JP4269142B2 - Novel alkaline laccase, its production method and its use - Google Patents

Description

【００２３】
本明細書では、「酵素が安定である」とは、ある処理をした場合に、酵素が、約８５％以上、好ましくは約９０％以上、より好ましくは約９５％以上の相対残存活性を有することをいう。
【００２４】
上記特性を有するアルカリラッカーゼは、これまで知られていない。
【００２５】
本発明のアルカリラッカーゼの代表例であるＰｈｏｍａ ｓｐ．Ｍａ２株由来の酵素は、７〜１１のｐＨ範囲で、好ましくはｐＨ８．３〜１０．６、より好ましくはｐＨ９．０〜１０．０のｐＨ範囲で、複素環系化合物または芳香族アミンの酸化反応を行い得、ｐＨ１０付近に至適ｐＨがあり、アルカリ性域での酸化反応を触媒するという特長を有する（図６）。また、至適温度は約５０℃であり（図４）、さらには、様々な一定温度で３０分間の加熱処理を施した後の活性は、例えば６０℃においてはほぼ９５％の残存活性を示した（図５）。さらには、様々なｐＨのバッファー中で３０℃、３時間の処理を施した後の残存活性は、広範囲のｐＨにおける安定性を示した（図７）。これらの結果は、弱アルカリ性からアルカリ性の範囲のｐＨ域で、中低温の様々な溶液中での酸化反応を保証する。
【００２６】
また、本発明のアルカリラッカーゼは、従来の酸性域に至適反応ｐＨを有する酵素と共に組み合わせて用いることも可能である。つまり、従来知られている酸性域に至適反応ｐＨを有するラッカーゼと本発明のアルカリラッカーゼを組み合わせて用いることで、酸性からアルカリ性の広範囲のｐＨ域においてラッカーゼ反応を行うことが可能となる。このような目的で酵素を混合して用いるとき、酸性域に至適反応ｐＨを有するラッカーゼの活性量と本発明のアルカリラッカーゼの活性量との混合比率は、好ましくは１：２０〜２０：１、より好ましくは１：２〜２：１にして用いることができる。このようにして広範囲のｐＨ域においてラッカーゼ反応を達成するためにも、本発明のアルカリラッカーゼは有用である。
【００２７】
（２）アルカリラッカーゼの起源
本発明の代表例のアルカリラッカーゼは、フォーマ属の糸状菌、特に平成１４年１２月１３日に工業技術院生命工学工業技術研究所（生命研と略称する）に寄託したＰｈｏｍａ ｓｐ．Ｍａ２株（受託番号：ＦＥＲＭ Ｐ−１９１５２）（以下、Ｍａ２株と略称する）により生産される。Ｍａ２株は、発明者が土壌、腐朽物、ならびにその他等からラッカーゼを生産する微生物を広くスクリーニングした結果、新たにマアジの腸内から単離された新菌株である。
【００２８】
本発明のアルカリラッカーゼ生産菌は、例えば、以下のようにして取得され得る。土壌、腐朽物、海水、淡水、海浜または河原で見られる泡塊、底泥、活性汚泥、魚類内臓などの試料を採取する。採取した試料を、生理食塩水または滅菌水とよく混合した後に静置し、上澄み液を得る。次に上澄み液を２００μＬ採取して染料を含有するアルカリ性の固形培地に塗り広げ、２８℃にて数日間静置培養する。出現した微生物コロニーの周囲に、染料の脱色ハローが観察されるコロニーを、アルカリラッカーゼ生産菌として分離する。
【００２９】
分離されるアルカリラッカーゼ生産菌は、当業者に周知の方法を用いて、生産するアルカリラッカーゼについてさらに詳細に特徴付けられ得る。
【００３０】
本発明者により単離されたＭａ２株の、分類学的性状を表２及び表３示す。表中に記載されるＯＡ〔碾割り燕麦 ６０ｇ、寒天 １２．５ｇ、蒸留水 １Ｌ〕、ＰＤＡ〔馬鈴薯浸出液 ２００ｇ、葡萄糖 ２０ｇ、寒天 １５ｇ、蒸留水 １Ｌ〕、ＭＥＡ〔麦芽エキス ３０ｇ、寒天 １５ｇ、蒸留水 １Ｌ〕は、培地の種類を表す。Ｍａ２株は、表２及び表３に記載のコロニー性状・分生子形成構造体の形態的特徴から（Ａｒｘ，Ｊ．Ａ．ｖｏｎ（１９７４） Ｔｈｅ ｇｅｎｅｒａ ｏｆ ｆｕｎｇｉ ｓｐｏｒｕｌａｔｉｎｇ ｉｎ ｐｕｒｅ ｃｕｌｔｕｒｅ，Ａ．Ｒ．Ｇａｎｔｎｅｒ Ｖｅｒｌａｇ ＫＧ，Ｖａｄｕｚ，Ｇｅｒｍａｎｙ，３１５頁、Ｄｏｍｓｃｈ，Ｋ．Ｈ．，Ｇａｍｓ，Ｗ．およびＡｎｄｅｒｓｏｎ，Ｔ．Ｈ．（１９９３）Ｃｏｍｐｅｎｄｉｕｍ ｏｆ ｓｏｉｌ ｆｕｎｇｉ，第１巻、ＩＨＷ−Ｖｅｒｌａｇ，Ｅｃｈｉｎｇ，Ｇｅｒｍａｎｙ，再版，８６０頁、およびＭａｌｌｏｃｈ，Ｄ．（１９８１）Ｍｏｕｌｄｓ−ｔｈｅｉｒ ｉｓｏｌａｔｉｏｎ，ｃｕｌｔｉｖａｔｉｏｎ，ａｎｄ ｉｄｅｎｔｉｆｉｃａｔｉｏｎ，Ｕｎｉｖｅｒｓｉｔｙ ｏｆ Ｔｏｒｏｎｔｏ Ｐｒｅｓｓ，Ｔｏｒｏｎｔｏ，ＵＳＡ，９７頁）、Ｐｈｏｍａ属と同定され、Ｐｈｏｍａ ｓｐ．Ｍａ２と命名された。
【００３１】
【表２】

【００３２】
【表３】

【００３３】
（３）アルカリラッカーゼを生産させるための培養条件
本発明のアルカリラッカーゼを生産する糸状菌を培養する代表的な条件を説明する。本発明の糸状菌は、液体培養または固体培養のいずれでも、培養され得る。本発明の糸状菌を培養する培地としては、特に限定されず、通常用いられる合成培地や有機炭素源、及び有機窒素源或いは無機窒素源を含む、液体培地または固体培地が使用可能である。炭素源の例としては、本発明の糸状菌が同化し得るものなら何でもよく、葡萄糖、蔗糖、乳糖、果糖、ソルビトール、糖蜜、澱粉等の糖類、ふすま、みかんパルプなどが単独で、または組み合わせて用いられ得る。窒素源の例としては、おから、アミノ酸、各種ペプトン類、脱脂大豆粉、コーンスティープリカー、尿素、酵母エキス、肉エキス、麦芽エキスなどの有機窒素源の他、硝石、硝安、硫安等の無機窒素源も、単独で、または組み合わせて使用し得る。必要に応じて、芒硝、瀉痢塩、緑礬、胆礬、皓礬といった硫酸塩、リン酸塩、カリウム、カルシウム、マンガン、コバルト等の無機塩類、ビタミン類、核酸関連化合物類等が添加され得る。また、酵素の生産性を高めるために適切量の生産誘導物質を添加してもよい。これらの培地成分は、本菌の生育を阻害しない濃度であればよく、炭素源は０．１〜２０重量％、好ましくは１〜１０重量％、窒素源は０．１〜１０重量％、好ましくは１〜５重量％である。また、培地のｐＨをアルカリ側にする場合、予め別殺菌しておいた苛性ソーダ、炭酸ソーダ、または重曹を含む溶液の適当量を、殺菌済みの培地に添加することによりｐＨ調整を達し得る。
【００３４】
培地のｐＨを、５．０から１２．０、好ましくは、６．０〜１０．０に調整し、滅菌して使用する。培養温度は、糸状菌類が生育し得る温度であればよく、実用上、１０〜約４０℃、好ましくは２５〜３５℃である。培養は、通常の中温菌の培養に準じ、液体培養にあっては通気培養或いは振盪培養、また固体培養にあっては静置培養により行えばよい。培養の継続時間は、種々の培養条件によって異なるが、目的とするラッカーゼが十分産生する期間であり、通気または振盪培養の場合は、通常２〜７日間が好ましい。
【００３５】
（４）培養液からのアルカリラッカーゼの調製
本発明のアルカリラッカーゼは、液体培養の場合、代表的には、菌体外に分泌される。従って、当該酵素を培地中に生産・蓄積せしめた後、下記の方法により培養液からのアルカリラッカーゼの調製を達成し得る。
【００３６】
すなわち、培養停止後、固液分離（例：濾布または濾紙などによる濾過、遠心分離、膜分離等）により菌体等の不溶物を除き、アルカリラッカーゼを含む培養濾液を回収する。次いで、透析、塩析、膜濃縮、各種カラムクロマトグラフィー、等電点沈殿、非水溶媒による沈殿、活性炭による吸着・脱色等といった、一般的に用いられるタンパク質の分別・分離法を、単独使用もしくは併用することにより該培養濾液から本発明のアルカリラッカーゼを調製し得る。例えば、上記で得られた培養濾液を限外濾過、硫安分画後、透析、各種クロマトグラフィーに順次供試することにより、アルカリラッカーゼの高活性画分が調製され得る。
【００３７】
（５）アルカリラッカーゼ活性の測定
本発明において、アルカリラッカーゼ活性は、アルカリラッカーゼが触媒する、ＭＢＴＨ（正式名：３−メチル−２−ベンゾチアゾリノンヒドラゾン）と、ホモバニリルアルコールとの酸化縮合による発色反応系において、酵素添加後の５６０ｎｍにおける吸光度の変化を調べることにより測定され得る。
【００３８】
詳細な測定方法は、次の通りである。１．５マイクロモルのホモバニリルアルコールおよび１．０マイクロモルのＭＢＴＨ含む１１５．４ｍｍｏｌ／Ｌ トリス−塩酸緩衝液（ｐＨ９．０）１０４０μＬを３０℃にて５分間プレインキュベートした後、酵素液１６０μＬを混合・攪拌する。混合・攪拌して３０秒後、測定を開始する。測定開始から３０秒後までの５６０ｎｍにおける吸光度の経時的変化を追跡する。１分間に吸光度を１増加させる酵素活性を１単位（Ｕ；ユニット）としてアルカリラッカーゼの活性値を算出する。
【００３９】
【実施例】
以下の実施例にて、本発明をさらに詳細に説明するが、これらはなんら本発明を限定するものではない。
【００４０】
実施例１
（Ｐｈｏｍａ ｓｐ．Ｍａ２株の培養）
１００ｍＬの培地（５％ グルコース、０．５％ ポリペプトン、０．１％ イーストエキス、０．２％ ＫＨ_２ＰＯ_４、０．１％Ｋ_２ＨＰＯ_４、０．０２％ＭｇＳＯ_４・７Ｈ_２Ｏ、ｐＨ７．０）を含む５００ｍＬ容丸フラスコに、Ｐｈｏｍａ ｓｐ． Ｍａ２株の一白金耳を接種し、３０℃で７日間、振盪培養した。次いで、この培養液１ｍＬを、１００ｍＬの上記培地を含む５００ｍＬ容丸フラスコに植菌し、３０℃で５日間、振盪培養した。
【００４１】
実施例２
（本アルカリラッカーゼの部分精製）
上記実施例で得た培養液に対し遠心分離（１２，０００ｒｐｍ×２０ｍｉｎ；４℃）を行い固液分離後、上清を濾別する。回収した濾液を合わせ培養濾液とした。
【００４２】
培養濾液をＵＦ（限外濾過）膜にて濃縮し、分子量３０，０００以上の画分として濃縮した。ＵＦ濃縮液を苛性ソーダでｐＨ７．０に調整後、硫安塩析（９０％飽和；４℃、一晩静置）を行い、次いで０．７％（Ｗ／Ｖ；Ｖ＝塩析前の液量）に相当する量のセライトを添加し、よく懸濁する。該懸濁液の濾別により、塩析ケーキが得られる。
【００４３】
低温下で塩析ケーキを９０％飽和硫安で数回洗淨した後、適当量の２０ｍｍｏｌ／Ｌ トリス−塩酸緩衝液（ｐＨ７．５）を添加し、よく攪拌する。この添加・攪拌操作により、塩析ケーキに含まれる塩析タンパク質が溶解されるので、懸濁液を濾過する。得た濾液に対し２０ｍｍｏｌ／Ｌ トリス−塩酸緩衝液（ｐＨ７．５）で透析し、これを透析内液とした。透析内液をＵＦ膜により濃縮し、これを部分精製液とした。
【００４４】
上記各工程における本アルカリラッカーゼの比活性（Ｕ／ ２８０ｎｍでの吸光度）、収率及び精製度を表４に示す。
【００４５】
【表４】

【００４６】
実施例３
（単一性の確認）
実施例２に記載の部分精製液の一部を、ポリアクリルアミドゲル等電点電気泳動に供試し、泳動後のゲルに対しＣＢＢ Ｒ−３５０（製品名：Ｃｏｏｍａｓｓｉｅ Ｔａｂｌｅｔｓ，ＰｈａｓｔＧｅｌ Ｂｌｕｅ Ｒ−３５０＜アマシャムバイオサイエンス社製；以下、“Ａ．Ｂ．社製”と略記＞）を用いた染色によるタンパク質の検出、およびホモバニリルアルコールおよびＭＢＴＨを用いる上記発色反応系によるアルカリラッカーゼの活性検出を行った
その結果、ｐＨ５以下の酸性側で検出された均一なタンパク質バンドとアルカリラッカーゼ活性バンドとの位置が一致し、実施例１〜２の工程を経て調製した当該部分精製液に一種類のアルカリラッカーゼが含まれることを確認した。
【００４７】
実施例４
（本アルカリラッカーゼの等電点）
実施例２に記載の部分精製液の一部を等電点電気泳動（以下、ＩＥＦと略記）に供試した。なおＩＥＦ専用装置としてＲｏｔｏｆｏｒ Ｃｏｍｂｉｎａｔｉｏｎ Ｓｙｓｔｅｍ（Ｂｉｏ−Ｒａｄ Ｌａｂｏｒａｔｏｒｉｅｓ社製）を、両性担体としてＡｍｐｈｏｌｉｎｅ，ｐｒｅｂｌｅｎｄｅｄ ｐＨ３．５−９．５（Ａ．Ｂ．社製；以下、“Ａｍｐｈｏｌｉｎｅ”と略記）を使用した。
【００４８】
先ず、部分精製液を４％、Ａｍｐｈｏｌｉｎｅを４％含む水溶液５０ｍＬに対して当該電気泳動を行い（初泳動）、アルカリラッカーゼ活性が強く検出された分画液、Ｎｏ．３（ｐＨ４．２）及びＮｏ．４（ｐＨ４．４）を足し合わせて活性画分とした。次に、部分精製液（２ｍＬ）および活性画分（全量）を含有する水溶液約６０ｍＬに対して、ＩＥＦを再度実施した（再泳動）。再泳動により得た分画液について、ｐＨ値および活性値をそれぞれ測定した。その結果を図１に示す。図１からわかるように、本アルカリラッカーゼの等電点は約３．７である。
【００４９】
実施例５
（本アルカリラッカーゼの分子量）
実施例２に記載の部分精製液の一部を、ＧＦＣ（ゲル濾過クロマトグラフィー）カラムに供試し、本アルカリラッカーゼの分子量を測定した。なおＧＦＣカラムとしてＨｉＰｒｅｐ ２６／６０ Ｓｅｐｈａｃｒｙｌ Ｓ−１００ ＨＲ（Ａ．Ｂ．社製；以下、本カラムと略記）を用いた。
【００５０】
２０ｍｍｏｌ／Ｌ トリス−塩酸緩衝液（１５０ｍｍｏｌ／Ｌ ＮａＣｌ含有；ｐＨ７．５）で予め平衡化しておいた本カラムに部分精製液をアプライした後、平衡化に用いたのと同一の緩衝液を用い流速０．９ｍＬ／ｍｉｎにて溶出を行った。溶出液の分画（０．９８ｍＬ／ｔｕｂｅ）の開始は、通液量が８６ｍＬに達した時点以降とし、その後、得られた分画液について、２８０ｎｍにおける吸光度およびアルカリラッカーゼ活性を測定した。アルカリラッカーゼ活性の測定は、室温（２６℃）で２０分間の上記発色反応後の呈色強度について、以下のように点数評価することによって目視的に行った：無色透明 ０点；非常に薄い紫色 ０．５点；薄い紫色（３段階評価） １．０点、１．５点、２．０点；紫色（２段階評価） ２．５点、３．０点；濃い紫色 ３．５点；黒紫色 ４．０点。その結果を、図２に示す。
【００５１】
次いで本カラムでの標準物質の溶出位置に基づいて予め作成しておいた分子量標準曲線（図３）に活性ピークの溶出位置を当てはめ、分子量を算出した。その結果、本アルカリラッカーゼの分子量は約４２〜４３ｋＤａであった。なお標準物質としてブルーデキストラン２０００、ウシ血清アルブミン、鶏卵オボアルブミン、ウシ膵臓キモトリプシノーゲンＡ、ウシ膵臓リボヌクレアーゼＡ（以上、Ａ．Ｂ．社製）を用いた。
【００５２】
実施例６
（本アルカリラッカーゼの作用適温範囲および熱安定性）
（１）作用適温の範囲
実施例２で得られた部分精製液の本アルカリラッカーゼの活性を、上記発色反応系において種々の温度条件（３０、４０、４５、５０、６０、７０℃）下で測定した。結果を図４に示す。図中の横軸は反応温度（℃）を表し、縦軸は最大活性値を１００とした場合の相対活性（％）を表す。本アルカリラッカーゼの至適温度は、約５０℃である。
【００５３】
（２）熱安定性
実施例２で得られた部分精製液の本アルカリラッカーゼを、種々の温度条件（０、３０、４０、５０、６０、６５、７０、８０、９０℃）下にて、トリス−塩酸緩衝液（ｐＨ７．５）中で３０分間インキュベートして前処理をした後、上記発色反応系にて残存活性を測定した。結果を図５に示す。図中の横軸は処理温度（℃）を表し、縦軸は最大活性値を１００とした場合の相対残存活性（％）を表す。本アルカリラッカーゼは、処理温度条件が６５℃の場合において酵素活性の約７３％、７０℃の場合においてその約５６％以上が保持され、安定である。本アルカリラッカーゼは、処理温度条件が６０℃では、酵素活性は約９５％保持される。
【００５４】
実施例７
（本アルカリラッカーゼの至適ｐＨおよび安定ｐＨ範囲）
（１）至適ｐＨ
上記発色反応系にて使用する１００ｍｍｏｌ／Ｌ トリス−塩酸緩衝液（ｐＨ９．０）の代わりに、ｐＨ領域に応じ、酢酸ソーダ−塩酸緩衝液、酢酸緩衝液、リン酸カリ緩衝液、トリス−塩酸緩衝液、グリシン−食塩−苛性ソーダ緩衝液を用いてｐＨを１．０〜１１．１（１．０、２．０、３．０、４．０、５．０、６．０、７．０、７．５、８．３、９．０、１０．０、１０．４、１０．６、１１．０、１２．５）に調整し、実施例２で得られた部分精製液の本アルカリラッカーゼの活性を測定した。結果を図６に示す。図中の横軸は反応ｐＨを表し、縦軸は最大活性値を１００とした場合の相対活性（％）を表す。本アルカリラッカーゼの至適ｐＨは、約１０である。
【００５５】
（２）安定ｐＨ範囲
実施例２で得られた部分精製液の本アルカリラッカーゼのｐＨを、種々の緩衝液にて１．０〜１２．５（１、２、３、４、５、６、７、８、９、１０、１１、１２．５）に調整し、３０℃で３時間インキュベートしてｐＨ処理液を得た。次いで、ｐＨ処理液各々について上記発色系にて残存活性を調べた。結果を図７に示す。図中の横軸は処理ｐＨを表し、縦軸は最大活性値を１００とした場合の相対残存活性（％）を表す。本アルカリラッカーゼの安定ｐＨ範囲は、４．０〜９．０である。
【００５６】
実施例８
（Ｍｙｒｏｔｈｅｃｉｕｍ ｖｅｒｕｃｃａｒｉａ由来アルカリラッカーゼとの比較：至適ｐＨ）
実施例７（１）に記載の方法において、部分精製液の替わりにＭｙｒｏｔｈｅｃｉｕｍ ｖｅｒｕｃｃａｒｉａ ２４Ｇ−４株（以下、２４Ｇ−４株と略称する）由来アルカリラッカーゼの部分精製液を用い、当該酵素の至適ｐＨを上記発色反応系の改変方法（反応液中のエタノール終濃度：１．５％）にて調べた。結果を図８に示す。図中の横軸は反応ｐＨを表し、縦軸は最大活性値を１００とした場合の相対活性（％）を表す。上記反応条件下での、２４Ｇ−４株由来アルカリラッカーゼの至適ｐＨは、約８．５である。
【００５７】
実施例７（１）に記載の方法において、部分精製液の替わりに２４Ｇ−４株由来アルカリラッカーゼの部分精製液を用いて、上記発色反応系にて使用する１００ｍｍｏｌ／Ｌ トリス−塩酸緩衝液のｐＨ値を８．０〜８．９（８．０、８．３、８．５、８．７、８．９）に調整した条件で、当該酵素の至適ｐＨを調べた。結果を図９に示す。図中の横軸は反応ｐＨを表し、縦軸は最大活性値を１００とした場合の相対活性（％）を表す。２４Ｇ−４株由来アルカリラッカーゼの上記発色反応系での至適ｐＨは、約８．３である。
【００５８】
実施例９
（アルカリラッカーゼの基質特異性▲１▼：ｐ−フェニレンジアミンに対する酸素消費量）
実施例２に記載の部分精製液をアルカリラッカーゼ粗酵素液として用い、ｐ−フェニレンジアミンを基質として供試し、基質の酸化に伴う酸素消費性をクラーク型酸素電極により電気信号に変換する酸素電極法を用いて調べた。詳細には、粗酵素液（０．８４Ｕ）を含む、１０１ｍｍｏｌ／Ｌ トリス−塩酸緩衝液（ｐＨ８．０） １４８５μＬを、３０℃で１０分間、空気で平衡化した。次いで、ｐ−フェニレンジアミン溶液 １５μＬ（基質含量：１．５μｍｏｌ）を添加することにより反応を開始し、酸素消費量を測定した。ブランク値は、部分精製液を２０ｍｍｏｌ／Ｌ トリス−塩酸緩衝液（ｐＨ７．５）に替えることにより調べられた。その結果、０．０１１ｍＶ／１０ｍｉｎの電圧変化があることを確認した。
【００５９】
実施例１０
（アルカリラッカーゼの基質特異性▲２▼：各基質に対する酸素消費活性）
実施例２に記載の部分精製液をアルカリラッカーゼ粗酵素液として用い、基質特異性を調べた。詳細には、７．５μｍｏｌの基質を含む、２３１ｍｍｏｌ／Ｌグリシン−食塩−苛性ソーダ緩衝液（ｐＨ１０．０） １３００μＬを、５０℃で１０分間、空気で平衡化した。次いで、粗酵素液２００μＬ（酵素含量：０．０８Ｕ）を添加することにより反応を開始し、酸素消費量を測定した。基質として、フェノール系化合物類（シリンガ酸、ホモバニリルアルコール）、芳香族アミン系化合物類（ｐ−フェニレンジアミン、バニリルアミン）、複素環系化合物類（ＭＢＴＨ、４−アミノアンチピリン、ＡＢＴＳ（正式名：２，２‘−アジノ−ビス（３−エチルベンゾチアゾリン−６−スルホン酸））、その他（アスコルビン酸）を用いた。ブランク値は、部分精製液を２０ｍｍｏｌ／Ｌ トリス−塩酸緩衝液（ｐＨ７．５）に替えることにより調べられた。その結果を表５に示す。表５から、部分精製アルカリラッカーゼは、フェノール系化合物より複素環系化合物及び芳香族アミン系化合物類に強く作用することが判る。
【００６０】
【表５】

【００６１】
実施例１１
（アルカリラッカーゼの基質特異性▲３▼：各基質に対する酸化呈色性）
実施例２に記載の部分精製液をアルカリラッカーゼ粗酵素液として用い、本アルカリラッカーゼが触媒する基質酸化に伴う呈色反応について吸収スペクトルの経時的変化を調べた（スキャニング法適用）。詳細には、１．２μｍｏｌの基質を含む、１１１．１ｍｍｏｌ／Ｌ グリシン−食塩−苛性ソーダ緩衝液（ｐＨ１０．０）１０８０μＬをキュベット内に入れ、５０℃で５分間、予温を行った後、粗酵素液１２０μＬ（酵素含量：０．０４Ｕ）を添加し、よく攪拌する。粗酵素液添加３０秒後にベースライン補正をしてから、１回目のスキャニングを開始した（粗酵素液添加２分後）。２回目以降のスキャニングは、１回目のスキャニングの開始から４分間隔（すなわち、粗酵素液添加６、１０、１４、１８、２２、２６、３０分後）で行った。ブランクのスペクトルは、上記条件において部分精製液を２０ｍｍｏｌ／Ｌ トリス−塩酸緩衝液（ｐＨ７．５）に替えることにより調べられた。基質として、ハイドロキノン、ホモバニリルアルコール、メタネフリン、ｐ−アミノフェノール、バニリルアミン、ｐ−フェニレンジアミン、ＭＢＴＨ、３−アミノ−１Ｈ−１，２，４−トリアゾールを用いた。なお、スキャニングの条件は、走査波長域を２００〜７００ｎｍ、走査波長間隔を５ｎｍ、走査時間を約４０秒とした。
【００６２】
結果は、上記被検基質に対する各々の吸収スペクトルの経時的変化について各基質ごとに全て重ね合わせた形で示した（図１０〜１７）。いずれの図も一番下のスペクトルグラフから順に、１回目〜最終回目のグラフに相当する。これらの図から、一部を除いて経時的に吸収値が上がっていき基質の酸化呈色反応が進行していることが分かると同時に、部分精製アルカリラッカーゼは、フェノール系化合物類より複素環系化合物類及び芳香族アミン系化合物類に強く作用することが示唆される。
【００６３】
【発明の効果】
本発明によれば、アルカリ性域に至適ｐＨを有し、かつアルカリ性条件下で耐熱性に優れた新規アルカリラッカーゼ、ならびにその生産方法および生産菌が提供される。本発明のアルカリラッカーゼは、従来のアルカリラッカーゼに較べてより高いアルカリ性域に至適ｐＨを有し、且つアルカリ性条件下で耐熱性に優れているため、洗剤及び染毛剤の製造、パルプ製造処理工程等に有用である。本発明のアルカリラッカーゼは、糸状菌の培養によって生産されるため、安価に大量にアルカリラッカーゼを提供することが出来、工業用途または試薬に広く利用され得る。
【図面の簡単な説明】
【図１】図１は、本発明のアルカリラッカーゼの等電点電気泳動の結果を示すグラフを示す図である。
【図２】図２は、ゲル濾過クロマトグラフィーによる本発明のアルカリラッカーゼ溶出の結果を示すグラフを示す図である。
【図３】図３は、ゲル濾過クロマトグラフィーによる本発明のアルカリラッカーゼの溶出ピークに基づいて算出された、本発明のアルカリラッカーゼの分子量を示すグラフを示す図である。
【図４】図４は、本発明のアルカリラッカーゼの至適温度を示すグラフを示す図である。
【図５】図５は、本発明のアルカリラッカーゼの熱安定性を示すグラフを示す図である。
【図６】図６は、本発明のアルカリラッカーゼの至適ｐＨを示すグラフを示す図である。
【図７】図７は、本発明のアルカリラッカーゼのｐＨ安定性を示すグラフを示す図である。
【図８】図８は、従来のＭｙｒｏｔｈｅｃｉｕｍ ｖｅｒｕｃｃａｒｉａ ２４Ｇ−４株由来アルカリラッカーゼの至適ｐＨを示すグラフを示す図である。
【図９】図９は、ｐＨ８．０〜９．０にて測定した、従来のＭｙｒｏｔｈｅｃｉｕｍ ｖｅｒｕｃｃａｒｉａ ２４Ｇ−４株由来アルカリラッカーゼの至適ｐＨを示すグラフを示す図である。
【図１０】図１０は、本発明のアルカリラッカーゼのハイドロキノンに対する酸化呈色についての、吸収スペクトルの経時的変化を示す図である。
【図１１】図１１は、本発明のアルカリラッカーゼのホモバニルアルコールに対する酸化呈色についての、吸収スペクトルの経時的変化を示す図である。
【図１２】図１２は、本発明のアルカリラッカーゼのメタネフリンに対する酸化呈色についての、吸収スペクトルの経時的変化を示す図である。
【図１３】図１３は、本発明のアルカリラッカーゼのｐ−アミノフェノールに対する酸化呈色についての、吸収スペクトルの経時的変化を示す図である。
【図１４】図１４は、本発明のアルカリラッカーゼのバニリルアミンに対する酸化呈色についての、吸収スペクトルの経時的変化を示す図である。
【図１５】図１５は、本発明のアルカリラッカーゼのｐ−フェニレンジアミンに対する酸化呈色についての、吸収スペクトルの経時的変化を示す図である。
【図１６】図１０は、本発明のアルカリラッカーゼのＭＢＴＨに対する酸化呈色についての、吸収スペクトルの経時的変化を示す図である。
【図１７】図１０は、本発明のアルカリラッカーゼの３−アミノ−１Ｈ−１，２，４−トリアゾールに対する酸化呈色についての、吸収スペクトルの経時的変化を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention provides a novel alkaline laccase having an optimum pH in an alkaline region and excellent heat resistance, and a novel alkaline laccase such as a former (Poma) A method for producing a filamentous fungus culture belonging to the genus and a filamentous fungus producing a novel alkaline laccase.
[0002]
[Prior art]
Conventionally, laccase and polyphenol oxidase (hereinafter abbreviated as oxidase) are known as enzymes that oxidize aromatic compounds and heterocyclic compounds using molecular oxygen. Oxidase is widely distributed in microorganisms such as bacteria, filamentous fungi, and basidiomycetes.
[0003]
In addition, a low molecular chemical substance that can be a relatively stable reaction intermediate radical among substrates that are oxidized by oxidase, particularly laccase, is called laccase mediator or redox mediator, and by undergoing oxidation of laccase, It causes phenomena such as enhancement of laccase oxidation or oxidation of non-substrate chemicals of laccase. For example, it has been clarified that laccase can be indirectly oxidized in the presence of a redox mediator with a group of hardly biodegradable organic pollutant chemicals that are difficult to decompose or cannot be decomposed by laccase alone (Bressler DC). Et al:Biotechnol.  Lett.,22, 119-125 (2000); Srebotnik E .; Et al:J. et al.  Biotechnol.,81179-188 (2000)).
[0004]
Thus, it has been found that laccase has a catalytic ability for various chemical reactions resulting from the radical species of the reaction intermediate produced by the oxidation of the substrate. Therefore, the development of applications that apply this catalytic ability has been actively carried out by researchers all over the world. For example, dyeing and discharging, prevention of color transfer during washing, pulp and fibers Bleaching, decolorization of colored waste liquid, decomposition of refractory biodegradable compounds, detoxification of toxic chemicals, removal of bitter taste of food, disappearance of mold odor of wine cork, prevention of turbidity of juice, promotion of in vivo digestion of livestock feed, Production of non-adhesive wood board, phenol resin, artificial lacquer paint, adhesive, desulfurization of fossil fuel, clinical analysis based on luminescence and color development, biosensor, synthesis of organic compounds, etc.
[0005]
However, the optimal pH of action of most laccases that can be produced on an industrial scale is acidic in spite of the fact that the optimal pH of action is often desired to be in the alkaline range in the development of the above applications. Since it is in the weakly acidic range from the range, few have been put into practical use.
[0006]
For example, it is desirable to use laccase having an optimum pH in the alkaline region for preventing color transfer during hair dyeing or washing. Moreover, also in the lignin removal in a kraft pulp manufacturing process, it is important from the point of cost reduction to use the laccase which has optimal pH in an alkaline region. As described above, laccase is often used in an alkaline region.
[0007]
On the other hand, several microorganisms producing oxidase whose optimum pH is present in the alkaline range are known, Stillella spp., Sagenomella spp., Stachiridium spp. (Above, JP-A-10-174583 (Patent Document 1)), Milosecium spp. (JP 2001-69973 (Patent Document 2), WO 97/00948 (Patent Document 3), JP 11-299482 (Patent Document 4), JP 11-299483 (Patent Document 5)) Microorganisms belonging to are known. Moreover, although the oxidase which is said to have an optimum pH derived from animals and plants in the alkaline region has been reported, it is difficult to produce a large amount from these animals and plants at low cost, and for industrial use An oxidase having an optimum pH derived from microorganisms in an alkaline region is desired.
[0008]
Currently, among the oxidases produced by the above-mentioned known microorganisms, laccase considered to act on the most alkaline side is derived from the genus Miloseces. However, the optimum pH of the laccase derived from the genus Miloseces described in JP-A No. 2001-69973 (Patent Document 2) is about 8.3 (measured by a color reaction system using oxidative condensation of MBTH and homovanillyl alcohol). As for heat resistance, it is completely deactivated by treatment at 70 ° C. for 1 hour. This also applies to the laccase described in JP-A-11-299482 (Patent Document 4) that is similar in properties to the laccase described in JP-A-2001-69973 (Patent Document 2) (eg, 70 ° C., Completely deactivated after 0.5 hour treatment). That is, until now, no laccase having high activity in the alkaline region and heat resistance and a microorganism producing the same have not been found. Here, the “alkaline region” refers to a range in which the optimum pH is 9.0 or more in the color development reaction system.
[0009]
[Patent Document 1]
Japanese Patent Laid-Open No. 10-174583
[Patent Document 2]
JP 2001-69973 A
[Patent Document 3]
International Publication No. 97/00948
[Patent Document 4]
JP-A-11-299482
[Patent Document 5]
JP-A-11-299483
[0010]
[Problems to be solved by the invention]
The present invention is intended to solve the above-mentioned conventional problems, and the object is to produce a novel alkaline laccase having an optimum pH in an alkaline region and excellent in heat resistance under alkaline conditions, and production thereof. It is to provide a method and producing bacteria. Until now, there is no known laccase having an optimum pH in an alkaline region and having high heat resistance under alkaline conditions, and a microorganism producing the same.
[0011]
[Means for Solving the Problems]
The present inventorPomaFrom the newly isolated filamentous fungi belonging to the genus), a novel alkaline laccase was found, and based on this, the present invention was completed.
The alkaline laccase of the present invention has the following properties:
(1) Molecular weight: about 42,000-43,000;
(2) Optimal pH: about 10; and
(3) Substrate specificity: Strongly acts on heterocyclic compounds or aromatic amines.
[0012]
As used herein, the term “strongly acting” means that when laccase activity is measured, the activity in an oxidation reaction when a phenolic compound is used as a substrate is about 3 times, preferably about 4 times, more preferably about It means that it can catalyze and decompose the oxidation reaction of heterocyclic compounds or aromatic amines with an activity 5 times, even more preferably about 8 times, most preferably about 10 times or more higher.
[0013]
Preferably, the alkaline laccase has the following properties:
(4) Range of optimum working temperature: the optimum temperature is 50 ° C;
(5) Thermal stability: stable to about 60 ° C. when held at pH 7.5 for 30 minutes;
(6) Stable pH range: when treated at 30 ° C. for 3 hours, pH 4.0-9.0; and
(7) Isoelectric point: about 3.7;
Have The alkaline laccase is preferably a filamentous fungus belonging to the genus Forma, more preferablyPoma  sp. Produced by Ma2 strain (Accession number: FERM P-19152).
[0014]
The method for producing the alkaline laccase of the present invention has the following characteristics:
(1) Molecular weight: about 42,000-43,000;
(2) Optimal pH: about 10; and
(3) substrate specificity: acts strongly on heterocyclic compounds or aromatic amines;
A step of culturing a filamentous fungus belonging to the genus Forma that produces an alkaline laccase having the following: a step of separating and purifying the alkaline laccase from the culture.
[0015]
Preferably, the alkaline laccase has the following properties:
(4) Range of optimum working temperature: the optimum temperature is 50 ° C;
(5) Thermal stability: stable to about 60 ° C. when held at pH 7.5 for 30 minutes;
(6) Stable pH range: when treated at 30 ° C. for 3 hours, pH 4.0-9.0; and
(7) Isoelectric point: about 3.7;
Have The alkaline laccase is preferably a filamentous fungus belonging to the genus Forma, more preferablyPoma  sp. Produced by Ma2 strain (Accession number: FERM P-19152).
[0016]
The filamentous fungus of the present invention has the following characteristics:
(1) Molecular weight: about 42,000-43,000;
(2) Optimal pH: about 10; and
(3) substrate specificity: acts strongly on heterocyclic compounds or aromatic amines;
To produce an alkaline laccase having
[0017]
Preferably, the alkaline laccase has the following properties:
(4) Range of optimum working temperature: the optimum temperature is 50 ° C;
(5) Thermal stability: stable to about 60 ° C. when held at pH 7.5 for 30 minutes;
(6) Stable pH range: when treated at 30 ° C. for 3 hours, pH 4.0-9.0; and
(7) Isoelectric point: about 3.7;
Have The filamentous fungus belongs to the genus Forma, especiallyPoma  sp. It is Ma2 strain (accession number: FERM P-19152).
[0018]
This achieves the above object.
[0019]
DETAILED DESCRIPTION OF THE INVENTION
The alkaline laccase of the present invention is typically derived from filamentous fungi. The “alkaline laccase derived from filamentous fungi” as used herein is not limited to alkali laccase prepared from a natural filamentous fungus culture, and is a method known per se (eg, irradiation with ultraviolet rays, X-rays, γ rays, etc., Oxidase having the same properties as the enzyme, prepared from a culture of alkaline laccase high-productivity mutants derived from those strains by drug treatment, culture on a drug-containing medium, genetic engineering techniques, etc.) Are also included. Preferably Forma (Poma) Derived from filamentous fungi belonging to the genus, particularly preferably former (Poma) Genus Ma2 strain (Accession number: FERM P-19152).
[0020]
Hereinafter, the present invention will be described in detail.
[0021]
(1) Alkaline laccase
Former which is a representative example of the present invention (Poma) Alkaline laccase produced by the genus Ma2 strain has various characteristics shown in Table 1 below.
[0022]
[Table 1]

[0023]
As used herein, “enzyme is stable” means that the enzyme has a relative residual activity of about 85% or more, preferably about 90% or more, more preferably about 95% or more after a certain treatment. That means.
[0024]
No alkaline laccase having the above properties has been known so far.
[0025]
It is a representative example of the alkaline laccase of the present inventionPoma  sp. The enzyme derived from the Ma2 strain is oxidized in the heterocyclic compound or aromatic amine in the pH range of 7 to 11, preferably in the pH range of 8.3 to 10.6, more preferably in the pH range of pH 9.0 to 10.0. The reaction can be carried out, has an optimum pH in the vicinity of pH 10, and has a feature of catalyzing an oxidation reaction in an alkaline region (FIG. 6). The optimum temperature is about 50 ° C. (FIG. 4), and the activity after 30 minutes of heat treatment at various constant temperatures shows a residual activity of almost 95% at 60 ° C., for example. (FIG. 5). Furthermore, the residual activity after 3 hours of treatment at 30 ° C. in various pH buffers showed stability over a wide range of pH (FIG. 7). These results assure the oxidation reaction in various medium and low temperature solutions in the pH range from weak alkaline to alkaline.
[0026]
The alkaline laccase of the present invention can also be used in combination with an enzyme having an optimum reaction pH in the conventional acidic range. That is, by using a combination of a laccase having an optimum reaction pH in a known acidic range and the alkaline laccase of the present invention, a laccase reaction can be performed in a wide pH range from acidic to alkaline. When an enzyme is mixed and used for such a purpose, the mixing ratio of the active amount of the laccase having the optimum reaction pH in the acidic region and the active amount of the alkaline laccase of the present invention is preferably 1:20 to 20: 1. More preferably, it can be used at 1: 2 to 2: 1. Thus, the alkaline laccase of the present invention is also useful for achieving a laccase reaction in a wide pH range.
[0027]
(2) Origin of alkaline laccase
Alkaline laccase, a representative example of the present invention, was deposited with the former genus filamentous fungi, in particular, the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (abbreviated as Life Research Institute) on December 13, 2002.Poma  sp. It is produced by Ma2 strain (Accession number: FERM P-19152) (hereinafter abbreviated as Ma2 strain). The Ma2 strain is a new strain newly isolated from the intestine of maji, as a result of extensive screening of microorganisms that produce laccase from soil, decayed products, and others.
[0028]
The alkaline laccase-producing bacterium of the present invention can be obtained, for example, as follows. Collect samples such as soil, decay, sea water, fresh water, beaches or riverbanks, lump, bottom mud, activated sludge, fish internal organs. The collected sample is mixed well with physiological saline or sterilized water and then allowed to stand to obtain a supernatant. Next, 200 μL of the supernatant is collected, spread on an alkaline solid medium containing a dye, and allowed to stand at 28 ° C. for several days. A colony in which a decoloring halo of the dye is observed around the appearing microbial colony is isolated as an alkaline laccase-producing bacterium.
[0029]
The isolated alkaline laccase producing bacteria can be characterized in more detail for the alkaline laccase to be produced using methods well known to those skilled in the art.
[0030]
Tables 2 and 3 show the taxonomic characteristics of the Ma2 strain isolated by the present inventors. OA [60% buckwheat soba, 12.5g agar, 1L distilled water], PDA [200g potato leachate, 20g sucrose, 15g agar, 1L distilled water], MEA [30g malt extract, 15g agar, distilled Water 1L] represents the type of medium. From the morphological characteristics of the colony characterization and conidia formation structures described in Table 2 and Table 3 (Arx, JA von (1974) The genera of fungi spouting in pure culture, A. R. Gantner. Verlag KG, Vaduz, Germany, page 315, Domsch, KH, Gams, W. and Anderson, TH (1993) Compendium of sol fungi, Volume 1, IHW-Verlag, Eching, Germany, Edition. 860, and Malloch, D. (1981) Moulds-their isolation, culture, and identification, University of T oronto Press, Toronto, USA, page 97),PomaIdentified as a genus,Poma  sp. It was named Ma2.
[0031]
[Table 2]

[0032]
[Table 3]

[0033]
(3) Culture conditions for producing alkaline laccase
The typical conditions for culturing the filamentous fungus producing the alkaline laccase of the present invention will be described. The filamentous fungus of the present invention can be cultured in either liquid culture or solid culture. The medium for culturing the filamentous fungus of the present invention is not particularly limited, and a liquid medium or solid medium containing a commonly used synthetic medium, organic carbon source, and organic nitrogen source or inorganic nitrogen source can be used. As an example of the carbon source, anything that can be assimilated by the filamentous fungus of the present invention may be used. Can be used. Examples of nitrogen sources include okara, amino acids, various peptones, defatted soy flour, corn steep liquor, urea, yeast extract, meat extract, malt extract, and other organic nitrogen sources, as well as inorganic substances such as nitrate, ammonium nitrate, and ammonium sulfate. Nitrogen sources can also be used alone or in combination. If necessary, inorganic salts such as sulfate, phosphate, potassium, calcium, manganese, cobalt, vitamins, nucleic acid-related compounds, etc. are added. obtain. In addition, an appropriate amount of a production inducer may be added to increase enzyme productivity. These medium components only have to have a concentration that does not inhibit the growth of the bacterium, the carbon source is 0.1 to 20% by weight, preferably 1 to 10% by weight, and the nitrogen source is 0.1 to 10% by weight, preferably Is 1 to 5% by weight. When the pH of the medium is set to the alkali side, the pH can be adjusted by adding an appropriate amount of a solution containing caustic soda, sodium carbonate, or sodium bicarbonate previously sterilized to the sterilized medium.
[0034]
The pH of the medium is adjusted to 5.0 to 12.0, preferably 6.0 to 10.0, and sterilized before use. The culture temperature may be any temperature at which filamentous fungi can grow, and is practically 10 to about 40 ° C, preferably 25 to 35 ° C. The culture may be carried out according to the usual culture of mesophilic bacteria, by aeration culture or shaking culture for liquid culture, and stationary culture for solid culture. Although the duration of the culture varies depending on various culture conditions, it is a period in which the target laccase is sufficiently produced. In the case of aeration or shaking culture, 2 to 7 days is usually preferable.
[0035]
(4) Preparation of alkaline laccase from the culture solution
In the case of liquid culture, the alkaline laccase of the present invention is typically secreted outside the cells. Therefore, after the enzyme is produced and accumulated in the medium, preparation of alkaline laccase from the culture solution can be achieved by the following method.
[0036]
That is, after the culture is stopped, insoluble matters such as cells are removed by solid-liquid separation (eg, filtration with filter cloth or filter paper, centrifugation, membrane separation, etc.), and the culture filtrate containing alkaline laccase is recovered. Next, commonly used protein fractionation / separation methods such as dialysis, salting out, membrane concentration, various column chromatography, isoelectric precipitation, precipitation with non-aqueous solvent, adsorption / decolorization with activated carbon, etc. When used in combination, the alkaline laccase of the present invention can be prepared from the culture filtrate. For example, a highly active fraction of alkaline laccase can be prepared by subjecting the culture filtrate obtained above to ultrafiltration, ammonium sulfate fractionation, dialysis, and various chromatographies sequentially.
[0037]
(5) Measurement of alkaline laccase activity
In the present invention, the alkaline laccase activity is determined by the enzyme addition in a color reaction system by oxidative condensation of MBTH (official name: 3-methyl-2-benzothiazolinone hydrazone) catalyzed by alkaline laccase and homovanillyl alcohol. It can be measured later by examining the change in absorbance at 560 nm.
[0038]
The detailed measurement method is as follows. After preincubation of 105.4 μL of 115.4 mmol / L Tris-HCl buffer (pH 9.0) containing 1.5 μmol homovanillyl alcohol and 1.0 μmol MBTH at 30 ° C., 160 μL enzyme solution Are mixed and stirred. Measurement is started 30 seconds after mixing and stirring. The change with time in absorbance at 560 nm from the start of measurement to 30 seconds later is followed. The activity value of alkaline laccase is calculated with the enzyme activity that increases the absorbance by 1 per minute as 1 unit (U; unit).
[0039]
【Example】
The following examples further illustrate the present invention in detail but are not intended to limit the present invention in any way.
[0040]
Example 1
(Poma  sp. Culture of Ma2 strain)
100 mL medium (5% glucose, 0.5% polypeptone, 0.1% yeast extract, 0.2% KH₂PO₄, 0.1% K₂HPO₄0.02% MgSO₄・ 7H₂O, pH 7.0) in a 500 mL round flaskPoma  sp. One platinum loop of Ma2 strain was inoculated and cultured with shaking at 30 ° C. for 7 days. Next, 1 mL of this culture solution was inoculated into a 500 mL round flask containing 100 mL of the above medium, and cultured with shaking at 30 ° C. for 5 days.
[0041]
Example 2
(Partial purification of this alkaline laccase)
Centrifugation (12,000 rpm × 20 min; 4 ° C.) is performed on the culture solution obtained in the above Example, and after solid-liquid separation, the supernatant is filtered. The collected filtrate was combined to obtain a culture filtrate.
[0042]
The culture filtrate was concentrated with a UF (ultrafiltration) membrane and concentrated as a fraction having a molecular weight of 30,000 or more. After adjusting the UF concentrate to pH 7.0 with caustic soda, ammonium sulfate salting out (90% saturation; 4 ° C., standing overnight) is performed, and then 0.7% (W / V; V = the amount of liquid before salting out) Add an amount of celite equivalent to) and suspend well. The salting out cake is obtained by filtering the suspension.
[0043]
The salted-out cake is washed several times with 90% saturated ammonium sulfate at a low temperature, and then an appropriate amount of 20 mmol / L Tris-HCl buffer (pH 7.5) is added and stirred well. Since the salting out protein contained in the salting out cake is dissolved by this addition / stirring operation, the suspension is filtered. The obtained filtrate was dialyzed with 20 mmol / L Tris-HCl buffer (pH 7.5), and this was used as a dialyzed internal solution. The dialyzed solution was concentrated with a UF membrane, and this was used as a partially purified solution.
[0044]
Table 4 shows the specific activity (absorbance at U / 280 nm), yield and degree of purification of the present alkaline laccase in each of the above steps.
[0045]
[Table 4]

[0046]
Example 3
(Check for unity)
A part of the partially purified solution described in Example 2 was subjected to polyacrylamide gel isoelectric focusing, and CBB R-350 (product name: Coomassie Tables, PhasGel Blue R-350 <Amersham) was applied to the gel after electrophoresis. The protein was detected by staining using Bioscience; hereinafter abbreviated as “AB”>, and the activity of alkaline laccase was detected by the color reaction system using homovanillyl alcohol and MBTH.
As a result, the positions of the uniform protein band and alkaline laccase activity band detected on the acidic side at pH 5 or lower coincided, and one kind of alkaline laccase was added to the partially purified solution prepared through the steps of Examples 1 and 2. Confirmed to be included.
[0047]
Example 4
(Isoelectric point of this alkaline laccase)
A part of the partially purified solution described in Example 2 was subjected to isoelectric focusing (hereinafter abbreviated as IEF). In addition, a Rotofor Combination System (manufactured by Bio-Rad Laboratories) was used as an IEF-dedicated device, and Amphorine, blended pH3.5-9.5 (manufactured by AB Corporation; hereinafter, abbreviated as “Ampine”) was used as an amphoteric carrier. .
[0048]
First, 50 mL of an aqueous solution containing 4% partially purified solution and 4% ampholine was subjected to the electrophoresis (initial migration), and a fraction solution in which alkaline laccase activity was strongly detected, No. 1 3 (pH 4.2) and No. 3 4 (pH 4.4) was added to obtain an active fraction. Next, IEF was performed again on about 60 mL of an aqueous solution containing the partially purified solution (2 mL) and the active fraction (total amount) (re-migration). About the fraction liquid obtained by re-electrophoresis, pH value and activity value were measured, respectively. The result is shown in FIG. As can be seen from FIG. 1, the isoelectric point of the alkaline laccase is about 3.7.
[0049]
Example 5
(Molecular weight of the alkaline laccase)
A part of the partially purified solution described in Example 2 was applied to a GFC (gel filtration chromatography) column, and the molecular weight of the alkaline laccase was measured. As the GFC column, HiPrep 26/60 Sephacryl S-100 HR (manufactured by AB Corporation; hereinafter abbreviated as this column) was used.
[0050]
A partially purified solution was applied to this column that had been equilibrated in advance with 20 mmol / L Tris-HCl buffer (containing 150 mmol / L NaCl; pH 7.5), and then the same buffer as that used for equilibration was used. Elution was performed at a flow rate of 0.9 mL / min. The eluate fractionation (0.98 mL / tube) was started after the time when the flow rate reached 86 mL, and then the absorbance at 280 nm and the alkaline laccase activity were measured for the obtained fraction solution. The alkaline laccase activity was measured visually by evaluating the color intensity after the above color reaction at room temperature (26 ° C.) for 20 minutes as follows: colorless and transparent 0 point; very light purple 0.5 points; light purple (3 grades) 1.0, 1.5, 2.0 points; purple (2 grades) 2.5, 3.0 points; dark purple 3.5 points; Black purple 4.0 points. The result is shown in FIG.
[0051]
Next, the elution position of the active peak was applied to a molecular weight standard curve (FIG. 3) prepared in advance based on the elution position of the standard substance in this column, and the molecular weight was calculated. As a result, the molecular weight of the alkaline laccase was about 42 to 43 kDa. Blue dextran 2000, bovine serum albumin, chicken egg ovalbumin, bovine pancreatic chymotrypsinogen A, and bovine pancreatic ribonuclease A (above AB) were used as standard substances.
[0052]
Example 6
(Operational temperature range and thermal stability of the alkaline laccase)
(1) Optimum temperature range
The activity of this alkaline laccase in the partially purified solution obtained in Example 2 was measured under various temperature conditions (30, 40, 45, 50, 60, 70 ° C.) in the color development reaction system. The results are shown in FIG. The horizontal axis in the figure represents the reaction temperature (° C.), and the vertical axis represents the relative activity (%) when the maximum activity value is 100. The optimum temperature of the alkaline laccase is about 50 ° C.
[0053]
(2) Thermal stability
The alkaline laccase of the partially purified liquid obtained in Example 2 was subjected to tris-hydrochloric acid buffer solution (0, 30, 40, 50, 60, 65, 70, 80, 90 ° C.) under various temperature conditions. After pretreatment by incubation for 30 minutes in pH 7.5), the residual activity was measured in the color reaction system. The results are shown in FIG. The horizontal axis in the figure represents the treatment temperature (° C.), and the vertical axis represents the relative residual activity (%) when the maximum activity value is 100. The alkaline laccase is stable with about 73% of the enzyme activity when the treatment temperature is 65 ° C. and about 56% or more when the temperature is 70 ° C. The alkaline laccase retains about 95% of the enzyme activity at a treatment temperature of 60 ° C.
[0054]
Example 7
(Optimal pH and stable pH range of the alkaline laccase)
(1) Optimum pH
Instead of the 100 mmol / L Tris-HCl buffer (pH 9.0) used in the color reaction system, sodium acetate-hydrochloric acid buffer, acetic acid buffer, potassium phosphate buffer, Tris-hydrochloric acid are used depending on the pH range. The pH is adjusted to 1.0 to 11.1 (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 using a buffer solution, glycine-salt-caustic soda buffer solution. , 7.5, 8.3, 9.0, 10.0, 10.4, 10.6, 11.0, 12.5), and the alkali of the partially purified solution obtained in Example 2 The activity of laccase was measured. The results are shown in FIG. The horizontal axis in the figure represents the reaction pH, and the vertical axis represents the relative activity (%) when the maximum activity value is 100. The optimum pH of the alkaline laccase is about 10.
[0055]
(2) Stable pH range
The pH of the alkaline laccase of the partially purified solution obtained in Example 2 was adjusted to 1.0 to 12.5 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.5) and incubated at 30 ° C. for 3 hours to obtain a pH treatment solution. Next, the residual activity of each pH treatment solution was examined using the color development system. The results are shown in FIG. The horizontal axis in the figure represents the treatment pH, and the vertical axis represents the relative residual activity (%) when the maximum activity value is 100. The stable pH range of the alkaline laccase is 4.0 to 9.0.
[0056]
Example 8
(Myrothecium  verucariaComparison with alkaline laccase derived from: optimal pH)
In the method described in Example 7 (1), instead of the partially purified solutionMyrothecium  verucaria  Using a partially purified solution of alkaline laccase derived from 24G-4 strain (hereinafter abbreviated as 24G-4 strain), the pH of the enzyme was adjusted to the above-described method for modifying the color reaction system (final ethanol concentration in the reaction solution: 1. 5%). The results are shown in FIG. The horizontal axis in the figure represents the reaction pH, and the vertical axis represents the relative activity (%) when the maximum activity value is 100. Under the above reaction conditions, the optimum pH of the 24G-4 strain-derived alkaline laccase is about 8.5.
[0057]
In the method described in Example 7 (1), instead of the partially purified solution, a partially purified solution of 24G-4 strain-derived alkaline laccase was used, and the 100 mmol / L Tris-HCl buffer solution used in the above color reaction system was used. Under the conditions where the pH value was adjusted to 8.0 to 8.9 (8.0, 8.3, 8.5, 8.7, 8.9), the optimum pH of the enzyme was examined. The results are shown in FIG. The horizontal axis in the figure represents the reaction pH, and the vertical axis represents the relative activity (%) when the maximum activity value is 100. The optimum pH of the 24G-4 strain-derived alkaline laccase in the color development reaction system is about 8.3.
[0058]
Example 9
(Substrate specificity of alkaline laccase (1):p-Oxygen consumption relative to phenylenediamine)
Using the partially purified solution described in Example 2 as an alkaline laccase crude enzyme solution,p-Phenylenediamine was used as a substrate, and the oxygen consumption due to oxidation of the substrate was examined using an oxygen electrode method in which a Clark-type oxygen electrode was used to convert it into an electrical signal. Specifically, 1485 μL of 101 mmol / L Tris-HCl buffer (pH 8.0) containing a crude enzyme solution (0.84 U) was equilibrated with air at 30 ° C. for 10 minutes. Thenp-The reaction was started by adding 15 μL of a phenylenediamine solution (substrate content: 1.5 μmol), and the oxygen consumption was measured. The blank value was examined by replacing the partially purified solution with 20 mmol / L Tris-HCl buffer (pH 7.5). As a result, it was confirmed that there was a voltage change of 0.011 mV / 10 min.
[0059]
Example 10
(Substrate specificity of alkaline laccase (2): Oxygen consumption activity for each substrate)
Substrate specificity was investigated using the partially purified solution described in Example 2 as the alkaline laccase crude enzyme solution. Specifically, 1300 μL of 231 mmol / L glycine-salt-caustic soda buffer (pH 10.0) containing 7.5 μmol of substrate was equilibrated with air at 50 ° C. for 10 minutes. Subsequently, the reaction was started by adding 200 μL of a crude enzyme solution (enzyme content: 0.08 U), and the oxygen consumption was measured. As substrates, phenolic compounds (syringic acid, homovanillyl alcohol), aromatic amine compounds (p-Phenylenediamine, vanillylamine), heterocyclic compounds (MBTH, 4-aminoantipyrine, ABTS (official name: 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), others (ascorbine The blank value was examined by replacing the partially purified solution with 20 mmol / L Tris-HCl buffer (pH 7.5), and the results are shown in Table 5. From Table 5, the partially purified alkali was used. It can be seen that laccase acts more strongly on heterocyclic compounds and aromatic amine compounds than phenol compounds.
[0060]
[Table 5]

[0061]
Example 11
(Substrate specificity of alkaline laccase (3): Oxidative coloration for each substrate)
Using the partially purified solution described in Example 2 as an alkaline laccase crude enzyme solution, the change over time in the absorption spectrum of the color reaction accompanying the substrate oxidation catalyzed by the alkaline laccase was examined (scanning method applied). Specifically, 1080 μL of 111.1 mmol / L glycine-salt-caustic soda buffer (pH 10.0) containing 1.2 μmol of substrate was placed in a cuvette, pre-warmed at 50 ° C. for 5 minutes, Add 120 μL of enzyme solution (enzyme content: 0.04 U) and stir well. After correcting for the baseline 30 seconds after the addition of the crude enzyme solution, the first scanning was started (2 minutes after the addition of the crude enzyme solution). The second and subsequent scans were performed at intervals of 4 minutes from the start of the first scan (that is, after 6, 10, 14, 18, 22, 26, 30 minutes after addition of the crude enzyme solution). The blank spectrum was examined by replacing the partially purified solution with 20 mmol / L Tris-HCl buffer (pH 7.5) under the above conditions. As substrates, hydroquinone, homovanillyl alcohol, metanephrine,p-Aminophenol, vanillylamine,p-Phenylenediamine, MBTH, 3-amino-1H-1,2,4-Triazole was used. The scanning conditions were a scanning wavelength range of 200 to 700 nm, a scanning wavelength interval of 5 nm, and a scanning time of about 40 seconds.
[0062]
The results are shown in such a form that all the absorption spectra with respect to the test substrate over time are overlapped for each substrate (FIGS. 10 to 17). Each figure corresponds to the first to final graphs in order from the bottom spectrum graph. From these figures, it can be seen that, with some exceptions, the absorption value increased with time, and the oxidation color reaction of the substrate progressed. At the same time, partially purified alkaline laccase was more heterocyclic than phenolic compounds. It is suggested that they act strongly on compounds and aromatic amine compounds.
[0063]
【The invention's effect】
According to the present invention, a novel alkaline laccase having an optimum pH in an alkaline region and excellent in heat resistance under alkaline conditions, a production method thereof, and a production fungus are provided. The alkaline laccase of the present invention has an optimum pH in a higher alkaline range than conventional alkaline laccases and is excellent in heat resistance under alkaline conditions. Useful for processes and the like. Since the alkaline laccase of the present invention is produced by culturing filamentous fungi, it can provide a large amount of alkaline laccase at a low cost, and can be widely used for industrial applications or reagents.
[Brief description of the drawings]
FIG. 1 is a graph showing the results of isoelectric focusing of the alkaline laccase of the present invention.
FIG. 2 is a graph showing the results of elution of an alkaline laccase of the present invention by gel filtration chromatography.
FIG. 3 is a graph showing the molecular weight of the alkaline laccase of the present invention calculated based on the elution peak of the alkaline laccase of the present invention by gel filtration chromatography.
FIG. 4 is a graph showing the optimum temperature of the alkaline laccase of the present invention.
FIG. 5 is a graph showing the thermal stability of the alkaline laccase of the present invention.
FIG. 6 is a graph showing the optimum pH of the alkaline laccase of the present invention.
FIG. 7 is a graph showing the pH stability of the alkaline laccase of the present invention.
[Fig. 8] Fig. 8 shows a conventional configuration.Myrothecium  verucaria  It is a figure which shows the graph which shows the optimal pH of 24G-4 stock | strain origin alkaline laccase.
FIG. 9 shows a conventional measurement measured at pH 8.0 to 9.0.Myrothecium  verucaria  It is a figure which shows the graph which shows the optimal pH of 24G-4 stock | strain origin alkaline laccase.
FIG. 10 is a graph showing the change in absorption spectrum over time for the oxidation coloration of the alkaline laccase of the present invention to hydroquinone.
FIG. 11 is a graph showing the change in absorption spectrum over time for the oxidation coloration of the alkaline laccase of the present invention to homovanyl alcohol.
FIG. 12 is a graph showing the time course change of the absorption spectrum for the oxidative coloration of the alkaline laccase of the present invention to metanephrine.
FIG. 13 shows the alkaline laccase of the present invention.p-It is a figure which shows the time-dependent change of the absorption spectrum about the oxidation coloring with respect to aminophenol.
FIG. 14 is a graph showing the change in absorption spectrum over time for the oxidation coloration of the alkaline laccase of the present invention to vanillylamine.
FIG. 15 shows the alkaline laccase of the present invention.pIt is a figure which shows the time-dependent change of the absorption spectrum about the oxidation coloring with respect to-phenylenediamine.
FIG. 10 is a graph showing the time course change of the absorption spectrum for the oxidative coloration of the alkaline laccase of the present invention with respect to MBTH.
FIG. 10 shows 3-amino-1 of the alkaline laccase of the present invention.HIt is a figure which shows the time-dependent change of the absorption spectrum about the oxidation coloring with respect to -1,2,4-triazole.

Claims (7)

The following characteristics:
(1) Molecular weight: about 42,000-43,000;
Alkaline laccase produced by filamentous fungi belonging to the genus Forma , having (2) optimum pH: about 10; and (3) substrate specificity: strongly acting on heterocyclic compounds or aromatic amines. The following characteristics:
(4) Range of optimum working temperature: the optimum temperature is 50 ° C;
(5) Thermal stability: stable to about 60 ° C. when held at pH 7.5 for 30 minutes;
(6) Stable pH range: when treated at 30 ° C. for 3 hours, pH 4.0-9.0; and (7) Isoelectric point: about 3.7;
The alkaline laccase according to claim 1, comprising: Pharma sp. Ma2 strain (Accession Number: FERM P-19152) is produced, alkaline laccase of claim 1. The following characteristics:
(1) Molecular weight: about 42,000-43,000;
(2) Optimal pH: about 10; and (3) Substrate specificity: strongly acting on heterocyclic compounds or aromatic amines;
A method for producing an alkaline laccase having a method comprising: culturing a filamentous fungus belonging to the genus Forma that produces the alkaline laccase; and separating and purifying the alkaline laccase from the culture. The alkaline laccase has the following characteristics:
(4) Range of optimum working temperature: the optimum temperature is 50 ° C;
(5) Thermal stability: stable to about 60 ° C. when held at pH 7.5 for 30 minutes;
(6) Stable pH range: when treated at 30 ° C. for 3 hours, pH 4.0-9.0; and (7) Isoelectric point: about 3.7;
The method of claim 4 , comprising: The filamentous fungus is Poma sp. The method according to claim 4 , wherein the strain is Ma2 strain (Accession number: FERM P-19152). The following characteristics:
(1) Molecular weight: about 42,000-43,000;
(2) Optimal pH: about 10; and (3) Substrate specificity: strongly acting on heterocyclic compounds or aromatic amines;
A filamentous fungi you produce alkaline laccase with, Phoma sp belonging to the former (Phoma) genus. Ma2 strain (Accession number: FERM P-19152) .

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