JP4085486B2 - A novel lysine residue-specific enzyme-producing bacterium - Google Patents

Description

【００１７】
また、グラム陰性桿菌同定キット ID 32GN（ビオメリュー社製）を用いた自動細菌検査装置ATB Expression（ビオメリュー社製）による同定試験及び腸内細菌以外のグラム陰性菌同定キットAPI 20NE（ビオメリュー社製）を用いた同定結果は以下の通りである。
ATB Expressionによる資化性試験：陽性；Ｎ−アセチルグルコサミン、クエン酸ナトリウム、３−ヒドロキシ酪酸及びＬ−プロリン。陰性；ラムノース、Ｄ−リボース、イノシトール、シュークロース、イタコン酸、スベリン酸、マロン酸ナトリウム、酢酸ナトリウム、乳酸、Ｌ−アラニン、Ｄ−マンニトール、グルコース、サリシン、Ｄ−メリビオース、Ｌ−フコース、Ｄ−ソルビトール、Ｌ−アラビノース、プロピオン酸、ｎ−カプリン酸、ｎ−吉草酸、Ｌ−ヒスチジン塩酸塩、５−ケトグルコン酸、グリコーゲン、ｍ−ヒドロキシ安息香酸、２−ケトグルコン酸、ｐ−ヒドロキシ安息香酸及びＬ−セリン。
また、ATB Expressionによるエスクリンの加水分解試験、ＤＮＡの分解試験及びレシチナーゼ試験は全て陽性であった。
【００１８】
API 20NEによる資化性試験：陽性；グルコース、Ｄ−マンノース、Ｎ−アセチル−Ｄ−グルコサミン、マルトース、ＤＬ−リンゴ酸及びクエン酸ナトリウム。陰性；Ｌ−アラビノース、Ｄ−マンニトール、グルコン酸カリウム、ｎ−カプリン酸、アジピン酸及び酢酸フェニル。
また、API 20NEによる硝酸塩還元試験、インドール生成試験、グルコース発酵性試験、アルギニンジヒドロラーゼ試験及び尿素分解試験は陰性であり、エスクリン分解試験、ゼラチン液化試験、ＰＮＰＧ試験及びオキシダーゼ試験は陽性であった。
【００１９】
この結果、本菌株はシュークロースを資化しなかったが、アクロモバクター リティカス M497-1はシュークロースを資化することから、これらは別の菌株であることが示唆された。資化性の異同を観るため、更に両菌をバイオローグシステム（Biology社、Microstation システム、USA）による９５種類の炭素源の資化性について比較検討した。
【００２０】
その結果、α−シクロデキストリン、ギ酸、フェニルエチルアミン、プトレッシン、２−アミノエタノール、２,３−ブタンジオール、セバシン酸及びγ−アミノ酪酸の８種の炭素源について、キサントモナス属IB-9374では陽性、アクロモバクター リティカス M497-1では陰性と、相違が観られた。
この結果より、キサントモナス属IB-9374は、アクロモバクター リティカス M497-1とは異なる菌株であることが明らかとなった。
【００２１】
アクロモバクター リティカス M497-1の他に本菌株と類似の生化学的性質を有するものにステノトロフォモナス マルトフィリア（Stenotrophomonas maltophilia）がある。この菌株の生育には、メチオニン及びグルタミン酸が必要であるという報告がなされている。そこで、メチオニン及びグルタミン酸の存在、非存在による生育への影響を調べた。その結果を図１及び図２に示す。尚、図１中の−○−は、メチオニンを培地に加えたときの、また、--○--はメチオニンを培地に加えないときのキサントモナス属IB-9374の生育曲線を夫々示し、−◆−は、メチオニンを培地に加えたときの、また、--◆--はメチオニンを培地に加えないときの、アクロモバクター リティカス M497-1の生育曲線を夫々示す。図２中の−○−は、グルタミン酸を培地に加えたときの、また、--○--はグルタミン酸を培地に加えないときのキサントモナス属IB-9374の生育曲線を夫々示し、−◆−は、グルタミン酸を培地に加えたときの、また、--◆--はグルタミン酸を培地に加えないときのアクロモバクター リティカス M497-1の生育曲線を夫々示す。
【００２２】
この結果から、メチオニン及びグルタミン酸によるキサントモナス属IB-9374への生育の影響は認められなかった。この結果より、キサントモナス属IB-9374は、ステノトロフォモナス マルトフィリアとも別の菌であることが判明した。
【００２３】
さらに、本菌株の１６Ｓ−ｒＤＮＡ塩基配列分析を行い、既知の微生物の塩基配列との相同性を検討したところ、ステノトロフォモナス マルトフィリア LMG 11087［（Stenotrophomonas maltophilia LMG 11087）、旧名：キサントモナスマルトフィリア（Xanthomonas maltophilia）］、キサントモナス バシコラ LMG 736^T（Xanthomonas vasicola LMG 736^T）、キサントモナス アルボリコラ 747^T（Xanthomonas arboricola 747^T）、キサントモナス カムペストリス LMG 568^T（Xanthomonas campestris LMG 568^T）、キサントモナス カッサバエ LMG 673^T（Xanthomonas cassavae LMG 673^T）、キサントモナス ホルトルム LMG 733^T（Xanthomonas hortorum LMG 733^T）、キサントモナス オリイザエ LMG 5047^T（Xanthomonas oryzae LMG 5047^T）、キサントモナス ピシ LMG 847^T（Xanthomonas pisi LMG 847^T）、キサントモナス ベシカトリア LMG 911^T（Xanthomonas vesicatoria LMG 911^T）及びキサントモナス カムペストリス pv.カムペストリス Xc17（Xanthomonas campestris pv.campestris Xc17）の１０種が、本菌株の16S-rDNAの塩基配列と高い相同性（９３％以上）を示した。尚、塩基配列分析の相同性は、各種細菌の16S-rDNA配列を用い、遺伝子解析ソフトウェアGENETYX-MAC7.3（SOFTWARE DEVELOPMENT社製）で計算を行った。
【００２４】
以上の結果より、本菌株はキサントモナス属の新菌種であると判断し、本菌株をキサントモナス属IB-9374と命名した。
【００２５】
実施例３．各種培地におけるキサントモナス属IB-9374のXan.Lys-C産生能の検討種々の培地を用い、キサントモナス属IB-9374のXan.Lys-C産生能を検討した。即ち、酵母エキス、カサミノ酸、肉エキス、ミルクカゼイン、ソイビーンパウダー、シュークロース、グルコース、ポリペプトン、KH₂PO₄、K₂HPO₄、MgSO₄・7H₂Oを所定濃度含有する各種培地でキサントモナス属IB-9374を３０℃所定時間培養した後、培養上清を採取し、培養上清のアミダーゼ活性を測定して、その値よりXan.Lys-C産生能を比較した。
【００２６】
その結果、０.５％ミルクカゼイン、１.０％シュークロース、１.０％肉エキス、０.０１％KH₂PO₄、０.０１％K₂HPO₄及び０.０１％MgSO₄・7H₂Oを含有するｐＨ７.２の培地（以下、Ｂ培地と呼ぶ）が最も好ましいことが判った。Ｂ培地を用いると、１５０時間で培養上清１ｍＬ当たり０.１単位（ｕ）、２５０時間で０.１８ｕの最も高いアミダーゼ活性を示した。Ｂ培地を用いた場合のキサントモナス属IB-9374の生育曲線（−○−）と所定時間培養後の培養上清のアミダーゼ活性を示す曲線（--◇--）を図３に示す。尚、対照として、アクロモバクターリティカス M497-1を同じＢ培地中で培養した場合の生育曲線（−□−）と所定時間培養後の培養上清のアミダーゼ活性を示す曲線（--×--）を図３に併せて示す。 図３の結果から、アクロモバクター リティカス M497-1のそれと比較した場合、培養１００時間で約５倍、２５０時間で約４倍の高い活性を示すことがわかる。また、図３の結果より、本発明のキサントモナス 属IB-9374が、アクロモバクター リティカス M497-1と異なる菌株であることが判明した。
【００２７】
実施例４．
実施例１で単離した菌を培養して得られるXan.Lys-Cを以下の手法により精製した。
（１）培養液１０Ｌを５０〜９０％の硫安分画し、CM-トヨパール処理、DEAE-セルロース処理、AHセファロース 4Bカラムクロマトグラフィー、STIアフィニティークロマトグラフィー、セファデックス G-50カラムクロマトグラフィーの６段階の操作で精製を行った。
操作方法の詳細を以下に示す。尚、精製操作は、全て４℃で行った。
【００２８】
▲１▼培養濾液の硫安分画
１５０時間培養した培養濾液９２１０ｍＬに５０％飽和度（温度４℃）になるように硫安を加えた。次にこれをバッチ式遠心分離（３１,０００ｇ、２０分、４℃）して得られた上清液１０,２９５ｍＬに９０％飽和度となるように硫安を加えた。続いて、上記と同様に遠心分離し、得られた沈殿を適当量の１０ｍＭ トリス塩酸緩衝液（ｐＨ７.０；以下、緩衝液Ａと呼ぶ）に溶解させた後、緩衝液Ａに対して４℃で７２時間透析した。
【００２９】
▲２▼CM-トヨパール 650M処理
透析して得られた酵素液１１２０ｍＬを緩衝液Ａで平衡化したCM-トヨパール 650M 約５２６.７ｇ（ｗｅｔ）に加えて室温で穏やかに１時間攪拌した。次いで、ガラスフィルター（直径１１.５cm、２５Ｇ−３）で樹脂を除去し、更に、こ のフィルター上の樹脂を緩衝液Ａ（ｐＨ７.０）１０００ｍＬで洗浄し、濾液と 洗浄液とを合わせ、未吸着画分であるXan.Lys-Cを含む酵素液２４００ｍＬを得 た。
【００３０】
▲３▼DEAE-セルロース SH処理
▲２▼で得られた褐色の酵素液２４００ｍＬをダイヤフローセル（膜：ＰＭ−１０）で１２００ｍＬに濃縮後、１０ｍＭ トリス塩酸緩衝液（ｐＨ８.０；以下、緩衝液Ｂと呼ぶ）で充分透析した。その後、この酵素液１２５０ｍＬに緩衝液Ｂで平衡化したDEAE-セルロース SH 約４７５ｇ（ｗｅｔ）を加えて、室温で穏やかに攪拌しながら着色タンパク質をはじめとする他の不純タンパク質を吸着させた。１時間後、ガラスフィルター（直径１１.５cm、２６Ｇ−３）で樹脂を除去し、次いで緩衝液Ｂ１０００ｍＬで樹脂を洗浄した。濾液と洗浄液とを合わせたXan.Lys-Cを含む溶液２４００ｍＬをダイヤフローセルにより２３７ｍＬに濃縮し、２ｍＭ トリス塩酸緩衝液（以下、緩衝液Ｃと呼ぶ）に充分透析した。
【００３１】
▲４▼AH セファロース 4B カラムクロマトグラフィー
▲３▼で得られた透析した酵素液２７０ｍＬを緩衝液Ｃで充分平衡化したAH セファロース 4B カラムクロマトグラフィー（４.１×１８cm）に吸着させた。同緩衝液で充分洗浄後、緩衝液Ｃ（ｐＨ８.０）１０００ｍＬと１.０Ｍ NaClを含む緩衝液Ｃ１０００ｍＬを用いて直線濃度勾配で溶出した。その結果、Xan.Lys-Cは完全に吸着され、０.５Ｍ NaClで溶出された。その活性画分を集め、５０ｍＭトリス塩酸緩衝液（ｐＨ８.０；以下、緩衝液Ｄと呼ぶ）に充分透析した酵素液７２０ｍＬをダイヤフローセルにより２８０ｍＬに濃縮した。
【００３２】
▲５▼STI セファロース 4B アフィニティーカラムクロマトグラフィー
▲４▼で得られた酵素液２８０ｍＬを２回に分けてクロマトグラフィーを行った。すなわち、酵素液１４０ｍＬを緩衝液Ｄで平衡化したSTI セファロース 4B アフィニティーカラム（３.２×６.０cm）に吸着させ、同緩衝液５００ｍＬで充分洗浄後、０.５Ｍ NaClを含む５０ｍＭ グリシン−塩酸緩衝液（ｐＨ４.５）で溶出させた。活性画分を集め、緩衝液Ｃに充分透析し、ダイヤフローセルにより濃縮する操作を２回行った。
尚、溶出時には分画用試験管に予め１Ｍ トリス塩酸緩衝液（ｐＨ８.０）０.１ｍＬを入れておいて分画を行った。
【００３３】
▲６▼セファデックス G-50 カラムクロマトグラフィー
▲５▼で得られた濃縮酵素液１０ｍＬを２回に分けて、ゲル濾過を行った。すなわち、濃縮酵素液５ｍＬを０.１Ｍ NaClを含む緩衝液Ｃで平衡化したセファデックス G-50 カラムクロマトグラフィー（２.０×９２cm）にかけ、Xan.Lys-Cの活性画分を集めた。残りの酵素液５ｍＬも同様にゲル濾過を行い、活性画分を集めて１回目の活性画分と合わせ、緩衝液Ｃに充分透析した。この透析した酵素液を、ダイヤフローセルにより濃縮し５ｍＬとして、−１５℃で冷凍保存した。
【００３４】
段階毎の回収率及び比活性等の値を表２に示した。
【００３５】
【表２】

【００３６】
最終的な回収率は約６８％となり、比活性は約９３０倍に上昇した。この時点で得られた酵素タンパク質量は、培養液１０Ｌ当たり０.１ｇで、アクロモバクター リティカス M497-1の培養液から生産されるＡＰＩのタンパク質量と比較すると約７倍量に相当した。
【００３７】
実施例５．キサントモナス属IB-9374が産生するXan.Lys-Cの性質の検討
Xan.Lys-Cについて、アミダーゼ活性、エステラーゼ活性又は／及びプロテイナーゼ活性を指標として、至適ｐＨ、至適温度、安定性、各種化合物の影響、アルカリ金属イオン及びアルカリ土類金属イオンの影響並びにタンパク質変性剤の影響を検討した。また、Xan.Lys-Cのアミノ酸組成及びＮ−末端アミノ酸配列についても検討した。
尚、アミダーゼ活性は、実施例１と同様にして測定した。また、エステラーゼ活性及びプロテイナーゼ活性の測定方法を以下に示す。
【００３８】
▲１▼エステラーゼ活性の測定法
石英セルに８０ｍＭ トリス塩酸緩衝液（ｐＨ８.０）１.５ｍＬ、５ｍＭ N-トシル-L-リシンメチルエステル（以下、Tos-Lys-OMeと略記する）０.２ｍＬ、純水１.２ｍＬを加えて３０℃で５分間保った後、酵素液０.１ｍＬを添加して反応させた。酵素添加と同時に５秒間攪拌し、２４７ｎｍでの吸光度変化を記録し、この結果に基づいて酵素活性を測定した。対照試料は、酵素溶液を純水に代えたものを用いた。
尚、酵素活性の単位は、上記の条件下で１分間当たりに１μｍｏｌのＮ−トシル−Ｌ−リシン（分子吸光係数９６０）を遊離する酵素活性を、１単位と定義した。
【００３９】
▲２▼プロテイナーゼ活性の測定法
反応用と対照用の２本の二股消化管の一方の股に酵素溶液０.２ｍＬ、他の股に基質緩衝液（０.８４％カゼインを含む６０ｍＭトリス塩酸緩衝液、ｐＨ９.０）１ｍＬを分注し、４０℃の恒温水槽中に５分間保った後、反応用二股消化管の両股内の液を良く混合した。そして、混合の時から正確に２０分間４０℃の恒温水槽に保って反応させた後、タンパク質沈殿試薬（０.１１Ｍトリクロロ酢酸と０.２２Ｍ酢酸ナトリウムを含む混合液を酢酸でｐＨ３.０に調整したもの）１ｍＬを加えて良く混合した。そして、再び４０℃の恒温水槽に３０分間保ち、沈殿の生成を完成させた後、濾紙（Advantec Ｎｏ.４Ａ）を用いて自然濾過させた。一方、対照用の二股消化管の液は、混合せずにそのまま同時間保った後、タンパク質沈殿試薬１ｍＬを酵素液に加えてからよく混合して反応用二股消化管と同様に処理した。得られた濾液試料は島津製分光光度計（ＵＶ−２５０型）を用いて、対照試料に対して２７５ｎｍにおける吸光度を測定した。
尚、酵素活性の単位は、上記の条件下（反応温度４０℃、反応ｐＨ９.０、基質濃度０.７％、反応時間２０分間）で１分間当たりにＬ−チロシン１μｍｏｌ相当量の生成物を与える酵素活性を、１単位と定義した。
【００４０】
（１）Xan.Lys-Cの至適ｐＨの検討
Bz-Lys-pNA、Tos-Lys-OMe及びカゼインに対するアミダーゼ活性、エステラーゼ活性及びプロテイナーゼ活性を指標とし、Xan.Lys-Cの至適ｐＨについて検討した。
アミダーゼ活性についての結果を図４に、エステラーゼ活性についての結果を図５に、プロテイナーゼ活性についての結果を図６に夫々示す。尚、図４に於いて、−●−は１００ｍＭトリス塩酸緩衝液（ｐＨ７.３−１０.０）を用いたときのアミダーゼ活性を、また、--○--は１００ｍＭ 2-アミノ-2-メチル-1,3-プロパンジオール−塩酸緩衝液（ｐＨ７.７−１０.６）を用いたときのアミダーゼ活性を夫々示す。また、図６に於いて、−●−は５０ｍＭ トリス塩酸緩衝液（ｐＨ７.０〜９.０）を用いたときのプロテイナーゼ活性を、また、--△--は５０ｍＭ ほう酸塩緩衝液（ｐＨ８.０〜１１.５）を用いたときのプロテイナーゼ活性を夫々示す。尚、図５のデータは１００ｍＭトリス塩酸緩衝液（ｐＨ７.１〜９.０）を用いて得られたものである。
【００４１】
その結果、アミダーゼ活性及びエステラーゼ活性の至適ｐＨは、それぞれ９.０〜９.６、８.０〜８.５であった。一方、プロテイナーゼ活性のそれは８.５〜１０.５と幅広い曲線が得られた。これらはＡＰＩのアミダーゼ活性（ｐＨ９.０〜９.５）、エステラーゼ活性（ｐＨ７.８〜８.２）及びプロテイナーゼ活性（ｐＨ８.５〜１０.７）の至適ｐＨと同じ傾向を示した。
（２）Xan.Lys-Cの至適温度
Bz-Lys-pNA、Tos-Lys-OMe及びカゼインに対するアミダーゼ活性、エステラーゼ活性及びプロテイナーゼ活性を指標とし、Xan.Lys-Cの至適温度を検討した。結果を図７に示す。尚、図７中の−●−はXan.Lys-Cのアミダーゼ活性を、--◆--はXan.Lys-Cのプロテイナーゼ活性を、--△--はXan.Lys-Cのエステラーゼ活性を夫々示す。
【００４２】
これらの結果から、何れの場合も５０℃で最大活性を示すことが判った。
【００４３】
（３）Xan.Lys-Cの安定性
▲１▼ｐＨ安定性
Bz-Lys-pNAに対するアミダーゼ活性を指標とし、ｐＨを変化させたときのXan.Lys-Cの安定性を検討した。
酵素（１μｇ）を所定ｐＨの各１００ｍＭの酢酸緩衝液、 トリス塩酸緩衝液、ＡＭＰ緩衝液及びリン酸緩衝液の夫々０.１ｍｌに入れ、４℃で２４時間放置後、Bz-Lys-pNAに対する活性の残存率（％）を測定した。
結果を図８に示す。尚、図８に於いて、−●−は１００ｍＭ酢酸緩衝液でのアミダーゼ活性を、−×−は１００ｍＭトリス塩酸緩衝液でのアミダーゼ活性を、−◆−は１００ｍＭ ＡＭＰ緩衝液でのアミダーゼ活性を、また、−◇−は１００ｍＭリン酸緩衝液でのアミダーゼ活性を夫々示す。
【００４４】
この結果から、Xan.Lys-Cの安定領域はｐＨ４.５〜１０.０であることが判る。これはＡＰＩの安定領域（ｐＨ４.０〜１０.７）と比べると少し狭い傾向が認められた。
【００４５】
▲２▼温度安定性
Bz-Lys-pNAに対するアミダーゼ活性を指標とし、温度を変化させたときのXan.Lys-Cの安定性を検討した。
酵素（０.６μｇ）を１００ｍＭのトリス塩酸緩衝液（ｐＨ８.０）０.１ｍｌに溶解したものを各種温度（２０〜６０℃）で３０分間放置後、急冷し、Bz-Lys-pNAに対する活性の残存率（％）を測定した。
結果を図９に示す。
【００４６】
この結果から、Xan.Lys-Cは４０℃までは安定であったが、６０℃で完全に失活することが判った。
【００４７】
（４）Xan.Lys-Cに対する各種化合物の影響
Bz-Lys-pNA及びTos-Lys-OMeに対するアミダーゼ活性、エステラーゼ活性を指標とし、Xan.Lys-Cに対する金属キレート試薬、ＳＨ試薬、及び還元剤等の影響を検討した。
即ち、酵素と各種のペプチダーゼ阻害剤を４０ｍＭ トリス塩酸緩衝液（ｐＨ８.０）で、３０分間３０℃で前処理した後、Xan.Lys-Cのアミダーゼ活性及びエステラーゼ活性を測定することにより、その影響を調べた。尚、アミダーゼ活性法を測定する場合にはBz-Lys-pNAが０.０５〜０.１６ｍＭの範囲になるように、エステラーゼ活性を測定する場合にはTos-Lys-OMeが０.０５〜０.５ｍＭの範囲になるようにした。
結果を表３に示す。尚、表３に於いて、ＥＤＴＡはエチレンジアミン−４酢酸（Ethylenediaminetetraacetic acid）の略、２−ＭＥは、２−メルカプトエタノールの略、ＮＥＭはN-エチルマレイミド（N-Ethylmaleimide）の略、ＭＩＡはモノイオド酢酸（Monoiodoacetic acid）の略、ＰＣＭＢはp-クロロメルクリ安息香酸（p-chloromercuribenzoic acid）の略、ＤＦＰはジイソプロピルフルオロリン酸（Diisopropyl fluorophosphate）の略、ＰＭＳＦはフェニルメタンスルホニルフルオリド（Phenylmethansulfonyl fluoride）の略、ＴＬＣＫはNα-トシル-L-リシンクロロメチルケトン（Nα-Tosyl-L-lysyl chloromethyl ketone）の略、ＴＡＣＫはNα-トシル-L-アルギニルクロロメチルケトン（Nα-Tosyl-L-arginyl chloromethyl ketone）の略、ＴＰＣＫはNα-トシル-L-フェニルアラニルクロロメチルケトン（Nα-Tosyl-L-phenylalanyl chloromethyl ketone）の略を表し、以下同様に略記する。
【００４８】
【表３】

【００４９】
表３の結果から、Xan.Lys-Cの活性が金属キレート試薬（ＥＤＴＡ、ｏ-フェナントロリン）、還元剤（システイン、2-ＭＥ）、及びＳＨ試薬（ＮＥＭ，ＭＩＡ，ＰＣＭＢ）によって阻害されないことより、Xan.Lys-Cは、メタルエンドペプチダーゼ及びシステインエンドペプチダーゼの何れでもないとみなされた。一方、ＤＦＰ及びＰＭＳＦがXan.Lys-Cのアミダーゼ活性及びエステラーゼ活性を強く阻害することから、Xan.Lys-Cはセリンエンドペプチダーゼであるとみなされた。更に、Xan.Lys-Cはトリプシンの活性中心にあるＨｉｓ残基に対して特異的に反応するＴＬＣＫによって阻害されたが、キモトリプシンのＨｉｓ残基に特異的であるＴＰＣＫによって阻害されなかったことから、Xan.Lys-Cはトリプシン様セリンエンドペプチダーゼあると認められた。また、トリプシンに対して、ＴＬＣＫよりも強力に阻害するＴＡＣＫによっては殆ど影響が認められなかった。このことはXan.Lys-Cがリシン残基に対して強い親和性を有していることを示唆している。
【００５０】
（５）Xan.Lys-Cに対する金属イオンの影響
Bz-Lys-pNA及びTos-Lys-OMeに対するアミダーゼ活性、エステラーゼ活性を指標とし、Xan.Lys-Cに対する金属イオンの影響を検討した。
即ち、酵素液と所定の金属イオンとを含有する４０ｍＭ トリスイオン酸緩衝液（ｐＨ８.０）を３０分間３０℃で前処理した後、Bz-Lys-pNA及びTos-Lys-OMeに対する残存活性を測定した。結果を表４に示す。
【００５１】
【表４】

【００５２】
Xan.Lys-Cのアミダーゼ活性及びエステラーゼ活性は１ｍＭ Ｃａ²⁺、Ｍｇ²⁺、Ｍｎ²⁺、Ｃｄ²⁺、Ｎｉ²⁺、Ｓｒ²⁺、Ｃｏ²⁺、０.１ｍＭ Ｈｇ²⁺によって殆ど影響は受けなかった。しかし、Xan.Lys-Cは１ｍＭ Ｂａ²⁺によって弱く、１ｍＭ Ｚｎ²⁺によって強く阻害され、特に後者の金属イオンの場合にはエステラーゼ活性に対して強い阻害が認められた。
【００５３】
（６）Xan.Lys-Cに対するアルカリ金属イオン及びアルカリ土類金属イオンの影響について
Bz-Lys-pNA及びTos-Lys-OMeに対するアミダーゼ活性、エステラーゼ活性を指標とし、Xan.Lys-Cに対するアルカリ金属イオン、Ｂａ²⁺、及びＮＨ₄ ⁺の影響を検討した。
即ち、３０℃のセル中に、８０ｍＭトリス塩酸緩衝液（ｐＨ８.０）１.５ｍｌ、各種濃度の金属水溶液１.０ｍＬ、各種濃度のTos-Lys-OMe又はBz-Lys-pNA０.４ｍＬを順次加えた後、酵素溶液０.２５μｇ／０.１ｍＬ（或いは１.０μｇ／０.１ｍＬ）をすばやく加え、３０℃で５分間反応させたものについての活性を調べた。その結果の一例としてＢａ²⁺のXan.Lys-Cに対する阻害作用のLineweaver-Burkプロットを図１０に示す。
その結果、何れの活性も、Ｂａ²⁺、ＮＨ₄ ⁺、Ｃｓ⁺、Ｒｂ⁺，Ｋ⁺、Ｎａ⁺、Ｌｉ⁺によってこの順序で拮抗的に阻害されることが明らかとなった。
【００５４】
また、阻害作用が認められた各種イオンの拮抗的阻害定数（Ｋｉ）をＤｉｘｏｎプロットにより算出した結果を、表５に示す。尚、表５にはＡＰＩについての結果も併せて示す。
【００５５】
【表５】

【００５６】
その結果、アルカリ金属イオンについては、阻害作用はイオン半径が小さくなるに従って強くなることが判る。しかし、アルカリ土類金属についてはＢａ²⁺のみに強い阻害が認められ、Ｍｇ²⁺、Ｃａ²⁺、Ｓｒ²⁺の影響は殆ど認められなかった（表５には未記載）。これらの結果は、ＡＰＩのそれと同じであった。
【００５７】
（７）Xan.Lys-Cに対するタンパク質変性剤の影響
Tos-Lys-OMeに対するエステラーゼ活性を指標とし、Xan.Lys-Cに対する尿素、塩酸グアニジン及びドデシル硫酸ナトリウム（以下、ＳＤＳと略記する）の影響を検討した。
即ち、各種濃度の尿素、塩酸グアニジン又はＳＤＳと、酵素（５μｇ）を含む４０ｍＭ トリス塩酸緩衝液（ｐＨ８.０）０.５ｍｌを３０分間３０℃で前処理した後、その溶液０.１ｍＬ（１μｇ）を、１ｍＭ Tos-Lys-OMeを含む４０ｍＭ トリス塩酸緩衝液（ｐＨ８.０）２.９ｍＬに加え５分間反応させ、エステラーゼ活性の残存率（％）を調べた。
尿素についての結果を図１１に、塩酸グアニジンについての結果を図１２に、ＳＤＳについての結果を図１３に夫々示す。
【００５８】
尚、４Ｍ尿素、０.２Ｍ塩酸グアニジン又は０.１％ＳＤＳを含む４０ｍＭ トリス塩酸緩衝液（ｐＨ８.０）中で酵素を、２０℃で２０分間前処理した場合のエステラーゼ活性の残存率は、それぞれ１００％、５０％、及び８５％と、ＡＰＩと同様の傾向を示した。
【００５９】
（８）Xan.Lys-Cの基質特異性の検討
Xan.Lys-Cの各種合成基質に対する特異性を検討した。
これらの結果を表６に示す。
尚、Kcat/Km値はLineweaver-Burkプロットから算出し、Kcat値は酵素の分子量を２８０００として算出した。
【００６０】
【表６】

【００６１】
表６の結果より、エステル基質の中では、Tos-Lys-OMeが本酵素によって最もよく加水分解され、そのKcat/Km値は、対応するTos-Arg-OMeのそれの約５００００倍の高い値を示した。また、Bz-Orn-OMeのKcat/Km値は、Tos-Arg-OMeのそれと比較すると、約１/１２００であり、あまり加水分解されなかった。これらのエステル基質に対するKcat/Km値をＡＰＩについてのそれと比較するとほぼ同じ値を示していた。
また、アニリド基質の中では、Bz-Lys-pNAとAc-Lys-pNAがよく加水分解され、Lys-pNAも若干加水分解された。一方、Bz-Arg-pNAとArg-pNAは、酵素濃度を８０倍以上高くして２４時間反応させてみたが、全く分解されなかった。更に、Suc-Ala-pNA、Boc-His-pNA、Ac-Phe-pNA、Bz-Tyr-pNA、Ac-Leu-pNAも同様に反応させたが、これらも全く加水分解されなかった（表６には未記載）。これらのアニリド基質に対するKcat/Km値もＡＰＩについてのそれとほぼ同じ値を示した。
以上の結果より、本酵素は、合成基質のリシルペプチド結合に対してＡＰＩと同等の加水分解能を有することが判明した。
【００６２】
（９）Xan.Lys-Cのアミノ酸組成
Xan.Lys-Cのアミノ酸組成分析を行った。Xan.Lys-Cのアミノ酸組成はＭｏｏｒｅとＳｔｅｉｎの方法に従って分析した。また、トリプトファン含量はＳｉｍｐｓｏｎの方法（R.J.Simpson,M.R.Neuberger and T.Y.Liu;J.Biol.Chem.,251,1936(1976)）、システイン及びシスチン含量ははHirsの方法（C.H.W.Hins;J.Biol.Chem.,219,611(19656)）による過ギ酸法及びSpencerとWoldの方法（R.L.Spencer and F.Wold;Anal.Biochem.,32,185(1969)）によるジメチルスルホキシド（ＤＭＳＯ）法によって定量をした。
【００６３】
Xan.Lys-Cの分子量を２７,８４９.５９（Electrospray−ＭＳ法より算出）と してアミノ酸残基数を求めた結果を表７に示す。尚、スレオニン及びセリンは、加水分解を２度行い、４８、７２、９６時間の加水分解物量より０時間に外挿し平均を取ったものを表記した。また、イソロイシンは、加水分解を２度行い、７２時間及び９６時間の平均より求め、システインは、２４時間の平均より求めた。それ以外のものは、加水分解を２度行い、２４、４８、７２、９６時間での平均値より求めた。また、比較として、［］内にＡＰＩのアミノ酸残基数を示す。
【００６４】
【表７】

【００６５】
その結果、Xan.Lys-Cのアミノ酸総数は２６５残基と算出された。
また、常法によりＮ−末端アミノ酸の種類を同定した結果、Xan.Lys-CのＮ−末端アミノ酸はグリシンであることが判った。
【００６６】
以上、酵素の性質をまとめると、分子量は２７,８５０、等電点は６.７６、カゼインに対する至適ｐＨは８.５〜１０.５、Bz-Lys-pNAに対する至適ｐＨは９.０〜９.６、Tos-Lys-OMeに対する至適ｐＨは８.０〜８.５であった。また、ｐＨ安定性領域は４.５〜１０.０、至適温度は５０℃、温度安定性は４０℃であり、０.１％ＳＤＳ含有時のタンパク加水分解率は８５％、４Ｍ尿素含有時は１００％、０.２Ｍ塩酸グアニジン含有時は５０％であった。Ｎ−末端アミノ酸はＧｌｙであり、阻害剤は、ＴＬＣＫ、ＤＦＰ、ＰＭＳＦ、Ｚｎ²⁺、Ｂａ²⁺、Ｋ⁺、Ｎａ⁺、Ｌｉ⁺であった。
【００６７】
これらの結果より、キサントモナス属IB-9374により産生されるXan.Lys-Cは、トリプシン様セリンエンドペプチダーゼに属し、リシン残基に対して高い親和性を有する酵素であると認められた。
【００６８】
【発明の効果】
上述した如く、本発明は、有用な酵素であるリシン残基特異的酵素を高効率で産生する微生物（菌）並びにこの酵素の取得方法を提供するものであり、本発明を用いればリシン残基特異的酵素を効率よく安価に取得することが可能となる。
【図面の簡単な説明】
【図１】実施例２で得られた、メチオニン添加・無添加培地中でのキサントモナス属IB-9374及びアクロモバクター リティカス M497-1の生育曲線を表した図である。
【図２】実施例２で得られた、グルタミン酸添加・無添加培地中でのキサントモナス属IB-9374及びアクロモバクター リティカス M497-1の生育曲線を表した図である。
【図３】実施例３で得られた、キサントモナス属IB-9374及びアクロモバクター リティカス M497-1の生育曲線及び培養上清のアミダーゼ活性を示した図である。
【図４】実施例５で得られた、ｐＨを変化させたときのリシン残基特異的酵素のアミダーゼ活性を示した図である。
【図５】実施例５で得られた、ｐＨを変化させたときのリシン残基特異的酵素のプロテイナーゼ活性を示した図である。
【図６】実施例５で得られた、ｐＨを変化させたときのリシン残基特異的酵素のプロテイナーゼ活性を示した図である。
【図７】実施例５で得られた、温度を変化させた時のリシン残基特異的酵素のアミダーゼ活性、エステラーゼ活性及びプロテイナーゼ活性を示した図である。
【図８】実施例５で得られた、リシン残基特異的酵素のｐＨ安定曲線である。
【図９】実施例５で得られた、リシン残基特異的酵素の温度安定曲線である。
【図１０】実施例５で得られた、リシン残基特異的酵素に対するＢａ²⁺の阻害作用のLineweaver-Burkプロットを示した図である。
【図１１】実施例５で得られた、リシン残基特異的酵素の尿素に対する安定性を示した図である。
【図１２】実施例５で得られた、リシン残基特異的酵素の塩酸グアニジンに対する安定性を示した図である。
【図１３】実施例５で得られた、リシン残基特異的酵素のドデシル硫酸ナトリウムによる安定性を示した図である。
【符号の説明】
図１に於いて、−○−は、メチオニンを培地に加えたときの、また、--○--はメチオニンを培地に加えないときのキサントモナス属IB-9374の生育曲線を夫々示す。また、−◆−は、メチオニンを培地に加えたときの、また、--◆--はメチオニンを培地に加えないときの、アクロモバクター リティカス M497-1の生育曲線を夫々示す。
図２に於いて、−○−は、グルタミン酸を培地に加えたときの、また、--○--はグルタミン酸を培地に加えないときのキサントモナス属IB-9374の生育曲線を夫々示す。また、−◆−は、グルタミン酸を培地に加えたときの、また、--◆--はグルタミン酸を培地に加えないときのアクロモバクター リティカス M497-1の生育曲線を夫々示す。
図３に於いて、−○−は、キサントモナス属IB-9374の生育曲線を示し、また、--◇--は、同培地でのキサントモナス属IB-9374培養上清のアミダーゼ活性を夫々示す。また、−□−は、アクロモバクター リティカス M497-1の生育曲線を、また、--×--は、アクロモバクター リティカス M497-1培養上清のアミダーゼ活性を夫々示す。
図４に於いて、−●−は１００ｍＭ トリス塩酸緩衝液（ｐＨ７.３−１０.０）を用いたときのアミダーゼ活性を、また、--○--は１００ｍＭ 2-アミノ-2-メチル-1,3-プロパンジオール−塩酸緩衝液（ｐＨ７.７−１０.６）を用いたときのアミダーゼ活性を夫々示す。
図６に於いて、−●−は５０ｍＭ トリス塩酸緩衝液（ｐＨ７.０〜９.０）を用いたときのプロテイナーゼ活性を、また、--△--は５０ｍＭ ほう酸塩緩衝液（ｐＨ８.０〜１１.５）を用いたときのプロテイナーゼ活性を夫々示す。
図７に於いて、−●−はリシン残基特異的酵素のアミダーゼ活性を、--◆--はリシン残基特異的酵素のプロテイナーゼ活性を、また、--△--はリシン残基特異的酵素のエステラーゼ活性を夫々示す。
図８に於いて、−●−は１００ｍＭ酢酸緩衝液でのアミダーゼ活性を、−×−は１００ｍＭ トリス塩酸緩衝液でのアミダーゼ活性を、−◆−は１００ｍＭ ＡＭＰ緩衝液でのアミダーゼ活性を、また、−◇−は１００ｍＭリン酸緩衝液でのアミダーゼ活性を夫々示す。BACKGROUND OF THE INVENTION
The present invention relates to a novel microorganism having a high ability to produce a lysine residue-specific enzyme and a method for obtaining the enzyme using the same.
[0001]
[Prior art]
As an enzyme that specifically cleaves the peptide bond on the C-terminal side of proteins and peptides, Achromobacter protease I [API, isolated from the culture solution of Achromobactor lyticus M497-1] EC 3.4.21.50, trade name lysyl endopeptidase (trademark registration No. 2255563, Wako Pure Chemical Industries, Ltd.)] is best known. This API is a kind of serine endopeptidase. It is a lysine residue-specific enzyme that cleaves all -Lys-X bonds including Lys-Pro bonds. It is extremely useful for preparation and synthesis of -Lys-X-compounds. This API is produced by culturing Achromobacter lyticus M497-1 bacteria, but has a problem that the enzyme cannot be made inexpensive due to poor production efficiency.
Therefore, a microorganism that efficiently produces a lysine residue-specific enzyme and a method that can be obtained at low cost are required.
[0002]
[Problems to be Solved by the Invention]
In view of the circumstances as described above, the problem to be solved by the present invention is to provide a microorganism that produces a lysine residue-specific enzyme with high efficiency and a method for obtaining the enzyme using this microorganism.
[0003]
[Means for Solving the Problems]
  The present invention was made for the purpose of solving the above-mentioned problems, and has the following properties, and is a microorganism having the deposit number FERM P-16928 that does not require methionine and glutamic acid for growth,lysineSpecifically cleaves the peptide bond on the C-terminal side of a residuelysineIt is an invention of a microorganism having the ability to produce a residue-specific enzyme.
a. Form: Neisseria gonorrhoeae
b. Size: 0.7 ~ 0.8 × 2.0 ~ 3.0μm
c. Gram staining: Negative
d. Spore: Negative
e. Motility: negative
f. Attitude toward oxygen: aerobic
g. Oxidase: positive
h. Catalase: positive
i. OF test: negative
j. Color of village: no characteristic pigment or light yellow
k. Quinone series: Q-8
l. GC content of intracellular DNA: 70 mol%
  Further, the present invention cultivates the microorganism and produces it from the culture solution.lysineSpecifically cleaves the peptide bond on the C-terminal side of a residuelysineCollecting residue-specific enzymeslysineIt is an invention of a method for obtaining a residue-specific enzyme.
  Furthermore, the present invention cultivates the above microorganism, and the culture solutionAmmonium sulfate fraction, DEAE- cellulose SH Column chromatography and AH Sepharose 4B It is obtained by column chromatography and has a molecular weight of 27850 Is,lysineSpecifically cleaves the peptide bond on the C-terminal side of a residuelysineIt is an invention of a residue-specific enzyme.
[0004]
That is, as a result of intensive studies on the properties of various fungi obtained from soil, the present inventors have found a new type of microorganism that produces a lysine residue-specific enzyme with high efficiency, and completed the present invention. It was.
[0005]
The microorganism of the present invention has been newly isolated and identified from soil by the present inventors, and has been named by the present inventors as Xanthomonas sp IB-9374.
[0006]
This freeze-dried product of the genus Xanthomonas IB-9374 was commissioned on August 3, 1998 to the Institute of Biotechnology, Tsukuba City, Ibaraki Pref. No. FERM P-16928.
[0007]
The medium composition of the genus Xanthomonas IB-9374 of the present invention may be either a natural medium or a synthetic medium as long as the microorganism can grow. The medium may be a solid or liquid medium. These media include, for example, sugars such as glucose, maltose and mannose as carbon sources, organic acids such as citric acid and malic acid, alcohols such as ethanol and glycerol, peptone as a nitrogen source, meat extract, In addition to general natural nitrogen sources such as yeast extract, protein hydrolyzate, and amino acids, various inorganic and organic ammonium salts are included. In addition, inorganic salts such as potassium, magnesium, iron, and calcium salts are required. Depending on the case, it is added appropriately. Further, Xanthomonas IB-9374 of the present invention can be cultured by a conventional method, but the culture temperature is usually 20 to 35 ° C, preferably 25 to 35 ° C, and the pH of the medium is usually 6 to 8 ° C. The pH is preferably 6.5 to 7.5. In addition, the culture is preferably performed under aerobic conditions by means such as shaking or aeration stirring, and the culture time is usually 150 to 250 hours.
[0008]
The method for obtaining a lysine residue-specific enzyme (hereinafter sometimes abbreviated as Xan.Lys-C) from Xanthomonas IB-9374 of the present invention is specifically carried out as follows. That is, Xanthomonas IB-9374 was cultured under the culture conditions as described above, and when the lysine residue-specific enzyme activity in the culture solution became high, the culture solution was filtered, and the obtained culture filtrate was at least ammonium sulfate. Fractionation (50-90%), DEAE-cellulose SH treatment, and AH Sepharose 4B column chromatography can give a lysine residue-specific enzyme, but preferably 6-step purification. That is, ammonium sulfate fraction of culture filtrate, CM-Toyopearl 650M treatment, DEAE-cellulose SH treatment, AH Sepharose 4B column chromatography, Soybean trypsin inhibitor (STI) Sepharose 4B affinity column chromatography, Sephadex G-50 Column chromatography is preferably performed in this order.
[0009]
Even when the lysine residue-specific enzyme of the present invention is collected using a 6-step purification process, API is obtained from Achromobacter lyticus M497-1 (T. Masaki, K. Nakamura, M. Isono and M.Soejima; Agric.Biol.Chem.,42 1443-1445 (1978), 7 steps), a lysine residue-specific enzyme can be obtained more easily and in a shorter time.
[0010]
In addition, the yield as the amount of enzyme protein corresponds to about 7 times the yield of API produced from Achromobacter lyticus M497-1 and having the same specificity as the present enzyme. In other words, the amount of bacteria required to obtain the same amount of lysine residue-specific enzyme can be reduced to one-seventh that when Achromobacter lyticus M497-1 is used as the production strain. Can be reduced.
[0011]
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
[0012]
【Example】
Example 1. Selection of novel lysine residue-specific enzyme highly efficient bacteria
The selection of the bacteria was performed as follows.
1 g of the collected soil was added to 10 mL of sterilized physiological saline and stirred for a while, then 1 mL of the supernatant was taken with a sterilized pipette, 9 mL of sterilized physiological saline was added, and the mixture was stirred again. This is diluted with sterilized physiological saline in order 10²-10⁹Double dilutions were prepared. Three drops of each diluted solution was dropped on an agar plate medium (2%) containing 1% wheat protein as a sole nutrient source, and stirred uniformly with a sterilized congeal bar. Next, after culturing at 30 ° C. for 2 to 3 days, a single colony producing a zona pellucida (halo) around the colony is selected from a large number of sufficiently grown colonies. Liquid medium (pH 7.2, 0.5% casein, 1.0% sucrose, 1.0% polypeptone, 0.01% KH in a test tube with cotton plug (25 mm x 200 mm)₂PO_Four, 0.01% K₂HPO_FourAnd 0.01% MgSO_Four・ 7H₂Contains O. (Hereinafter referred to as “A medium”) 15 mL, and cultured at 30 ° C. for 24 hours. After culture, the mixture was centrifuged (16,000 rpm × 10 minutes), and the resulting supernatant was used as a crude enzyme solution. The amidase activity of Nα-benzoyl-DL-lysine-p-nitroanilide hydrobromide (hereinafter abbreviated as BZ-lys-pNA) in the crude enzyme solution was examined. Microorganisms showing positive amidase activity were fished in 100 mL of medium A in a 500 mL Sakaguchi flask and cultured at 30 ° C. for 24 hours. Supernatant obtained by centrifugation after incubation is partially purified by ammonium sulfate fractionation and STI Sepharose 4B affinity column chromatography. Specificity of the resulting purified enzyme for glucagon (there is one lysine residue in the peptide chain) I examined the sex.
[0013]
As a result, it was found that only one of the 31,563 isolates produced a lysine residue-specific enzyme.
[0014]
For the amidase activity, BZ-lys-pNA is used as a substrate, and p-nitroaniline released by enzymatic action is measured at 405 nm (T. Tupy, UWiesbaver and Wintersberger; Hoppe-Seyler's Z.physiol. Chem.,329278 (1962)). That is, 0.15 mL of 2.5 mM BZ-lys-pNA aqueous solution and 1.3 mL of 0.2 M Tris-HCl buffer (pH 9.2) were dispensed into a test tube (1.5 × 13 cm), respectively, and a constant temperature of 30 ° C. After maintaining in a water bath for 5 minutes, 0.05 mL of the enzyme solution was added and reacted at the same temperature. After exactly 5 minutes, 0.5 mL of 45% acetic acid solution was added to stop the reaction, and the absorbance at 405 nm was measured. As a control sample, 0.15 mL of aqueous substrate solution, 1.3 mL of 0.2 M Tris-HCl buffer (pH 9.2), 0.5 mL of 45% acetic acid solution, and 0.05 mL of enzyme solution were sequentially added and mixed quickly. . The enzyme activity that liberates 1 μmol of p-nitroaniline (molecular extinction coefficient 9620) per minute under the above conditions was defined as 1 unit.
The specificity for glucagon was examined as follows.
70 μg of glucagon was dissolved in 0.1 ml of 50 mM Tris-HCl buffer (pH 9.2), and 10 μl (5.6 μg, 0.20 nmol) of an enzyme corresponding to 1/100 of the substrate was added at a molar ratio, The reaction was performed for 24 hours. After completion of the reaction, the decomposed product was heated with boiling water at 100 ° C. for 3 minutes to stop the reaction and freeze-dried. The lyophilized degradation product was fractionated by HPLC and purified. Each purified peptide was identified by amino acid composition analysis and N-terminal amino acid sequence analysis, and the molecular weight was measured by ESI-MS method. The amino acid composition analysis was identified by 835 type amino acid analyzer (manufactured by Hitachi), and the N-terminal amino acid sequence analysis was identified by Applied Biosystems 494 Protein Sequencer. In the ESI-MS method, molecular weight was measured by Perkin Elmer API300.
[0015]
Example 2
As a result of requesting the Japan Food Research Center to identify the separated and sorted cells, Xanthomonas maltophilia (formerly Pseudomonas maltophilia, now called Stenotrophomonas maltophilia) was identified, but its literature value (NRKrieg and JGHolt: "Bergey's Manual of Synstematic Bacteriology" Vol. 1 (1984) Williams & Wilkins, J. Swings, P. De Vos, M. Van den Mooter and J. De Ley: Int. J. Syst. Bacteriol.,33409 (1983) and this strain, it was revealed that the properties of motility, oxidase, etc. are slightly different.
The analysis results are shown in Table 1. In Table 1, the literature values of this strain, Achromobacter lyticus M497-1 (Japanese Patent Publication No. 46-42953) and the literature values of Xanthomonas maltophilia are also shown.
[0016]
[Table 1]

[0017]
In addition, an identification test using an automatic bacterial testing device ATB Expression (made by Biomelieu) using Gram negative bacillus identification kit ID 32GN (made by Biomelieu) and Gram negative bacteria identification kit API 20NE (made by Biomelieu) other than enteric bacteria The identification results used are as follows.
Utilization test by ATB Expression: positive; N-acetylglucosamine, sodium citrate, 3-hydroxybutyric acid and L-proline. Negative: rhamnose, D-ribose, inositol, sucrose, itaconic acid, suberic acid, sodium malonate, sodium acetate, lactic acid, L-alanine, D-mannitol, glucose, salicin, D-melibiose, L-fucose, D- Sorbitol, L-arabinose, propionic acid, n-capric acid, n-valeric acid, L-histidine hydrochloride, 5-ketogluconic acid, glycogen, m-hydroxybenzoic acid, 2-ketogluconic acid, p-hydroxybenzoic acid and L -Serine.
Moreover, the hydrolysis test of esculin by ATB Expression, the degradation test of DNA, and the lecithinase test were all positive.
[0018]
Utilization test by API 20NE: positive; glucose, D-mannose, N-acetyl-D-glucosamine, maltose, DL-malic acid and sodium citrate. Negative; L-arabinose, D-mannitol, potassium gluconate, n-capric acid, adipic acid and phenyl acetate.
Further, the nitrate reduction test, indole production test, glucose fermentation test, arginine dihydrolase test and urea decomposition test by API 20NE were negative, and the esculin decomposition test, gelatin liquefaction test, PNPG test and oxidase test were positive.
[0019]
As a result, this strain did not assimilate sucrose, but Achromobacter lyticus M497-1 assimilated sucrose, suggesting that these are different strains. In order to see the difference in assimilability, we further compared the assimilability of 95 types of carbon sources by the biolog system (Biology, Microstation system, USA).
[0020]
As a result, it was positive in Xanthomonas IB-9374 for eight carbon sources of α-cyclodextrin, formic acid, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, sebacic acid and γ-aminobutyric acid, Achromobacter lyticus M497-1 showed a difference from negative.
From these results, it was revealed that Xanthomonas sp. IB-9374 is a strain different from Achromobacter lyticus M497-1.
[0021]
In addition to Achromobacter lyticus M497-1, Stenotrophomonas maltophilia has biochemical properties similar to this strain. It has been reported that methionine and glutamic acid are required for the growth of this strain. Then, the influence on growth by the presence or absence of methionine and glutamic acid was examined. The results are shown in FIGS. In FIG. 1,-○-indicates the growth curve of Xanthomonas IB-9374 when methionine is added to the medium, and-○-indicates the growth curve of Xanthomonas IB-9374 when methionine is not added to the medium. -Indicates the growth curve of Achromobacter lyticus M497-1 when methionine is added to the medium, and-♦-indicates when methionine is not added to the medium. In FIG. 2, -o- indicates the growth curve of Xanthomonas IB-9374 when glutamic acid is added to the medium, and --o-- indicates that when glutamic acid is not added to the medium. The growth curves of Achromobacter lyticus M497-1 when glutamic acid is added to the medium and when no glutamic acid is added to the medium are shown.
[0022]
From this result, the influence of growth on Xanthomonas IB-9374 by methionine and glutamic acid was not recognized. From these results, it was revealed that Xanthomonas IB-9374 is a different bacterium from Stenotrophomonas maltophilia.
[0023]
Furthermore, the 16S-rDNA base sequence analysis of this strain was conducted and the homology with the base sequence of a known microorganism was examined. (Xanthomonas maltophilia)], Xanthomonas bacilola LMG 736^T(Xanthomonas vasicola LMG 736^T), Xanthomonas arboricola 747^T(Xanthomonas arboricola 747^T), Xanthomonas campestris LMG 568^T(Xanthomonas campestris LMG 568^T), Xanthomonas cassava fly LMG 673^T(Xanthomonas cassavae LMG 673^T), Xanthomonas hortrum LMG 733^T(Xanthomonas hortorum LMG 733^T), Xanthomonas oryzae LMG 5047^T(Xanthomonas oryzae LMG 5047^T), Xanthomonas pishi LMG 847^T(Xanthomonas pisi LMG 847^T), Xanthomonas vesicatoria LMG 911^T(Xanthomonas vesicatoria LMG 911^T) And 10 species of Xanthomonas campestris pv. Campestris Xc17 (Xanthomonas campestris pv. Campestris Xc17) showed high homology (93% or more) with the 16S-rDNA base sequence of this strain. In addition, the homology of the base sequence analysis was calculated using gene analysis software GENETYX-MAC7.3 (manufactured by SOFTWARE DEVELOPMENT) using 16S-rDNA sequences of various bacteria.
[0024]
From the above results, this strain was judged to be a new species of Xanthomonas, and this strain was named Xanthomonas IB-9374.
[0025]
Example 3 Examination of Xan.Lys-C production ability of Xanthomonas IB-9374 in various media Using various media, the Xan.Lys-C production ability of Xanthomonas IB-9374 was examined. That is, yeast extract, casamino acid, meat extract, milk casein, soy bean powder, sucrose, glucose, polypeptone, KH₂PO_Four, K₂HPO_Four, MgSO_Four・ 7H₂After culturing Xanthomonas IB-9374 at 30 ° C for a predetermined time in various media containing O at a predetermined concentration, the culture supernatant is collected, the amidase activity of the culture supernatant is measured, and Xan.Lys-C production is determined from the value. The performance was compared.
[0026]
As a result, 0.5% milk casein, 1.0% sucrose, 1.0% meat extract, 0.01% KH₂PO_Four, 0.01% K₂HPO_FourAnd 0.01% MgSO_Four・ 7H₂It was found that a medium with pH 7.2 containing O (hereinafter referred to as B medium) was most preferable. Using B medium, the highest amidase activity was 0.1 units (u) per mL of culture supernatant at 150 hours and 0.18 u at 250 hours. FIG. 3 shows a growth curve of Xanthomonas IB-9374 (-○-) and a curve (-◇-) showing the amidase activity of the culture supernatant after culturing for a predetermined time when B medium is used. As a control, a growth curve (-□-) when Achromobacter lyticus M497-1 was cultured in the same B medium and a curve (--x-) showing the amidase activity of the culture supernatant after culturing for a predetermined time. -) Is also shown in FIG. From the results shown in FIG. 3, it can be seen that when compared with that of Achromobacter lyticus M497-1, the activity is about 5 times higher at 100 hours of culture and about 4 times higher at 250 hours. From the results shown in FIG. 3, it was found that Xanthomonas sp. IB-9374 of the present invention is a strain different from Achromobacter lyticus M497-1.
[0027]
Example 4
Xan.Lys-C obtained by culturing the bacteria isolated in Example 1 was purified by the following method.
(1) 10L of culture solution is fractioned 50 to 90% ammonium sulfate, CM-Toyopearl treatment, DEAE-cellulose treatment, AH Sepharose 4B column chromatography, STI affinity chromatography, Sephadex G-50 column chromatography Purification was performed by
Details of the operation method are shown below. All purification operations were performed at 4 ° C.
[0028]
(1) Ammonium sulfate fraction of culture filtrate
Ammonium sulfate was added to 9210 mL of the culture filtrate cultured for 150 hours so as to achieve 50% saturation (temperature: 4 ° C.). Next, ammonium sulfate was added to 10,295 mL of the supernatant obtained by batch centrifugation (31,000 g, 20 minutes, 4 ° C.) so as to achieve 90% saturation. Subsequently, the mixture was centrifuged as described above, and the resulting precipitate was dissolved in an appropriate amount of 10 mM Tris-HCl buffer (pH 7.0; hereinafter referred to as buffer A), and then 4 times with respect to buffer A. Dialyzed at 72 ° C. for 72 hours.
[0029]
▲ 2 ▼ CM-Toyopearl 650M treatment
1120 mL of the enzyme solution obtained by dialysis was added to about 526.7 g (wet) of CM-Toyopearl 650M equilibrated with buffer A and gently stirred at room temperature for 1 hour. Next, the resin is removed with a glass filter (diameter: 11.5 cm, 25G-3), and the resin on the filter is further washed with 1000 mL of buffer A (pH 7.0), and the filtrate and the washing solution are combined. 2400 mL of an enzyme solution containing Xan.Lys-C, which is an adsorption fraction, was obtained.
[0030]
(3) DEAE-cellulose SH treatment
2400 mL of the brown enzyme solution obtained in (2) was concentrated to 1200 mL with a Diaflow cell (membrane: PM-10), and then dialyzed sufficiently with 10 mM Tris-HCl buffer (pH 8.0; hereinafter referred to as Buffer B). . Thereafter, about 475 g (wet) of DEAE-cellulose SH equilibrated with buffer B was added to 1250 mL of this enzyme solution, and other impure proteins including colored protein were adsorbed while gently stirring at room temperature. After 1 hour, the resin was removed with a glass filter (diameter 11.5 cm, 26G-3), and then the resin was washed with 1000 mL of buffer B. 2400 mL of a solution containing Xan.Lys-C, which is a combination of the filtrate and the washing solution, was concentrated to 237 mL using a Diaflow cell, and sufficiently dialyzed against 2 mM Tris-HCl buffer (hereinafter referred to as Buffer C).
[0031]
(4) AH Sepharose 4B column chromatography
270 mL of the dialyzed enzyme solution obtained in (3) was adsorbed on AH Sepharose 4B column chromatography (4.1 × 18 cm) which had been sufficiently equilibrated with buffer C. After thoroughly washing with the same buffer, elution was performed with a linear concentration gradient using 1000 mL of Buffer C (pH 8.0) and 1000 mL of Buffer C containing 1.0 M NaCl. As a result, Xan.Lys-C was completely adsorbed and eluted with 0.5M NaCl. The active fractions were collected, and 720 mL of an enzyme solution sufficiently dialyzed in 50 mM Tris-HCl buffer (pH 8.0; hereinafter referred to as buffer D) was concentrated to 280 mL using a Diaflow cell.
[0032]
(5) STI Sepharose 4B affinity column chromatography
280 mL of the enzyme solution obtained in (4) was chromatographed in two portions. Specifically, 140 mL of the enzyme solution was adsorbed on an STI Sepharose 4B affinity column (3.2 × 6.0 cm) equilibrated with Buffer D, washed thoroughly with 500 mL of the same buffer, and then 50 mM glycine-hydrochloric acid containing 0.5 M NaCl. Elute with buffer (pH 4.5). The active fraction was collected, dialyzed sufficiently against buffer C, and concentrated twice using a Diaflow cell.
At the time of elution, 0.1 mL of 1 M Tris-HCl buffer (pH 8.0) was put in advance in a test tube for fractionation, and fractionation was performed.
[0033]
(6) Sephadex G-50 column chromatography
10 mL of the concentrated enzyme solution obtained in (5) was divided into two portions and subjected to gel filtration. That is, 5 mL of concentrated enzyme solution was subjected to Sephadex G-50 column chromatography (2.0 × 92 cm) equilibrated with buffer C containing 0.1 M NaCl, and active fractions of Xan.Lys-C were collected. The remaining 5 mL of the enzyme solution was similarly subjected to gel filtration, and the active fraction was collected, combined with the first active fraction, and sufficiently dialyzed against buffer C. The dialyzed enzyme solution was concentrated with a Diaflow cell to 5 mL, and stored frozen at -15 ° C.
[0034]
Values such as recovery rate and specific activity at each stage are shown in Table 2.
[0035]
[Table 2]

[0036]
The final recovery rate was about 68%, and the specific activity increased about 930 times. The amount of the enzyme protein obtained at this time was 0.1 g per 10 L of the culture solution, which corresponds to about 7 times the amount of API protein produced from the culture solution of Achromobacter lyticus M497-1.
[0037]
Example 5 FIG. Study on the properties of Xan.Lys-C produced by Xanthomonas IB-9374
For Xan.Lys-C, using amidase activity, esterase activity or / and proteinase activity as indicators, optimal pH, optimal temperature, stability, effects of various compounds, effects of alkali metal ions and alkaline earth metal ions, and proteins The effect of denaturing agents was examined. In addition, the amino acid composition and N-terminal amino acid sequence of Xan.Lys-C were also examined.
The amidase activity was measured in the same manner as in Example 1. Moreover, the measuring method of esterase activity and proteinase activity is shown below.
[0038]
(1) Measuring method of esterase activity
To a quartz cell, add 1.5 mL of 80 mM Tris-HCl buffer (pH 8.0), 0.2 mL of 5 mM N-tosyl-L-lysine methyl ester (hereinafter abbreviated as Tos-Lys-OMe), and 1.2 mL of pure water. After maintaining at 30 ° C. for 5 minutes, 0.1 mL of the enzyme solution was added and allowed to react. Simultaneously with the addition of the enzyme, the mixture was stirred for 5 seconds, the change in absorbance at 247 nm was recorded, and the enzyme activity was measured based on this result. As the control sample, an enzyme solution was used instead of pure water.
The unit of enzyme activity was defined as 1 unit of enzyme activity that liberates 1 μmol of N-tosyl-L-lysine (molecular extinction coefficient 960) per minute under the above conditions.
[0039]
(2) Method for measuring proteinase activity
0.2 mL of enzyme solution is added to one crotch of two bifurcated digestive tracts for reaction and control, and 1 mL of substrate buffer (60 mM Tris-HCl buffer solution containing 0.84% casein, pH 9.0) is added to the other crotch. The solution was dispensed and kept in a constant temperature water bath at 40 ° C. for 5 minutes, and then the liquid in both thighs of the reaction bifurcated digestive tract was well mixed. After the mixing, the reaction was carried out in a constant temperature bath at 40 ° C. for exactly 20 minutes, and then the protein precipitation reagent (mixture containing 0.1M trichloroacetic acid and 0.22M sodium acetate was adjusted to pH 3.0 with acetic acid. 1 mL was added and mixed well. Then, it was kept again in a constant temperature water bath at 40 ° C. for 30 minutes to complete the formation of a precipitate, and then naturally filtered using a filter paper (Advantec No. 4A). On the other hand, the liquid in the bifurcatal digestive tract for control was kept for the same time without mixing, and then 1 mL of the protein precipitation reagent was added to the enzyme solution and mixed well, and then treated in the same manner as the bifurcatal digestive tract for reaction. The obtained filtrate sample was measured for absorbance at 275 nm with respect to a control sample using a spectrophotometer (UV-250 type) manufactured by Shimadzu.
The unit of enzyme activity is the product corresponding to 1 μmol of L-tyrosine per minute under the above conditions (reaction temperature 40 ° C., reaction pH 9.0, substrate concentration 0.7%, reaction time 20 minutes). The enzyme activity provided was defined as 1 unit.
[0040]
(1) Examination of optimum pH of Xan.Lys-C
The optimum pH of Xan.Lys-C was examined using amidase activity, esterase activity and proteinase activity for Bz-Lys-pNA, Tos-Lys-OMe and casein as indices.
FIG. 4 shows the results for amidase activity, FIG. 5 shows the results for esterase activity, and FIG. 6 shows the results for proteinase activity. In FIG. 4,-●-indicates amidase activity when 100 mM Tris-HCl buffer (pH 7.3-10.0) is used, and-○-indicates 100 mM 2-amino-2-. The amidase activity when methyl-1,3-propanediol-hydrochloric acid buffer (pH 7.7 to 10.6) is used is shown. In FIG. 6,-●-indicates proteinase activity when 50 mM Tris-HCl buffer (pH 7.0 to 9.0) is used, and --Δ-- indicates 50 mM borate buffer (pH 8). The proteinase activity when 0.01 to 11.5) is used. The data shown in FIG. 5 was obtained using a 100 mM Tris-HCl buffer (pH 7.1 to 9.0).
[0041]
As a result, the optimum pHs for the amidase activity and the esterase activity were 9.0 to 9.6 and 8.0 to 8.5, respectively. On the other hand, a wide curve of proteinase activity of 8.5 to 10.5 was obtained. These showed the same tendency as the optimum pH of API amidase activity (pH 9.0 to 9.5), esterase activity (pH 7.8 to 8.2) and proteinase activity (pH 8.5 to 10.7).
(2) Optimal temperature of Xan.Lys-C
The optimum temperature of Xan.Lys-C was examined using amidase activity, esterase activity and proteinase activity for Bz-Lys-pNA, Tos-Lys-OMe and casein as indices. The results are shown in FIG. In FIG. 7,-●-indicates the amidase activity of Xan.Lys-C,-◆-indicates the proteinase activity of Xan.Lys-C, and-△-indicates the esterase activity of Xan.Lys-C. Respectively.
[0042]
From these results, it was found that in any case, the maximum activity was exhibited at 50 ° C.
[0043]
(3) Stability of Xan.Lys-C
(1) pH stability
Using the amidase activity for Bz-Lys-pNA as an index, the stability of Xan.Lys-C when the pH was changed was examined.
Enzyme (1 μg) was placed in 0.1 ml each of 100 mM acetate buffer, Tris-HCl buffer, AMP buffer and phosphate buffer at a predetermined pH, and allowed to stand at 4 ° C. for 24 hours, and then against Bz-Lys-pNA. The residual rate of activity (%) was measured.
The results are shown in FIG. In FIG. 8,-●-indicates amidase activity in 100 mM acetate buffer, -X- indicates amidase activity in 100 mM Tris-HCl buffer,-♦-indicates amidase activity in 100 mM AMP buffer. In addition,-◇-indicates amidase activity in a 100 mM phosphate buffer, respectively.
[0044]
From this result, it can be seen that the stable region of Xan.Lys-C is pH 4.5 to 10.0. This was observed to be a little narrower than the stable region of API (pH 4.0 to 10.7).
[0045]
(2) Temperature stability
Using the amidase activity for Bz-Lys-pNA as an index, the stability of Xan.Lys-C when the temperature was changed was examined.
An enzyme (0.6 μg) dissolved in 0.1 ml of 100 mM Tris-HCl buffer (pH 8.0) is allowed to stand at various temperatures (20-60 ° C.) for 30 minutes and then rapidly cooled to show activity against Bz-Lys-pNA. The residual ratio (%) of was measured.
The results are shown in FIG.
[0046]
From this result, it was found that Xan.Lys-C was stable up to 40 ° C, but was completely deactivated at 60 ° C.
[0047]
(4) Effects of various compounds on Xan.Lys-C
Using the amidase activity and esterase activity for Bz-Lys-pNA and Tos-Lys-OMe as indicators, the effects of metal chelating reagents, SH reagents, reducing agents, etc. on Xan.Lys-C were examined.
That is, after pretreating an enzyme and various peptidase inhibitors with 40 mM Tris-HCl buffer (pH 8.0) for 30 minutes at 30 ° C., the amidase activity and esterase activity of Xan.Lys-C were measured. The effect was investigated. When measuring the amidase activity method, Bz-Lys-pNA is in the range of 0.05 to 0.16 mM, and when measuring the esterase activity, Tos-Lys-OMe is 0.05 to 0. The range was set to 5 mM.
The results are shown in Table 3. In Table 3, EDTA is an abbreviation for ethylenediaminetetraacetic acid, 2-ME is an abbreviation for 2-mercaptoethanol, NEM is an abbreviation for N-ethylmaleimide, and MIA is a monoiodide. Abbreviation for acetic acid (Monoiodoacetic acid), PCMB is an abbreviation for p-chloromercuribenzoic acid, DFP is an abbreviation for diisopropyl fluorophosphate, PMSF is a phenylmethanesulfonyl fluoride (Phenylmethansulfonyl fluoride) Abbreviation, TLCK is an abbreviation for Nα-Tosyl-L-lysyl chloromethyl ketone, TACK is Nα-Tosyl-L-arginyl chloromethyl ketone (Nα-Tosyl-L-arginyl chloromethyl ketone) abbreviation of ketone), TPCK is Nα-Tosyl-L-phenylalanyl chloromethyl ketone It represents substantially, similarly abbreviated below.
[0048]
[Table 3]

[0049]
From the results of Table 3, it can be seen that the activity of Xan.Lys-C is not inhibited by metal chelating reagents (EDTA, o-phenanthroline), reducing agents (cysteine, 2-ME), and SH reagents (NEM, MIA, PCMB). Xan.Lys-C was considered neither a metal endopeptidase nor a cysteine endopeptidase. On the other hand, since DFP and PMSF strongly inhibit the amidase activity and esterase activity of Xan.Lys-C, Xan.Lys-C was considered to be a serine endopeptidase. Furthermore, Xan.Lys-C was inhibited by TLCK, which reacts specifically with the His residue in the active center of trypsin, but not by TPCK, which is specific for the His residue of chymotrypsin. Xan.Lys-C was found to be a trypsin-like serine endopeptidase. Moreover, almost no influence was recognized by TACK which inhibits trypsin more strongly than TLCK. This suggests that Xan.Lys-C has a strong affinity for lysine residues.
[0050]
(5) Effect of metal ions on Xan.Lys-C
Using the amidase activity and esterase activity for Bz-Lys-pNA and Tos-Lys-OMe as indicators, the influence of metal ions on Xan.Lys-C was examined.
That is, after pretreating a 40 mM Tris ionic acid buffer solution (pH 8.0) containing an enzyme solution and a predetermined metal ion at 30 ° C. for 30 minutes, the residual activity against Bz-Lys-pNA and Tos-Lys-OMe was observed. It was measured. The results are shown in Table 4.
[0051]
[Table 4]

[0052]
Xan.Lys-C has amidase activity and esterase activity of 1 mM Ca.²⁺, Mg²⁺, Mn²⁺, Cd²⁺, Ni²⁺, Sr²⁺, Co²⁺0.1 mM Hg²⁺Was almost unaffected. However, Xan.Lys-C is 1 mM Ba²⁺Weakly by 1mM Zn²⁺In particular, in the case of the latter metal ion, strong inhibition of esterase activity was observed.
[0053]
(6) Effect of alkali metal ions and alkaline earth metal ions on Xan.Lys-C
Bz-Lys-pNA and Tos-Lys-OMe with amidase activity and esterase activity as indicators, alkali metal ions with respect to Xan.Lys-C, Ba²⁺And NH_Four ⁺The effect of was examined.
That is, 1.5 ml of 80 mM Tris-HCl buffer (pH 8.0), 1.0 mL of various aqueous metal solutions, and 0.4 mL of various concentrations of Tos-Lys-OMe or Bz-Lys-pNA are sequentially placed in a cell at 30 ° C. After the addition, 0.25 μg / 0.1 mL of the enzyme solution (or 1.0 μg / 0.1 mL) was quickly added, and the activity of the mixture reacted at 30 ° C. for 5 minutes was examined. As an example of the result, Ba²⁺FIG. 10 shows a Lineweaver-Burk plot of the inhibitory action on Xan.Lys-C.
As a result, any activity is²⁺, NH_Four ⁺, Cs⁺, Rb⁺, K⁺, Na⁺, Li⁺Was found to be antagonistically inhibited in this order.
[0054]
In addition, Table 5 shows the results of calculating the antagonistic inhibition constant (Ki) of various ions in which the inhibitory action was recognized, using the Dixon plot. Table 5 also shows the results for the API.
[0055]
[Table 5]

[0056]
As a result, it can be seen that for alkali metal ions, the inhibitory action increases as the ionic radius decreases. However, for alkaline earth metals, Ba²⁺Only strong inhibition was observed, Mg²⁺, Ca²⁺, Sr²⁺No effect was observed (not shown in Table 5). These results were the same as those of the API.
[0057]
(7) Effect of protein denaturant on Xan.Lys-C
Using the esterase activity for Tos-Lys-OMe as an index, the effects of urea, guanidine hydrochloride and sodium dodecyl sulfate (hereinafter abbreviated as SDS) on Xan.Lys-C were examined.
Specifically, 0.5 ml of 40 mM Tris-HCl buffer (pH 8.0) containing various concentrations of urea, guanidine hydrochloride or SDS and enzyme (5 μg) was pretreated for 30 minutes at 30 ° C., and then 0.1 mL (1 μg) of the solution. ) Was added to 2.9 mL of 40 mM Tris-HCl buffer (pH 8.0) containing 1 mM Tos-Lys-OMe and reacted for 5 minutes, and the residual rate (%) of esterase activity was examined.
FIG. 11 shows the results for urea, FIG. 12 shows the results for guanidine hydrochloride, and FIG. 13 shows the results for SDS.
[0058]
The residual rate of esterase activity when the enzyme was pretreated at 20 ° C. for 20 minutes in 40 mM Tris-HCl buffer (pH 8.0) containing 4 M urea, 0.2 M guanidine hydrochloride or 0.1% SDS, The trend was similar to that of API at 100%, 50%, and 85%, respectively.
[0059]
(8) Examination of substrate specificity of Xan.Lys-C
The specificity of Xan.Lys-C for various synthetic substrates was examined.
These results are shown in Table 6.
The Kcat / Km value was calculated from the Lineweaver-Burk plot, and the Kcat value was calculated with the molecular weight of the enzyme as 28000.
[0060]
[Table 6]

[0061]
From the results shown in Table 6, among the ester substrates, Tos-Lys-OMe was most hydrolyzed by this enzyme, and its Kcat / Km value was about 50,000 times higher than that of the corresponding Tos-Arg-OMe. showed that. Moreover, the Kcat / Km value of Bz-Orn-OMe was about 1/1200 compared with that of Tos-Arg-OMe, and it was not hydrolyzed so much. Comparing the Kcat / Km values for these ester substrates with those for the API showed similar values.
Among the anilide substrates, Bz-Lys-pNA and Ac-Lys-pNA were well hydrolyzed, and Lys-pNA was also slightly hydrolyzed. On the other hand, Bz-Arg-pNA and Arg-pNA were reacted for 24 hours at an enzyme concentration of 80 times or more, but were not decomposed at all. Furthermore, Suc-Ala-pNA, Boc-His-pNA, Ac-Phe-pNA, Bz-Tyr-pNA and Ac-Leu-pNA were reacted in the same manner, but these were not hydrolyzed at all (Table 6). Not described). The Kcat / Km values for these anilide substrates also showed approximately the same values as for API.
From the above results, it was found that this enzyme has a hydrolytic ability equivalent to that of API with respect to the lysyl peptide bond of the synthetic substrate.
[0062]
(9) Amino acid composition of Xan.Lys-C
The amino acid composition analysis of Xan.Lys-C was performed. The amino acid composition of Xan.Lys-C was analyzed according to the method of Moore and Stein. The tryptophan content was determined by the method of Simpson (R.J.Simpson, M.R.Neuberger and T.Y.Liu; J. Biol. Chem.,251, 1936 (1976)), cysteine and cystine contents are determined by the method of Hirs (C.H.W.Hins; J. Biol. Chem.,219611 (19656)) and the method of Spencer and Wold (R.L.Spencer and F.Wold; Anal.Biochem.,32, 185 (1969)) and quantified by the dimethyl sulfoxide (DMSO) method.
[0063]
Table 7 shows the results of calculating the number of amino acid residues with the molecular weight of Xan.Lys-C being 27,849.59 (calculated by Electrospray-MS method). In addition, threonine and serine were expressed by taking the average after extrapolating at 0 hour from the amount of hydrolyzate after 48, 72, and 96 hours after hydrolysis. In addition, isoleucine was hydrolyzed twice and obtained from an average of 72 hours and 96 hours, and cysteine was obtained from an average of 24 hours. Other than that, it hydrolyzed twice and calculated | required from the average value in 24, 48, 72, and 96 hours. For comparison, the number of amino acid residues of API is shown in [].
[0064]
[Table 7]

[0065]
As a result, the total number of amino acids of Xan.Lys-C was calculated to be 265 residues.
As a result of identifying the type of N-terminal amino acid by a conventional method, it was found that the N-terminal amino acid of Xan.Lys-C was glycine.
[0066]
As described above, the enzyme properties are summarized as follows: molecular weight is 27,850, isoelectric point is 6.76, optimum pH for casein is 8.5 to 10.5, and optimum pH for Bz-Lys-pNA is 9.0. The optimum pH for ˜9.6, Tos-Lys-OMe was 8.0-8.5. The pH stability region is 4.5 to 10.0, the optimum temperature is 50 ° C., the temperature stability is 40 ° C., and the protein hydrolysis rate when containing 0.1% SDS is 85%, containing 4M urea. The time was 100%, and when 0.2M guanidine hydrochloride was contained, it was 50%. N-terminal amino acid is Gly and inhibitors are TLCK, DFP, PMSF, Zn²⁺, Ba²⁺, K⁺, Na⁺, Li⁺Met.
[0067]
From these results, it was confirmed that Xan.Lys-C produced by Xanthomonas IB-9374 belongs to a trypsin-like serine endopeptidase and is an enzyme having a high affinity for lysine residues.
[0068]
【The invention's effect】
As described above, the present invention provides a microorganism (fungus) that produces a lysine residue-specific enzyme, which is a useful enzyme, with high efficiency, and a method for obtaining this enzyme. It becomes possible to obtain a specific enzyme efficiently and inexpensively.
[Brief description of the drawings]
1 is a graph showing the growth curves of Xanthomonas sp. IB-9374 and Achromobacter lyticus M497-1 in a methionine-added / non-added medium obtained in Example 2. FIG.
FIG. 2 is a graph showing the growth curves of Xanthomonas sp. IB-9374 and Achromobacter lyticus M497-1 obtained in Example 2 in a medium with or without glutamic acid.
FIG. 3 is a graph showing the growth curve and amidase activity of the culture supernatant of Xanthomonas sp. IB-9374 and Achromobacter lyticus M497-1 obtained in Example 3.
4 shows the amidase activity of a lysine residue-specific enzyme obtained in Example 5 when the pH is changed. FIG.
FIG. 5 shows the proteinase activity of a lysine residue-specific enzyme obtained in Example 5 when the pH is changed.
FIG. 6 shows the proteinase activity of a lysine residue-specific enzyme obtained in Example 5 when the pH is changed.
7 shows the amidase activity, esterase activity and proteinase activity of a lysine residue-specific enzyme obtained in Example 5 when the temperature is changed. FIG.
FIG. 8 is a pH stability curve of a lysine residue-specific enzyme obtained in Example 5.
9 is a temperature stability curve of a lysine residue-specific enzyme obtained in Example 5. FIG.
FIG. 10 shows Ba for lysine residue-specific enzyme obtained in Example 5.²⁺It is the figure which showed the Lineweaver-Burk plot of the inhibitory action of No ..
11 is a graph showing the stability of a lysine residue-specific enzyme to urea obtained in Example 5. FIG.
12 is a graph showing the stability of a lysine residue-specific enzyme to guanidine hydrochloride obtained in Example 5. FIG.
13 is a graph showing the stability of a lysine residue-specific enzyme obtained in Example 5 with sodium dodecyl sulfate. FIG.
[Explanation of symbols]
In FIG. 1, -o- indicates a growth curve of Xanthomonas IB-9374 when methionine is added to the medium, and --o-- indicates when methionine is not added to the medium. In addition,-♦-indicates a growth curve of Achromobacter lyticus M497-1 when methionine is added to the medium, and-◆-indicates when methionine is not added to the medium.
In FIG. 2, -o- indicates a growth curve of Xanthomonas IB-9374 when glutamic acid is added to the medium, and --o-- indicates no growth of glutamic acid to the medium. In addition,-♦-indicates a growth curve of Achromobacter lyticus M497-1 when glutamic acid is added to the medium, and-◆-indicates when Glutamic acid is not added to the medium.
In FIG. 3,-○-indicates the growth curve of Xanthomonas IB-9374, and-◇-indicates the amidase activity of the culture supernatant of Xanthomonas IB-9374 in the same medium, respectively. Further,-□-indicates a growth curve of Achromobacter lyticus M497-1, and --x-- indicates an amidase activity of the culture supernatant of Achromobacter lyticus M497-1.
In FIG. 4,-●-represents amidase activity when 100 mM Tris-HCl buffer (pH 7.3 to 10.0) was used, and-○-represents 100 mM 2-amino-2-methyl- The amidase activity when 1,3-propanediol-hydrochloric acid buffer (pH 7.7 to 10.6) is used is shown.
In FIG. 6,-●-indicates proteinase activity when 50 mM Tris-HCl buffer (pH 7.0 to 9.0) was used, and --Δ-- represents 50 mM borate buffer (pH 8.0). The proteinase activity when using ˜11.5) is shown.
In FIG. 7,-●-indicates amidase activity of a lysine residue-specific enzyme,-◆-indicates proteinase activity of a lysine residue-specific enzyme, and --Δ-- indicates lysine residue-specific enzyme. The esterase activity of the target enzyme is shown respectively.
In FIG. 8,-●-indicates amidase activity in 100 mM acetate buffer, -x- indicates amidase activity in 100 mM Tris-HCl buffer,-♦-indicates amidase activity in 100 mM AMP buffer, ,-◇-indicates amidase activity in a 100 mM phosphate buffer, respectively.

Claims (3)

It has the following characteristics, and a accession number FERM microorganisms P-16928, which does not require methionine and glutamic acid for growth, lysine residues specific to specifically cleave the peptide bond of the C-terminal side of lysine residues A microorganism that has the ability to produce enzymes.
a. Form: Neisseria gonorrhoeae b. Size: 0.7 ~ 0.8 × 2.0 ~ 3.0μm
c. Gram stainability: negative d. Spore: negative e. Motility: negative f. Attitude toward oxygen: aerobic g. Oxidase: positive h. Catalase: positive i. OF test: negative j. Set color: no characteristic pigment or pale yellowish k. Quinone series: Q-8
l. GC content of intracellular DNA: 70 mol% Culturing the microorganism according to claim 1, and recovering the lysine residues specific enzyme that specifically cleaves peptide bonds on the C-terminal side of lysine residues produced from the culture broth, lysine residue A method for obtaining a group-specific enzyme. The microorganism according to claim 1 is cultured, and the culture solution is obtained by subjecting in turn an ammonium sulfate fraction, DEAE- cellulose SH column chromatography treatment and AH Sepharose 4B column chromatography treatment, and has a molecular weight of 27850 . there, lysine residues specific enzyme that specifically cleaves peptide bonds on the C-terminal side of lysine residues.

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