, manganese sulfate tetrahydrate 0.6g, Bacteria were collected after shaking culture at 30 ° C for 4 days in ferric chloride hexahydrate 0.12g, zinc sulfate 0.06g / 100ml), and the cells were collected into a Saline-EDTA solution (0.1M EDTA, 15M NaCl, Suspend in 4 ml of pH 8.0, add 8 mg of lysozyme, shake for 1 hour at 37 ° C. and freeze.Next, add 10 ml of Tris-SDS solution (1% SDS, 0.1 M NaCl, 0.1 M Tris, pH 9.0). Gently shake, add proteinase K (Merck) (final concentration 1 mg) and shake for 1 hour at 37 ° C. Next, equidistant TE saturated phenol (TE: 10 mM Tris, 1 mM EDTA) After adding pH 8.0), the mixture was stirred and centrifuged, and the upper layer was collected. After adding twice the amount of ethanol, the DNA was wound with a glass rod and phenol was sequentially removed with 90%, 80%, and 70% ethanol. Dissolve the DNA in 3 ml of TE buffer, add ribonuclease A solution (100 ° C, heat-treated for 15 minutes) to a final concentration of 10 mg / ml and shake for 30 minutes at 37 ° C. After shaking at 37 ° C. for 30 minutes, an equal amount of TE saturated phenol was added and centrifuged, and the upper layer was separated from the upper layer and the lower layer (hereinafter, this operation is called phenol extraction) After repeating phenol extraction twice, The same amount of chloroform (containing 4% isoamyl alcohol) was added and the same extraction procedure was repeated (hereinafter this procedure is referred to as chloroform extraction), and then twice the amount of ethanol was added to the upper layer, and the DNA was wound up and collected with a glass rod. Obtain a chromosomal DNA preparation .
[0059]
(2) Probe preparation
The present applicants first succeeded in isolating ethylenediamine-N, N'-disuccinate: ethylenediamine lyase gene from Brevundimonas sp. TN-3, and revealed its amino acid sequence and gene sequence for the first time. (Described in JP-A-10-210984). Using the degenerate primer used in the publication, that is, synthetic DNAs having sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4 (referred to as primers # 1 and # 2, respectively), and obtained in step (1) PCR was performed using the obtained chromosomal DNA of MR-E001 strain as a template.
Primer # 1: ATGACICCIC AYAAYCCIGA YGC (SEQ ID NO: 3)
Primer # 2: CCDATYTGCAT YTTICCIGC RACIGAICCD ATYTC (SEQ ID NO: 4)
[0060]
MR-E001 strain chromosomal DNA 1 μl, 10-fold concentration of reaction buffer 10 μl, 10 mM dNTP 4 μl, primers # 1 and # 2 each 1 μl (corresponding to 100 pmol concentration), ExTaq (Takara Shuzo) 1 μl are added to make 100 μl did. This solution was incubated at 95 ° C. for 30 seconds (denaturation process), 55 ° C. for 30 seconds (annealing process), 72 ° C. for 2 minutes (extension process) for 30 cycles. After completion of the reaction, phenol extraction and chloroform extraction were performed, and DNA amplified by ethanol precipitation was recovered. After separation by 1.0% agarose gel electrophoresis, a DNA fragment of about 300 bp, which is thought to encode a part of the ethylenediamine-N, N′-disuccinate: ethylenediamine lyase gene of MR-E001 strain, was obtained. The DNA fragment thus obtained was labeled with a DIG DNA Labeling kit (manufactured by Roche Diagnostics) to obtain a probe.
[0061]
(3) Preparation of DNA library
MR-E001 strain chromosomal DNA (10 μl), 10-fold concentration restriction enzyme reaction buffer (5 μl), sterilized water (33 μl) and restriction enzyme KpnI (2 μl) were added and reacted at 37 ° C. for 16 hours, followed by ethanol precipitation. Agarose gel electrophoresis was performed, and a DNA fragment of 6.5 Kb to 5.5 Kb was excised from the gel and recovered using DNA PREP (Diatron). This DNA fragment was inserted into the KpnI site of Escherichia coli vector pUC18 using DNA Ligation Kit Ver. 1 (Takara Shuzo) to prepare a recombinant DNA library. The pUC18 fragment used for ligation was prepared as follows. Add 2 μl of pUC18 stock solution, 5 μl of 10-fold restriction enzyme buffer, 40 μl of sterilized water, 3 μl of restriction enzyme KpnI, react at 37 ° C. for 2 hours, phenol extraction and chloroform extraction, ethanol precipitation, and drying And dissolved in 50 μl of sterile water. Further, 1 μl of alkaline phosphatase (Takara Shuzo Co., Ltd.), 10 μl of 10-fold concentration buffer and 39 μl of sterilized water were added and reacted at 65 ° C., followed by phenol extraction and chloroform extraction, ethanol precipitation, drying and dissolution in sterile water.
[0062]
(4) Preparation of E. coli transformants and selection of recombinant DNA
E. coli strain JM109 was inoculated into 1 ml of LB medium (1% bactotryptone, 0.5% bacto yeast extract, 0.5% NaCl) and pre-cultured aerobically at 37 ° C. for 5 hours, and 0.4 ml of this culture was added to 40 ml of SOB medium ( 2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO _Four , 1 mM MgCl ₂ ), And cultured at 18 ° C. for 20 hours. The culture was collected by centrifugation, and then cooled TF solution (20 mM PIPES-KOH (pH 6.0), 200 mM KCl, 10 mM CaCl ₂ , 40 mM MnCl ₂ ) Was added and left at 0 ° C. for 10 minutes. Thereafter, the mixture was centrifuged again, the supernatant was removed, and the precipitated E. coli was suspended in 3.2 ml of cold TF solution, 0.22 ml of dimethyl sulfoxide was added, and the mixture was allowed to stand at 0 ° C. for 10 minutes. Add 10 μl of the solution (DNA library) containing the recombinant plasmid DNA prepared in step (3) to 200 μl of the competent cells prepared in this way, leave it at 0 ° C for 30 minutes, and then apply heat shock at 42 ° C for 30 seconds. After cooling at 0 ° C. for 2 minutes, SOC medium (20 mM glucose, 2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO _Four , 1 mM MgCl ₂ ) 1 ml was added and cultured with shaking at 37 ° C. for 1 hour. 200 μl of this was plated on LBAmp agar medium (LB medium containing ampicillin 100 mg / L, 1.5% agar) and cultured at 37 ° C. From the transformant colonies grown on the agar medium, a transformant considered to possess the ethylenediamine-N, N′-disuccinate: ethylenediamine lyase gene was selected by colony hybridization. That is, the transformant grown on the agar medium was transferred onto a nylon membrane (Biodyne A: manufactured by Nihon Pall Co., Ltd.), the cells were dissolved to fix the DNA, and then the probe prepared in step (2) ( The colonies having the target recombinant DNA were selected using DIG Luminescent Detection Kit (Roche Diagnostics).
[0063]
(5) Preparation of recombinant plasmid
The transformant selected in step (4) is cultured overnight at 37 ° C in 100 ml of LBAmp medium (LB medium containing ampicillin 100 mg / L). After collection, Flexi Prep (manufactured by Amersham Biosciences) is used. Used to recover plasmid DNA. The obtained recombinant plasmid DNA was named pEDS9001.
[0064]
(6) Construction of restriction enzyme map and identification of ethylenediamine-N, N'-disuccinate: ethylenediamine lyase gene region
The plasmid pEDS9001 obtained in step (5) was cleaved with several kinds of restriction enzymes to prepare a restriction enzyme map (FIG. 1). In addition, subcloning was performed as usual. That is, pEDS9001 was cleaved with the restriction enzyme BamHI and then subjected to agarose gel electrophoresis, and a DNA fragment of about 5.3 Kb was excised from the gel and recovered using DNA PREP (manufactured by Diatron). After self-ligation using DNA Ligation Kit Ver.1 (Takara Shuzo Co., Ltd.), E. coli JM109 strain is transformed to contain ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase gene A plasmid (pEDS9003) into which a fragment of about 2.6 Kb was inserted (FIG. 2) was obtained.
[0065]
(7) Determination of nucleotide sequence
The base sequence around the region identified in step (6) was determined using a fluorescence sequencer ALFII (Amersham Biosciences). As a result, a base sequence (SEQ ID NO: 2) encoding an open reading frame consisting of the amino acid sequence shown in SEQ ID NO: 1 was found.
[0066]
[Example 2] MR-E001 strain-derived wild-type ethylenediamine-N, N'-disuccinate: activity evaluation of transformants introduced with ethylenediamine lyase gene
Ethylenediamine-N, N'-disuccinic acid obtained in step (6) of Example 1: 2 μl of recombinant plasmid pEDS9003 having an ethylenediamine lyase gene 2 μl of 10-fold concentration restriction enzyme buffer, 15 μl of sterile water, restriction 1 μl of enzyme KpnI was added and reacted at 37 ° C. for 2 hours. Plasmid DNA was collected by ethanol precipitation. After drying, 17 μl of sterilized water, 2 μl of 10-fold restriction enzyme buffer, and 1 μl of restriction enzyme BamHI were added and reacted at 37 ° C. for 2 hours. A fragment of about 2.6 Kb was prepared from this reaction solution by agarose gel electrophoresis and inserted into the E. coli vector pUC119. Escherichia coli JM109 was transformed with the prepared ligation solution to obtain the target plasmid. The plasmid prepared here was named pEDS9020 (FIG. 3), and the transformant was named JM109 / pEDS9020. JM109 / pEDS9020 and JM109 / pEDS020 (described in JP-A-10-210984) as a control were inoculated into 1 ml of LBAmp medium and shake-cultured at 37 ° C. for 8 hours. The cells were cultured in LBAmp medium at 37 ° C. for 30 hours. The obtained culture was washed with 10 mM sodium phosphate buffer (pH 8.0) and then suspended in 2 ml of the same buffer. A part of the obtained cell suspension was suspended in 50 ml of an aqueous solution of pH 8.0 containing 342 mM fumaric acid and 171 mM ethylenediamine, and reacted at 30 ° C. A part of the reaction mixture (0.1 ml) was taken out at a time interval, and added to 0.9 ml of 0.42N NaOH aqueous solution to stop the reaction. After removing the cells by centrifugation, S, S-ethylenediamine-N, N'-disuccinic acid produced by HPLC was analyzed (WAKOSIL5C8 (manufactured by Wako Pure Chemical Industries, Ltd.)) [eluent: 10 mM tetrahydroxide- n-Butylammonium and 0.4mMCuSO _Four Containing 50 mM phosphoric acid; pH 2]). Under the above measurement conditions, per unit of JM109 / pEDS9020 and JM109 / pEDS020 cells (per OD630), with the amount of enzyme that produces 1 μmol of S, S-ethylenediamine-N, N'-disuccinic acid per minute under the above measurement conditions ) Were found to be 1.22 mU / ODml and 0.89 mU / ODml, respectively, and it was confirmed that MR-E001 strain-derived ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase has high enzyme activity. . PEDS9020 was deposited on February 5, 2003 as FERM P-19202 at the Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology) (1st-1, 1st East, 1st Street, Tsukuba, Ibaraki Prefecture).
[0067]
[Example 3] Acquisition of modified ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase gene
(1) Wild-type ethylenediamine-N, N'-disuccinic acid: Mutation introduction into ethylenediamine lyase gene
Based on the plasmid pEDS9020 obtained in Example 2, random mutation was introduced into the wild-type ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase gene. Mutation introduction utilized base substitution due to erroneous incorporation of nucleotides in PCR. Ethylenediamine-N, N'-disuccinic acid: oligonucleotide ED-01 (SEQ ID NO: 5) containing an ethylenediamine lyase gene start codon region and ED-02 (SEQ ID NO: 6) containing a region about 50 bp downstream from the gene stop codon As a primer for mutagenesis PCR, 100 μl of a PCR reaction solution having the following composition was prepared.
Primer:
ED-01: CG CCATGG CC CCGCATAACC CAGATGCCAC C (SEQ ID NO: 5)
(Underlined part is restriction enzyme NcoI cleavage recognition site)
ED-02: AAAC AAGCTT CGTCATGGCT ATCCCCTC (SEQ ID NO: 6)
(Underlined part is restriction enzyme HindIII cleavage recognition site)
Reaction solution composition:
Template DNA (pEDS9020 prepared in the above step) 1μl
10 × PCR Buffer (GIBCO) 10 μl
50 mM MgCl ₂ (GIBCO) 3μl
Primer ED-01 1μl
Primer ED-02 1μl
2.5 mM dNTP 2 μl each
10 mM dITP 2 μl
10 mM dBraUTP 2 μl
Sterile water 71 μl
Taq DNA polymerase (GIBCO) 1μl
[0068]
The reaction solution was incubated at 94 ° C. for 30 seconds (denaturation process) and 68 ° C. for 180 seconds (annealing process / extension process) for 30 cycles. After completion of the PCR, 10 μl of the reaction solution was electrophoresed on a 0.7% agarose gel to detect an amplified fragment of about 1.5 kb. In addition, the wild type ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase gene was amplified by ordinary PCR for use as a control for heat resistance evaluation. That is, 100 μl of a PCR reaction solution having the following composition was prepared.
Template DNA (pEDS9020) 1μl
10 × Pyrobest Buffer (Takara Shuzo) 10μl
Primer ED-01 1μl
Primer ED-02 1μl
5 mM dNTP 2 μl each
Sterile water 78μl
Pyrobest ^TM DNA polymerase (Takara Shuzo) 1μl
[0069]
The reaction solution was incubated at 94 ° C. for 30 seconds (denaturation process) and 68 ° C. for 180 seconds (annealing process / extension process) for 30 cycles. After completion of the PCR, 10 μl of the reaction solution was electrophoresed on a 0.7% agarose gel to detect an amplified fragment of about 1.5 kb.
[0070]
In primers ED-01 and ED-02, a restriction enzyme NcoI cleavage recognition site and a restriction enzyme HindIII cleavage recognition site were introduced (underlined portions of the base sequences of primers ED-01 and ED-02), and amplified DNA By cleaving the product with both restriction enzymes, it becomes possible to easily insert between the NcoI site and HindIII site of the expression vector pFY529V described later.
[0071]
(2) Construction of expression vector
In the screening process described later, in order to efficiently detect the residual activity of ethylenediamine-N, N′-disuccinate: ethylenediamine lyase after heat treatment, an E. coli expression vector pFY529V with high copy number and high expression efficiency was prepared (FIG. 4). Add 5 μl of expression vector pKK233-2 (manufactured by Amersham) with trc promoter to 1 μl of restriction enzyme NaeI, 1 μl of ScaI, 1 μl of 10 times concentration restriction enzyme reaction buffer and 2 μl of sterilized water, and perform the cleavage reaction at 37 ° C. for 12 hours went. After cleavage, electrophoresis is performed on 0.7% agarose gel to cut out a NaeI-ScaI fragment (1.2 kb) that does not contain the replication origin of the plasmid, and use DNA PREP (manufactured by Diatron) to contain the DNA fragment. 3 μl of TE solution (10 mM Tris, 1 mM EDTA, pH 8.0) was recovered. In parallel with this operation, 1 μl of restriction enzyme PvuII, 1 μl of ScaI, 1 μl of 10-fold concentration restriction enzyme reaction buffer and 5 μl of sterile water were added to 2 μl of pUC18, a high copy number vector, and the cleavage reaction was carried out at 37 ° C. for 12 hours. went. After cleavage, electrophoresis was performed on a 0.7% agarose gel, a PvuII-ScaI fragment (1.6 kb) containing the replication origin of the plasmid was excised, and DNA PREP (manufactured by Diatron) was used to prepare TE containing the DNA fragment. 1 μl was recovered. Both DNA fragments thus obtained were ligated with DNA Ligation Kit Ver.1 (Takara Shuzo). 3 μl of pKK233-2-derived NaeI-ScaI fragment solution, 1 μl of pUC18-derived PvuII-ScaI fragment solution, 16 μl of solution A in the kit and 4 μl of solution B in the kit were mixed, and a ligation reaction was performed at 16 ° C. for 16 hours. Using the reaction solution after the ligation reaction, E. coli JM109 was transformed by the method described in Example 1 (4). About 10 clones from the obtained transformant colonies were inoculated into 1.5 ml of LBAmp medium and cultured with shaking at 37 ° C. for 12 hours. After culturing, the culture was collected by centrifugation, and then plasmid DNA was extracted by using Flexi Prep (manufactured by Amersham Bioscience). The obtained plasmid DNA was cleaved with the restriction enzyme ScaI and then electrophoresed on a 0.7% agarose gel. The pKK233-2-derived NaeI-ScaI fragment (1.2 kb) and the pUC18-derived PvuII-ScaI fragment (1.6 kb) were correctly ligated. Was selected and named pFY529V and used as an expression vector for the mutation library.
[0072]
(3) Preparation of mutation library
The reaction solution containing the mutagenized ethylenediamine-N, N'-disuccinate: ethylenediamine lyase gene obtained by PCR in step (1) is purified by ethanol precipitation according to a conventional method, and the precipitate is reconstituted in 70 μl of sterile water. Suspended. A 10-fold concentration restriction enzyme reaction buffer (10 μl), restriction enzyme NcoI (10 μl), and HindIII (10 μl) were added, and a cleavage reaction was performed at 37 ° C. for 12 hours. After the cleavage reaction, phenol extraction and chloroform extraction were performed, followed by ethanol precipitation. The precipitate was resuspended in 100 μl of sterile water to obtain a mutant DNA fragment solution. In parallel with this operation, 67 μl of sterilized water, 10 μl of 10-fold concentration restriction enzyme reaction buffer, 10 μl of restriction enzyme NcoI and 10 μl of HindIII were added to 3 μl of the expression vector pFY529V prepared in step (2). A time cleavage reaction was performed. After cleavage, phenol extraction and chloroform extraction were performed, and after purification by ethanol precipitation, the precipitate was resuspended in 10 μl of sterilized water to obtain a cleaved pFY529V solution. Ligation of the mutant DNA fragment and the expression vector pFY529V was performed using DNA Ligation Kit Ver.1 (Takara Shuzo). 3 μl of the above mutant DNA fragment solution, 1 μl of the cut pFY529V solution, 16 μl of solution A in the kit, and 4 μl of solution B in the kit were mixed, and ligation reaction was performed at 16 ° C. for 16 hours. Using the reaction solution after the ligation reaction, E. coli JM109 was transformed by the method described in Example 1 (step (4)) to obtain transformants carrying various mutation-introduced EDDSase genes. The same operation as described above was performed using the wild-type ethylenediamine-N, N′-disuccinate: ethylenediamine lyase gene amplified in step (1) as an insert fragment. Plasmids were extracted from the resulting transformant colonies, and the nucleotide sequence was confirmed using a fluorescent sequencer ALF II (manufactured by Amersham Biosciences). As a result, wild-type ethylenediamine-determined in Example 1 (step (7)) N, N′-disuccinic acid: identical to the base sequence of ethylenediamine lyase (SEQ ID NO: 2) {except for the fourth base change (A → G) due to introduction of the NcoI site of primer ED-01}. This plasmid was named pEDTrc9003, and Escherichia coli containing the plasmid, that is, JM109 / pEDTrc9003, was used as a control in the screening step of ethylenediamine-N, N′-disuccinate: ethylenediamine lyase with improved heat resistance described in (4) described later. PEDTrc9003 was deposited on February 5, 2003 as FERM P-19201 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture).
[0073]
(4) Heat-resistant ethylenediamine-N, N'-disuccinic acid: screening for ethylenediamine lyase
Mutant ethylenediamine-N, N'-disuccinate obtained in step (3): JM109 transformant containing ethylenediamine lyase gene and JM109 / pEDTrc9003 as a control were dispensed in 1.5 ml aliquots into 48-well multi-dish Each was inoculated and liquid cultured at 37 ° C. for 12 hours. The obtained culture was subjected to a heat treatment at a temperature of 50 ° C. for 30 minutes, and then the residual ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity was measured by the method described in Example 2. As a result of screening about 10000 transformants obtained in step (3), JM109 / pEDTrc9003 carrying wild-type ethylenediamine-N, N'-disuccinate: ethylenediamine lyase no longer completely lost its activity. In contrast, 4 strains in which the enzyme activity remained were obtained.
[0074]
(5) Identification of mutation
Mutation-introduced ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase gene, which mutation was introduced at which site in the 4 heat resistance improvement candidate strains obtained in step (4) In order to confirm, the following analysis was performed. Recombinant plasmid DNA contained in the 4 strains with improved heat resistance was purified using Flexi Prep (Amersham Biosciences), and the resulting recombinant plasmid DNAs were pEDTrcI-2, pEDTrcI-23, and pEDTrcJ, respectively. -05, named pEDTrcK-01. Moreover, the mutant enzymes contained in the respective plasmids were named I-2, I-23, J-05, and K-01. The nucleotide sequence of the mutated ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase gene contained in these recombinant plasmid DNAs was determined using a fluorescent sequencer ALF II (Amersham Biosciences). As a result of comparative analysis of the determined nucleotide sequence of each mutated ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase gene and the wild-type ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase base sequence (SEQ ID NO: 2), In pEDTrcI-2, the 166th isoleucine residue (ATC) is threonine (ACC), in pEDTrcI-23 the 120th lysine residue (AAA) is glutamic acid (GAA), and in pEDTrcJ-05 the 166th isoleucine residue (ATC) was found to be a single mutant in which serine (AGC) was substituted, and in pEDTrcK-01, the 365th alanine residue (GCC) was substituted with valine (GTC). , Shows changes in the base sequence encoding each amino acid].
[0075]
(6) Evaluation of heat resistance of single mutant
Four types of single mutant ethylenediamine-N, N'-disuccinic acid identified in step (5): ethylenediamine lyase (I-2, I-23, J-05, K-01) Examined. Single mutant ethylenediamine-N, N'-disuccinate: 4 strains of E. coli carrying ethylenediamine lyase (JM109 / pEDTrcI-2, JM109 / pEDTrcI-23, JM109 / pEDTrcJ-05, JM109 / pEDTrcK-01) and wild type Ethylenediamine-N, N'-disuccinic acid: E. coli JM109 / pEDTrc9003 containing ethylenediamine lyase was inoculated into 1.5 ml of LBAmp medium and shake-cultured at 30 ° C for 8 hours. 400 μl each was inoculated into 40 ml of the LBAmp medium prepared in the above, and cultured with shaking at 37 ° C. for 12 hours. 1.5 ml is collected from the obtained culture, collected by centrifugation, washed with 50 mM borate buffer (pH 9.0), and suspended in 1.5 ml of the same buffer. A suspension was prepared. This cell suspension was subjected to ultrasonic crushing to obtain a crude enzyme extract. The obtained crude enzyme extract was heat-treated at 40 ° C., 45 ° C., 50 ° C., 55 ° C., 60 ° C. and 65 ° C. for 30 minutes, and then immediately cooled to 4 ° C. After heat treatment, ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity was measured by the method described in Example 2, and the ones kept at 4 ° C. without heat treatment were controlled for their relative activity values (100 %) As a relative relative activity.
[0076]
The results are shown in FIG. In FIG. 5, the vertical axis represents the residual ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity, and the horizontal axis represents the treatment temperature. Wild type ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase completely lost its enzyme activity at 50 ° C, whereas single mutants I-2, I-23, J-05, K-01 Have a residual relative activity of 75% at 50 ° C, 85% at 50 ° C, 12% at 60 ° C, and 12% at 55 ° C, respectively. It was confirmed that there was an improvement.
[0077]
[Example 4] Production of multiple mutants and evaluation of heat resistance
(1) Production of multiple mutants
Multiple mutants were prepared by combining substitutions of amino acid residues in the four types of single mutants whose heat resistance was confirmed in Example 3. The mutation sites of the single mutants I-2, I-23, J-05, and K-01 are the 166th isoleucine residue, 120th lysine residue, 166th isoleucine residue, and the 366th alanine residue, respectively. By cutting the ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase gene DNA fragment with restriction enzymes EcoT22I, AccI, and ClaI, DNA fragments containing each of these mutation sites can be obtained (Fig. 6). The fragmented single mutant DNA fragment was replaced with the corresponding DNA fragment derived from a different single mutant and religated to produce a chimeric enzyme gene having double or triple mutation (FIG. 6). For example, the double mutant Ch-4 is prepared by first cutting pEDTrcI-23 containing the gene encoding the single mutant I-23 with restriction enzymes NcoI and AccI, and the shorter DNA fragment resulting from the cleavage. (About 0.5 kb) was excised by agarose gel electrophoresis. At the same time, pEDTrcJ-05 containing the gene encoding the single mutant J-05 was cleaved with restriction enzymes NcoI and AccI, and the longer DNA fragment (about 3.8 kb, including the vector) resulting from the cleavage was subjected to agarose gel electrophoresis. It cut out by. Both DNA fragments were recovered using DNA PREP (manufactured by Diatron), ligated using DNA Ligation Kit Ver. 1 (manufactured by Takara Shuzo), and then transformed into E. coli JM109 according to a conventional method. A plasmid was extracted from the resulting transformant, cleaved with restriction enzymes NcoI and AccI, and confirmed to be correctly ligated by agarose gel electrophoresis. This plasmid DNA was designated as pEDTrcCh-4, and the chimeric enzyme was designated as Ch- It was named 4. Similarly, a plasmid DNA obtained by ligating a single mutant I-23-derived NcoI-AccI fragment (about 0.5 kb) and a single mutant I-2-derived NcoI-AccI fragment (about 3.8 kb, including the vector) is called pEDTrcCh -6, the chimeric enzyme was named Ch-6. Using a similar technique, plasmid DNA obtained by ligating a single mutant J-05-derived NcoI-ClaI fragment (about 1 kb) and a single mutant K-01-derived NcoI-ClaI fragment (about 3.3 kb, including vector) pEDTrcCh-1, with the chimeric enzyme Ch-1, linking the single mutant I-2 derived NcoI-ClaI fragment (approximately 1 kb) to the single mutant K-01 derived NcoI-ClaI fragment (approximately 3.3 kb, including vector) The plasmid DNA thus obtained was designated pEDTrcCh-2, and the chimeric enzyme was designated Ch-2. For the triple mutant, the single mutant I-23-derived NcoI-AccI fragment (approximately 0.5 kb), the single mutant J-05-derived AccI-ClaI fragment (approximately 0.5 kb), and the single mutant K-01-derived NcoI The plasmid DNA obtained by ligating the -ClaI fragment (about 3.3 kb, including the vector) is pEDTrcCh-8, the chimeric enzyme is Ch-8, and the single mutant I-23-derived NcoI-AccI fragment (about 0.5 kb) Plasmid DNA obtained by ligating the mutant I-2-derived AccI-ClaI fragment (approximately 0.5 kb) and the single mutant K-01-derived NcoI-ClaI fragment (approximately 3.3 kb, including the vector) is pEDTrcCh-10, a chimeric enzyme Was Ch-10. The structure of the chimeric enzyme thus obtained is shown in FIG.
[0078]
(2) Evaluation of heat resistance of multiple mutants
6 types of multiple mutant ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase (Ch-4, Ch-6, Ch-1, Ch-2, Ch-8, Ch-10) prepared in step (1) The heat resistance of was examined. Multiple mutant ethylenediamine-N, N'-disuccinate: 6 strains of E. coli carrying ethylenediamine lyase (JM109 / pEDTrcCh-4, JM109 / pEDTrcCh-6, JM109 / pEDTrcCh-1, JM109 / pEDTrcCh-2, JM109 / pEDTrcCh- 8, JM109 / pEDTrcCh-10), and wild-type ethylenediamine-N, N'-disuccinate: ethylenediamine lyase-bearing E. coli JM109 / pEDTrc9003 was inoculated into 1.5 ml of LBAmp medium and cultured with shaking at 30 ° C. for 8 hours. Thereafter, 400 μl of each culture was inoculated into 40 ml of LBAmp medium prepared in a 500 ml Erlenmeyer flask and cultured with shaking at 37 ° C. for 12 hours. 1.5 ml is collected from the obtained culture, collected by centrifugation, washed with 50 mM borate buffer (pH 9.0), and suspended in 1.5 ml of the same buffer. A suspension was prepared. This cell suspension was subjected to ultrasonic crushing to obtain a crude enzyme extract. The obtained crude enzyme extract was heat-treated at 40 ° C., 45 ° C., 50 ° C., 55 ° C., 60 ° C. and 65 ° C. for 30 minutes, and then immediately cooled to 4 ° C. After the heat treatment, the ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity was measured by the method described in Example 2, and the ones kept at 4 ° C. without heat treatment were used as controls for their relative activity values. Residual relative activity was determined. The results are shown in FIG. In FIG. 7, the vertical axis represents the residual ethylenediamine-N, N′-disuccinic acid: ethylenediamine lyase activity, and the horizontal axis represents the treatment temperature. Wild-type ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase completely lost its enzyme activity at 50 ° C, whereas multiple mutants Ch-4, Ch-6, Ch-1, Ch-2 , Ch-8 and Ch-10 have a residual relative activity of 72% at 60 ° C, 89% at 55 ° C, 56% at 60 ° C, 25% at 55 ° C, 90% at 60 ° C, and 97% at 55 ° C. It was confirmed that the heat resistance of these multiple mutants was improved compared to the wild type and single mutants.
[0079]
【The invention's effect】
According to the present invention, the Breven Dimonas Diminuta ( Brevundimonas diminuta ) MR-E001 strain-derived wild-type ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase base sequence and amino acid sequence are provided. Also provided are the base sequence and amino acid sequence of modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase derived from wild-type ethylenediamine-N, N′-disuccinate: ethylenediamine lyase. Furthermore, wild-type and modified ethylenediamine-N, N′-disuccinic acid: a recombinant DNA containing an ethylenediamine lyase gene, a transformant or a transductant containing the recombinant DNA, a transformant or a transductant A process for producing the diaminoalkylene-N, N′-disuccinic acid used is provided. According to the present invention, it is possible to efficiently produce diaminoalkylene-N, N′-disuccinic acid.
[0080]
[Sequence Listing]

[0081]
[Sequence Listing Free Text]
Sequence number 1: Xaa represents Met or deletion
Sequence number 3: Synthetic DNA
Sequence number 4: Synthetic DNA
Sequence number 5: Synthetic DNA
Sequence number 6: Synthetic DNA
[Brief description of the drawings]
1 is a restriction enzyme map of plasmid pEDS9001.
FIG. 2: Restriction enzyme map of plasmid pEDS9003.
FIG. 3 Restriction enzyme map of plasmid pEDS9020.
FIG. 4 is a construction diagram of an expression vector pFY529V.
FIG. 5 is a view showing evaluation of heat resistance of a single mutant.
FIG. 6 is a schematic diagram showing the structure of a single mutant and multiple mutants. □ indicates a mutation of T497C (Ile166Thr), Δ indicates a mutation of A358G (Lys120Glu), ○ indicates a mutation of T497G (Ile166Ser), and ◇ indicates a mutation of C1094T (Ala365Val).
FIG. 7 shows the evaluation of heat resistance of multiple mutants.

Claims (6)

In the amino acid sequence described in SEQ ID NO: 1, at least one of substitution of the 120th lysine residue with glutamic acid, substitution of the 166th isoleucine residue with serine or threonine, and substitution of the 365th alanine residue with valine A protein having an amino acid sequence having two substitutions and having ethylenediamine-N, N′-disuccinate: ethylenediamine lyase activity. A genetic DNA encoding the protein according to claim 1 . Recombinant DNA obtained by inserting the gene DNA of claim 2 into vector DNA. A transformant or transductant comprising the recombinant DNA according to claim 3 . Ethylenediamine-N, N'-disuccinic acid characterized by collecting ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase from the culture obtained by culturing the transformant or transductant according to claim 4 : Ethylenediamine lyase production method. Diaminoalkylene-N, N'-disuccinic acid is collected from the reaction product obtained by reacting fumaric acid and diamine in the presence of the transformant or transductant according to claim 4. A process for producing alkylene-N, N′-disuccinic acid.

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