JP3768755B2 - Efficient method for remediation of environment contaminated with organic compounds using microorganisms - Google Patents

Description

単位はppm、検出器の検出限界は0.1ppm、
ＮＤ：検出限界以下（≦0.1ppm）
Ahp：アルキルヒドロペルオキシドレダクターゼ
（実施例３）
JM1株のDCE分解における、共発現アルキルヒドロペルオキシドレダクターゼの効果（液体系）
上記のTCE分解と同じく、その他の塩素置換エチレンを分解する際にも、共発現させたアルキルヒドロペルオキシドレダクターゼにより、分解の効率化が達成できることを検証した。利用した菌は、実施例２に記載するアルキルヒドロペルオキシドレダクターゼ遺伝子組み換えJM1株であり、宿主のJM1株（FERM BP-5352）の分解処理速度を対比させた。
【００５８】
本例の試験においては、分解対象物質は、cis-1,2-ジクロロエチレン（cis-1,2-DCE）、trans-1,2-ジクロロエチレン、及び1,１-ジクロロエチレン（1,1-DCE）である。各ジクロロエチレンについて、実施例2と同様に、アルキルヒドロペルオキシドレダクターゼ遺伝子組み換えJM1株の菌懸濁液、宿主のJM1株の菌懸濁液、ならびに、対照としてM9培地のみをバイアル瓶中に注入した３本をそれぞれ用意した。各DCEについて、各バイアル瓶中の液中濃度が5.0ppmになるようにそれぞれ添加した。DCEを加えた後、各バイアル瓶をテフロンライナー付きブチルゴム栓及びアルミシールで密栓した。
【００５９】
これら密栓したバイアル瓶を30℃で振とうし、バイアル瓶気相部分のDCE濃度を一定時間後にガスクロマトグラフィーで測定した。測定の後、栓を外して一旦開放し、再び、初期と同程度の液中濃度となるDCE飽和水溶液所定量を加えた後に密栓し直した。ただし、対照のバイアル瓶は、初期状態のままとした。次いで、再密栓したバイアル瓶を30℃で振とうし、一定時間が経過した時点で、バイアル瓶気相部分のDCE濃度をガスクロマトグラフィーで測定した。
【００６０】
上記の測定と再密栓の操作を繰り返し、DCEの繰り返し分解における、共発現させたアルキルヒドロペルオキシドレダクターゼの効果を評価した。測定は基本的に実施例2と同様に行い、具体的には、1回目（1時間後）、2回目（1.5時間後、合計2.5時間後）、3回目（2時間後、合計4.5時間後）、4回目（2.5時間後、合計7時間後）、5回目（3時間後、合計10時間後）、6回目（12時間後、合計22時間後）の計６回の測定を行った。なお、この間、菌を維持するために、前記する1回目、3回目、5回目の測定を行う際、合計3回、エネルギー源としてホルムアルデヒドを各回菌懸濁液中において最終濃度1mMとなるように、バイアル瓶中に加えた。表２に、cis-1,2-DCEに関する測定結果を、表３に、cis-1,2-DCEに関する測定結果を、表４に、1,1-DCEに関する測定結果をそれぞれ示す。
【００６１】
表２に示す結果のとおり、アルキルヒドロペルオキシドレダクターゼを共発現していない菌では、3回目（合計4.5時間後）において、未分解のcis-1,2-DCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現してる菌では、未分解のcis-1,2-DCEは測定されていない。4回目（合計7時間後）以降、アルキルヒドロペルオキシドレダクターゼを共発現してる菌でも、未分解のcis-1,2-DCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現していない菌において測定される未分解cis-1,2-DCE量より、有意に低い量である。この比較から、cis-1,2-DCEの分解においても、共発現してるアルキルヒドロペルオキシドレダクターゼにより、明らかに分解処理の効率化、あるいは、分解効率低下の抑制が図られていると判断される。
【００６２】
表３に示す結果のとおり、アルキルヒドロペルオキシドレダクターゼを共発現していない菌では、３回目（合計4.5時間後）以降において、未分解のtrans-1,2-DCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現してる菌では、未分解のtrans-1,2-DCEは測定されていない。4回目（合計7時間後）以降、アルキルヒドロペルオキシドレダクターゼを共発現してる菌でも、未分解のtrans-1,2-DCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現していない菌において測定される未分解trans-1,2-DCE量より、有意に低い量である。この比較から、trans-1,2-DCEの分解においても、共発現してるアルキルヒドロペルオキシドレダクターゼにより、明らかに分解処理の効率化、あるいは、分解効率低下の抑制が図られていると判断される。
【００６３】
表４に示す結果のとおり、アルキルヒドロペルオキシドレダクターゼを共発現していない菌では、3回目（合計4.5時間後）以降において、未分解の1,1-DCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現してる菌では、未分解の1,1-DCEは測定されていない。4回目（合計7時間後）以降、アルキルヒドロペルオキシドレダクターゼを共発現してる菌でも、未分解の1,1-DCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現していない菌において測定される未分解1,1-DCE量より、有意に低い量である。この比較から、1,1-DCEの分解においても、共発現してるアルキルヒドロペルオキシドレダクターゼにより、明らかに分解処理の効率化、あるいは、分解効率低下の抑制が図られていると判断される。
【００６４】
従って、アルキルヒドロペルオキシドレダクターゼを共発現させることにより、微生物による各DCEの分解効率を高くでき、また、その高い効率を長期間維持できる効果が得られることが示された。
【００６５】
【表２】

単位はppm、検出器の検出限界は0.1ppm、
ＮＤ：検出限界以下（≦0.1ppm）
Ahp：アルキルヒドロペルオキシドレダクターゼ
【００６６】
【表３】

単位はppm、検出器の検出限界は0.1ppm、
ＮＤ：検出限界以下（≦0.1ppm）
Ahp：アルキルヒドロペルオキシドレダクターゼ
【００６７】
【表４】

単位はppm、検出器の検出限界は0.1ppm、
ＮＤ：検出限界以下（≦0.1ppm）
Ahp：アルキルヒドロペルオキシドレダクターゼ
本結果より、TCEの分解処理に加えて、JM1株（FERM BP-5352）を用いる液体系のDCEの分解処理においても、アルキルヒドロペルオキシドレダクターゼを共発現させる効果が示された。また、前記のアルキルヒドロペルオキシドレダクターゼを共発現させる効果自体は、DCEの種類、塩素置換の位置に実質的に依存していないことが分かる。
【００６８】
（実施例４）
KK01株のTCE分解における、共発現アルキルヒドロペルオキシドレダクターゼの効果（液体系)
実施例１に記載の方法.で調製したアルキルヒドロペルオキシドレダクターゼ遺伝子組み換えプラスミドをエレクトロポーレーション（Bio-Rad）法により、バルクホルデリア・セパシア KK01株（FERM BP-4235）に導入した。得られる形質転換株は、宿主のKK01株（FERM BP-4235）と同様にTCE分解能を有し、新たに導入された大腸菌由来のアルキルヒドロペルオキシドレダクターゼ遺伝子から、アルキルヒドロペルオキシドレダクターゼの発現がなされる。
【００６９】
エレクトロポーレーションに供する菌の培養条件、集菌条件などは、市販のエレクトロポーレーション装置に添付されるインストラクションに従った。装置の設定は、キャパシタンス25μF、抵抗値200Ωとし、供与するDNA量は1μgとした。また、導入菌株の懸濁液は、OD₆₀₀が10となるように菌体密度を調製した液を100μl用いた。キュベットは0.2mmギャップのものを用い（Bio-Rad）、印可電圧は2.5kVとした。ここでキュベットは4℃にあらかじめ冷却しておいたものを用いた。エレクトロポーレーションの際の時定数は4.7 msであった。
【００７０】
上記の条件で、アルキルヒドロペルオキシドレダクターゼ遺伝子組み換えプラスミドを導入したKK01株の形質転換体の選択は、上記実施例２と同じく、広宿主域プラスミドベクタpBBR122自体が有する選択マーカーを利用し、100μg/ml濃度のクロラムフェニコルを含有した寒天M9培地（0.5％グルタミン酸ナトリウム含有）を用いた。
【００７１】
寒天M9培地（0.1％酵母エキス及び2mMフェノール含有）上でコロニーを形成する菌株を、アルキルヒドロペルオキシドレダクターゼ遺伝子組み換えKK01株として選別した。採取したアルキルヒドロペルオキシドレダクターゼ遺伝子組み換えKK01株を、寒天を除き、前記寒天M9培地と同じ組成の液体M9培地200mlに接種し、500ml容振盪フラスコ中、25℃で振盪培養を行った。対数増殖後期にあたる、培養開始から２４時間を経過した時点で、菌体を遠心分離により集菌した。集菌した菌体を、等量の炭素源を含まないM9培地に再懸濁し、27.5ml容バイアル瓶に10mlの菌懸濁液を注入した。
【００７２】
また、別途、アルキルヒドロペルオキシドレダクターゼ遺伝子組み換えを施していないKK01株、すなわち、宿主のKK01株（FERM BP-4235）自体も、同様に培養、集菌、懸濁し、その10mlの菌懸濁液を27.5ml容バイアル瓶に注入した。
【００７３】
これら菌懸濁液を注入したバイアル瓶２本と、対照としてM9培地のみ10ml加えたバイアル瓶１本、合計3本のバイアル瓶に、各バイアル瓶中の液中濃度が12ppm程度になるようにTCE飽和水溶液所定量を加え、テフロンライナー付きブチルゴム栓及びアルミシールで密栓した。
【００７４】
これら密栓したバイアル瓶を30℃で振とうし、バイアル瓶気相部分のTCE濃度を一定時間後にガスクロマトグラフィーで測定した。測定の後、栓を外して一旦開放し、再び、初期と同程度の液中濃度となるTCE飽和水溶液所定量を加えた後に密栓し直した。ただし、対照のバイアル瓶は、初期状態のままとした。次いで、再密栓したバイアル瓶を30℃で振とうし、一定時間が経過した時点で、バイアル瓶気相部分のTCE濃度をガスクロマトグラフィーで測定した。
【００７５】
上記の測定と再密栓の操作を繰り返し、TCEの繰り返し分解における、共発現させたアルキルヒドロペルオキシドレダクターゼの効果を評価した。具体的には、1回目：1時間後、2回目：1時間後（合計2時間後）、3回目：1.5時間後（合計3.5時間後）、4回目：1.5時間後（合計5時間後）、5回目：1.5時間後（合計6.5時間後）、6回目：2時間後（合計8.5時間後）、7回目：2時間後（合計10.5時間後）、8回目：2時間後（合計12.5時間後）、9回目：10時間後（合計22.5時間後）の計９回の測定を行った。その他の操作は、上記実施例２に記載の手順に準じた。表５に、測定結果を示す。
【００７６】
【表５】

単位はppm、検出器の検出限界は0.1ppm、
ＮＤ：検出限界以下（≦0.1ppm）
Ahp：アルキルヒドロペルオキシドレダクターゼ
表５に示す結果のとおり、アルキルヒドロペルオキシドレダクターゼを共発現していない菌では、７回目（合計8.5時間後）において、未分解のTCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現してる菌では、未分解のTCEは測定されていない。8回目（合計12.5時間後）以降、アルキルヒドロペルオキシドレダクターゼを共発現してる菌でも、未分解のTCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現していない菌において測定される未分解TCE量より、有意に低い量である。この比較から、共発現してるアルキルヒドロペルオキシドレダクターゼにより、明らかに分解処理の効率化、あるいは、分解効率低下の抑制が図られていると判断される。従って、上記のJM1株と同じくKK01株（FERM BP-4235）においても、アルキルヒドロペルオキシドレダクターゼを共発現させることにより、微生物によるTCEの分解効率を高くでき、また、その高い効率を長期間維持できる効果が得られることが示された。本結果より、KK01株（FERM BP-4235）においても、液体系のTCEの分解処理におけるアルキルヒドロペルオキシドレダクターゼの効果が示された。
【００７７】
（実施例５）
アルキルヒドロペルオキシドレダクターゼ遺伝子の取得（２）
アルキルヒドロペルオキシドレダクターゼ遺伝子及びTCE分解酵素遺伝子とを組み込むプラスミドの構築
大腸菌HB101株を50mlのLB培地で37℃、終夜培養の後、常法により全DNAを調製した。この全DNAをテンプレートとして、以下に示すプライマ３およびプライマ４を用いてPCRを行い、アルキルヒドロペルオキシドレダクターゼ遺伝子の増幅を行った。
プライマ３
5'-AACTTCTAGATTTGATAATGGAAACGC-3'
プライマ４（プライマ２と同塩基配列）
5'-GCAATTGCAGGTGAAGCTTACTTC-3'
上述の実施例１に記載するPCR操作に準じ、LA-PCRキット（宝酒造）を用い、反応液組成、反応条件はキット添付のインストラクションに従った。PCRにより約2500bpの増幅産物を得ることができた。このPCR産物を制限酵素XbaIとHindIIIで完全分解し、特開平６−２９６７１１号公報に開示されているKK01株（FERM BP-4235）由来のTCE分解酵素遺伝子を含む発現ベクタpSE280（インビトロジェン）の制限酵素XbaIとHindIIIによる完全分解物に、ライゲーションキット（宝酒造）を用いてライゲーション反応を行った。得られるプラスミドは、大腸菌HB101株由来のアルキルヒドロペルオキシドレダクターゼ遺伝子およびKK01株（FERM BP-4235）由来のTCE分解酵素遺伝子を含むものとなる。このプラスミドを用いて、宿主のHB101株（宝酒造）を形質転換した。形質転換体の選択には、発現ベクタpSE280自体のアンピシリン耐性選択マーカーを利用し、100μg/ml濃度のアンピシリンを含有した寒天LB培地を用いた。
【００７８】
採取した形質転換体において、アルキルヒドロペルオキシドレダクターゼ遺伝子がKK01株（FERM BP-4235）由来のTCE分解酵素遺伝子を含む発現ベクタpSE280に設計通りに挿入されているかのチェックは、アルカリ法により回収した組み換えプラスミドを制限酵素XbaIとHindIIIで完全分解することにより行った。その確認を経て、アルキルヒドロペルオキシドレダクターゼ遺伝子が挿入された組み換えプラスミドを保有する形質転換体を得ることができた。この選別された形質転換体を培養し、培養菌体を破砕し、プラスミドを分離回収することで、アルキルヒドロペルオキシドレダクターゼ遺伝子およびKK01株（FERM BP-4235）由来のTCE分解酵素遺伝子を含む組み換えプラスミドを得ることができた。
【００７９】
（実施例６）
TCE分解遺伝子組み換え大腸菌のTCE分解における、共発現アルキルヒドロペルオキシドレダクターゼの効果（液体系)
実施例５の手法で調製した、KK01株（FERM BP-4235）由来のTCE分解酵素遺伝子及びアルキルヒドロペルオキシドレダクターゼ遺伝子組み換えプラスミドを大腸菌HB101株（宝酒造）に導入した形質転換体を用いて、TCE分解における、共発現アルキルヒドロペルオキシドレダクターゼの効果を評価した。
【００８０】
前記TCE分解酵素遺伝子およびアルキルヒドロペルオキシドレダクターゼ遺伝子組み換え大腸菌HB101株を寒天LB培地（100μg/mlアンピシリン含有）上に接種し、再度コロニーを選択した。コロニーを形成した菌株を、寒天を除き、前記寒天LB培地（100μg/mlアンピシリン含有）と同じ組成の液体LB培地200mlに接種し、500ml容振盪フラスコ中、37℃で振盪培養を行った。対数増殖後期にあたる、培養開始から12時間を経過する時点で、菌体を遠心分離により集菌し、等量の炭素源を含まないM9培地に再懸濁し、10mlの菌懸濁液を27.5ml容バイアル瓶に注入した。
また、アルキルヒドロペルオキシドレダクターゼ遺伝子で組み換えていないTCE分解遺伝子組み換えHB101株も同様に培養、集菌、懸濁し、10mlの菌懸濁液を27.5ml容バイアル瓶に注入した。これらのバイアル瓶と、対照としてM9培地のみ10ml加えたバイアル瓶合計3本に、バイアル瓶中の液中濃度が12ppm程度になるようにTCE飽和水溶液を加え、テフロンライナー付きブチルゴム栓及びアルミシールで密栓した。
【００８１】
これら密栓したバイアル瓶を30℃で振とうし、バイアル瓶気相部分のTCE濃度を一定時間後にガスクロマトグラフィーで測定した。測定の後、栓を外して一旦開放し、再び、初期と同程度の液中濃度となるTCE飽和水溶液所定量を加えた後に密栓し直した。ただし、対照のバイアル瓶は、初期状態のままとした。次いで、再密栓したバイアル瓶を30℃で振とうし、一定時間が経過した時点で、バイアル瓶気相部分のTCE濃度をガスクロマトグラフィーで測定した。
【００８２】
上記の測定と再密栓の操作を繰り返し、TCEの繰り返し分解における、共発現させたアルキルヒドロペルオキシドレダクターゼの効果を評価した。具体的には、1回目：1時間後、2回目：1時間後（合計2時間後）、3回目：1.5時間後（合計3.5時間後）、4回目：1.5時間後（合計5時間後）、5回目：1.5時間後（合計6.5時間後）、6回目：2時間後（合計8.5時間後）、7回目：2時間後（合計10.5時間後）、8回目：2時間後（合計12.5時間後）、9回目：10時間後（合計22.5時間後）の計９回の測定を行った。その他の操作は、上記実施例２に記載の手順に準じた。表６に、測定結果を示す。
【００８３】
【表６】

単位はppm、検出器の検出限界は0.1ppm、
ＮＤ：検出限界以下（≦0.1ppm）
Ahp：アルキルヒドロペルオキシドレダクターゼ
表６に示す結果のとおり、アルキルヒドロペルオキシドレダクターゼを共発現していない菌では、６回目（合計8.5時間後）において、未分解のTCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現してる菌では、未分解のTCEは測定されていない。7回目（合計10.5時間後）以降、アルキルヒドロペルオキシドレダクターゼを共発現してる菌でも、僅かに未分解TCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現していない菌において測定される未分解TCE量より、格段に低い量である。この比較から、共発現してるアルキルヒドロペルオキシドレダクターゼにより、明らかに分解処理の効率化、あるいは、分解効率低下の抑制が図られていると判断される。従って、上記のKK01株（FERM BP-4235）と同じくKK01株（FERM BP-4235）由来のTCE分解酵素遺伝子組み換えプラスミドにより形質転換された大腸菌HB101株においても、アルキルヒドロペルオキシドレダクターゼを共発現させることにより、微生物によるTCEの分解効率を高くでき、また、その高い効率を長期間維持できる効果が得られることが示された。
【００８４】
本結果より、TCE分解酵素遺伝子組み換え大腸菌においても、液体系のTCEの分解処理におけるアルキルヒドロペルオキシドレダクターゼの効果が示された。
【００８５】
（実施例７）
JM1株のTCE分解における、共発現アルキルヒドロペルオキシドレダクターゼの効果（土壌系）
上記実施例２に示した液体系と同様に、TCE汚染土壌浄化に対しても、JM1株のTCE分解において、共発現アルキルヒドロペルオキシドレダクターゼが効果示すことを下記のとおり評価した。
【００８６】
佐原通し砂（含水比13%、未滅菌）を68ml容バイアル瓶15本に各50g入れ、土壌中の濃度が5ppmとなるようにTCE飽和水溶液を加え、密栓して15℃で3日間静置して擬似TCE汚染土壌試料を作成した。
【００８７】
その後、実施例２の手順に準じ、調製したアルキルヒドロペルオキシドレダクターゼ遺伝子組み換えJM1株の菌懸濁液をバイアル瓶に5ml注入した。また、アルキルヒドロペルオキシドレダクターゼ遺伝子で組み換えていない宿主JM1株（FERM BP-5352）の菌懸濁液についても、同様に調製し、バイアル瓶に5ml注入した。さらに、対照として、もう1本にはM9培地のみ5ml添加した。なお、菌懸濁液の静置及びTCE分解実験は実際の土壌温度に近い20℃で行った。
【００８８】
TCE分解実験は、バイアル瓶に菌懸濁液を注入した後、土壌中の液体部分にTCEがすべて溶解した場合の濃度が10ppmとなるようにTCE飽和水溶液を加え、テフロンライナーブチルゴム栓及びアルミキャップで完全密封した。これら密栓したバイアル瓶を20℃で静置し、バイアル瓶気相部分のTCE濃度を一定時間後にガスクロマトグラフィーで測定した。測定の後、栓を外して一旦開放し、再び、初期と同程度の液中濃度となるTCEガスを加えた後に密封し直した。ただし、対照のバイアル瓶は、初期状態のままとした。次いで、再密封したバイアル瓶を20℃で静置し、一定時間が経過した時点で、バイアル瓶気相部分のTCE濃度をガスクロマトグラフィーで測定した。
【００８９】
上記の測定と再密栓の操作を繰り返し、TCEの繰り返し分解における、共発現させたアルキルヒドロペルオキシドレダクターゼの効果を評価した。具体的には、1回目：2時間後、2回目：3時間後（合計5時間後）、3回目：4時間後（合計9時間後）、4回目：5時間後（合計14時間後）、5回目：10時間後（合計24時間後）の計５回の測定を行った。なお、1回目、3回目、5回目のTCEを添加した後、密封する際に、エネルギー源としてのホルムアルデヒドを各最終濃度1mMとなるように加えた。その他の操作は、上記実施例２に記載の手順に準じた。表７に、測定結果を示す。
【００９０】
【表７】

単位はppm、検出器の検出限界は0.01ppm、
ＮＤ：検出限界以下（≦0.01ppm）
Ahp：アルキルヒドロペルオキシドレダクターゼ
表７に示す結果のとおり、アルキルヒドロペルオキシドレダクターゼを共発現していない菌では、３回目（合計９時間後）において、未分解のTCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現してる菌では、未分解のTCEは測定されていない。4回目（合計14時間後）以降、アルキルヒドロペルオキシドレダクターゼを共発現してる菌でも、若干の未分解TCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現していない菌において測定される未分解TCE量より、格段に低い量である。この比較から、共発現してるアルキルヒドロペルオキシドレダクターゼにより、明らかに分解処理の効率化、あるいは、分解効率低下の抑制が図られていると判断される。本結果より、JM1株を用いた土壌系のTCEの分解処理においても、共発現アルキルヒドロペルオキシドレダクターゼの効果が示された。すなわち、汚染土壌浄化においても、浄化処理が長期にわたる際にしばしば起こる微生物の示す分解活性の経時的な低下、失活を、アルキルヒドロペルオキシドレダクターゼを共発現させることで、効果的に抑制できる効果が示された。
【００９１】
（実施例８）
JM1株のTCE分解における、共発現アルキルヒドロペルオキシドレダクターゼの効果（気相系）
多孔質セルロース担体（平均ポアサイズ約500mm、直径約5mm）をオートクレーブにて滅菌し、室温以下に冷却したものをバイアル瓶3本に詰めた。実施例２に記載の手順に従い、調製したアルキルヒドロペルオキシドレダクターゼ遺伝子組み換えJM1株の菌懸濁液をバイアル瓶に15ml注入した。また、アルキルヒドロペルオキシドレダクターゼ遺伝子で組み換えていない宿主JM1株（FERM BP-5352）の菌懸濁液を別のバイアル瓶に15ml注入した。さらに、対照として、もう1本のバイアル瓶にはM9培地のみ１5ml添加した。
【００９２】
各バイアル瓶中に、TCE汚染空気（パーミエーターにて作成、ガス濃度100ppm）を1mlガスタイトシリンジで加え、密封した。25℃でTCE分解を行わせ、4時間経過した時点で、気相部分のTCE濃度をガスクロマトグラフィーで測定した。測定の後、栓を外して一旦開放し、再び、初期と同程度の気相濃度となるTCEガスを加えた後に密封し直した。ただし、対照のバイアル瓶は、初期状態のままとした。次いで、再密封したバイアル瓶中で25℃でTCE分解を行わせ、４時間が経過した時点で、バイアル瓶気相部分のTCE濃度をガスクロマトグラフィーで測定した。
【００９３】
4時間毎に、上記の測定と再密封の操作を繰り返し、TCEの繰り返し分解における、共発現させたアルキルヒドロペルオキシドレダクターゼの効果を評価した。具体的には、1回目：4時間後、2回目：4時間後（合計8時間後）、3回目：4時間後（合計12時間後）、4回目：4時間後（合計16時間後）、5回目：4時間後（合計20時間後）の計５回の測定を行った。なお、1回目、3回目、5回目のTCEガスを添加、密封する際に、エネルギー源としてのホルムアルデヒドを各最終濃度1mMとなるように加えた。その他の操作は、上記実施例２に記載の手順に準じた。表８に、測定結果を示す。表８に示すTCE濃度はガス濃度である。
【００９４】
【表８】

単位はppm（ガス濃度）、検出器の検出限界は10ppm、
ＮＤ：検出限界以下（≦10ppm）
Ahp：アルキルヒドロペルオキシドレダクターゼ
表８に示す結果のとおり、アルキルヒドロペルオキシドレダクターゼを共発現していない菌とアルキルヒドロペルオキシドレダクターゼを共発現してる菌ともに、４回目（合計１２時間後）以降において、未分解のTCEが測定されるが、アルキルヒドロペルオキシドレダクターゼを共発現してる菌において測定される未分解TCE量は、アルキルヒドロペルオキシドレダクターゼを共発現していない菌において測定される未分解TCE量と比較して、有意に低いものである。この比較から、共発現してるアルキルヒドロペルオキシドレダクターゼにより、明らかに分解処理の効率化、あるいは、分解効率低下の抑制が図られていると判断される。本結果より、JM1株を用いた気相系のTCEの分解処理、つまり汚染空気浄化においても、共発現アルキルヒドロペルオキシドレダクターゼの効果が示された。すなわち、汚染空気浄化においても、浄化処理が長期にわたる際にしばしば起こる微生物の示す分解活性の経時的な低下、失活を、アルキルヒドロペルオキシドレダクターゼを共発現させることで、効果的に抑制できる効果が示された。
【００９５】
【発明の効果】
本発明の汚染環境の修復方法では、分解活性を示す微生物において、アルキルヒドロペルオキシドレダクターゼを共発現させることにより、例えば、上述した塩素化エチレン化合物のような難分解性の揮発性有機化合物等で汚染された液体、土壌、及び空気の効率的な生物分解浄化処理が可能となる。特には、浄化処理が長期にわたる際に、アルキルヒドロペルオキシドレダクターゼを共発現させることにより、微生物の示す分解活性の経時的な低下、失活を効果的に抑制できる効果を持つ。従って、前記の分解活性の経時的な低下、失活を防ぐことに伴い、生物分解浄化処理の効率化が達成される利点を持つ。[0001]
BACKGROUND OF THE INVENTION
The present invention uses a microorganism to biodegrade and treat organic compounds that cause environmental pollution such as rivers, groundwater, lakes, and water and liquids such as industrial wastewater, soil, and air, and The present invention relates to a method for purifying a contaminated environment and repairing the environment using a method of biodegrading and treating an organic compound using the microorganism. In particular, when the organic compound that is a pollutant is a hardly decomposable organic compound, specifically, a halogenated hydrocarbon such as trichloroethylene or dichloroethylene, the organic compound that is the pollutant is continuously decomposed by microorganisms. The present invention relates to a method for improving processing efficiency.
[0002]
[Prior art]
In recent years, environmental pollution due to volatile organic chlorine compounds that are harmful to humans and difficult to decompose has become a major problem. In particular, volatile organochlorine compounds such as tetrachloroethylene (PCE), trichlorethylene (TCE), and dichloroethylene (DCE) have been used for a long time as solvents and solvents in various industries. Along with these corporate activities, these used organic compounds have been disposed of and treated with great care, but unfortunately, a very small amount of these organic compounds escapes the recovery process and is There were not a few leaks / dissipated from the facilities.
[0003]
For example, volatile organochlorine compounds such as tetrachlorethylene (PCE), trichlorethylene (TCE), and dichloroethylene (DCE) are difficult to decompose and therefore gradually accumulate, and in the soil of industrial areas in Japan and overseas, It is thought that contamination by persistent organic chlorinated compounds in the region has spread to a considerable extent. Actually, many cases detected by environmental surveys have been reported. These volatile organochlorine compounds are said to remain in the soil and dissolve in groundwater by rainwater, etc., and spread throughout the surrounding area. These persistent organochlorine compounds are suspected to be carcinogenic and are stable in the environment, and in particular, may contaminate groundwater used as a source of drinking water. It is also a problem.
[0004]
Thus, it is an extremely important issue from the viewpoint of environmental conservation to remove and decompose volatile organic chlorine compounds to purify aqueous media such as contaminated groundwater, soil, and the surrounding gas phase. Although development of technologies necessary for purification (for example, adsorption treatment with activated carbon, decomposition treatment with light and heat, etc.) has already been promoted, many of the current purification technologies are in terms of cost and operability, It is not necessarily practical.
[0005]
On the other hand, a microbial decomposition method has recently been reported for volatile organic chlorine compounds such as TCE which are stable in the environment. Advantages of the method of decomposing and treating using microorganisms: 1) In the biodegradation treatment using microorganisms, volatile organochlorine compounds can be decomposed to harmless substances by selecting the microorganisms to be used. 2) Basic 3) that special chemicals are not required, and 3) the labor and cost required for maintenance can be reduced.
[0006]
Many microorganisms that decompose volatile organochlorine compounds such as TCE have been found, and efforts to search for new strains exhibiting degrading activity are continuing. Examples of microorganisms already reported and described in the literature include, for example, Welchia alkenophila sero 5 (USP 4877736, ATCC 53570), Welchia alkenophila sero 33 (USP 4877736, ATCC 53571), Methylocystis sp. M (Agric. Biol. Chem., 53, 2903 (1989), Biosci. Biotech. Biochem., 56, 486 (1992), 56, 736 (1992)), Methylosinus trichosprium OB3b (Am. Chem. Soc. Natl Meet. Dev. Environ. Microbiol., 29, 365 (1989), Appl. Environ. Microbiol., 55, 3155 (1989), Appl. Biochem. Biotechnol., 28, 877 (1991), JP 02-92274 Publication, JP-A-03-292970), Methylomonas sp. MM2 (Appl. Environ. Microbiol., 57, 236 (1991)), Alcaligenes denitrificans ssp. Xylosoxidans JE75 (Arch. Microbiol., 154, 410 (1990)) ), Alcaligenes eutrophus JMP134 (Appl. Environ. Microbiol., 56, 1179 (1990)), Alcaligenes eutrophus FERM-13761 (JP 07-123976 A), Pseudomonas aeruginosa JI104 (special No. 07-236895), Mycobacterium vaccae JOB5 (J. Gen. Microbiol., 82, 163 (1974), Appl. Environ. Microbiol., 54, 2960 (1989), ATCC 29678), Pseudomonas putida BH (Sewerage Association) Journal, 24, 27 (1987)), Pseudomonas sp. Strain G4 (Appl. Environ. Microbiol., 52, 383 (1986), 53, 949 (1987), 54, 951 (1989), 56, 279 (1990), 57, 193 (1991), USP 4925802, ATCC 53617, this strain was initially classified as Pseudomonas cepacia, but later changed to Pseudomonas sp.), Pseudomonas mendocina KR-1 ( Bio / Technol., 7, 282 (1989)), Pseudomonas putida F1 (Appl. Environ. Microbiol., 54, 1703 (1988), 54, 2578 (1988)), Pseudomonas fluorescens PFL12 (Appl. Environ. Microbiol. , 54, 2578 (1988)), Pseudomonas putida KWI-9 (JP 06-70753), Pseudomonas cepacia KK01 (JP 06-227769), Nitrosomonas europaea (Appl. Environ. Microbiol., 56, 1169) (1990)) Lactobacillus vaginalis sp.nov (Int. J. Syst. Bacteriol., 39, 368 (1989), ATCC 49540), Nocardia corallina B-276 (JP 08-70881 A, FERM BP-5124, ATCC 31338) It has been reported. The above-mentioned TCE-degrading bacteria have their strain name, deposit number, and depositing institution listed in each attached document, so that the bacteria can be identified, and the proper procedures are followed to obtain distribution from these depositing institutions. Therefore, it can be easily obtained.
[0007]
However, when these degrading bacteria are used for actual environmental purification treatment, it is more important to optimize the expression of volatile organochlorine compounds such as TCE and continuously maintain the decomposing activity. Become. The addition of various inducers has been found to be effective for the expression of the degradation activity of these degrading bacteria, and a method for expressing and maintaining the degradation activity by adding an inducer to the system is used. . For example, in an environmental purification process using phenol, toluene, methane or the like as an inducer, the depletion of these inducers directly causes the decomposition of volatile organochlorine compounds such as TCE. Therefore, in order to maintain the concentration of the inducer above a certain level, it is necessary to continuously supply the inducer. On the other hand, when comparing the substrate affinity between volatile organochlorine compounds such as TCE, which is originally the target substance for decomposition and purification, and these inducers, it is often the case that the target substance itself for the decomposition and purification itself. Can be significantly lower than the inducer. In this situation, since the inducer is added to the system, there is a conflicting problem that efficient decomposition and purification becomes difficult. Furthermore, in an actual treatment site, precise control of the inducer concentration is not desired, and there are essential problems regarding the stability and reproducibility of the purification treatment.
[0008]
In addition, it has been pointed out that the added inducer may flow out into the environment. For example, aromatic compounds such as phenol and toluene are very effective inducers for certain strains, but their release into the environment is out of the question due to their high toxicity. Although methane is also an effective inducer, it is a flammable gas, and it is very difficult to introduce it into the environment and control its concentration. The danger is also inherent.
[0009]
Thus, in actual environmental purification using decomposing microorganisms, continuous expression of decomposing activity using an inducer and an increase in decomposition and purification efficiency have difficult problems that are incompatible with each other. In addition, since environmental purification has been performed, there is also a risk of causing another environmental pollution, which has been a major issue that hinders its spread in practical use of environmental purification processing using microorganisms.
[0010]
In order to avoid this problem, Nelson et al. Developed a method using tryptophan as a decomposition inducer of volatile organic chlorine compounds (Japanese Patent Laid-Open No. 4-502277). However, tryptophan is an expensive substance, although adding toxicity and danger, which are inherent problems with the above-mentioned inducer addition, is an expensive substance, and adding excess carbon and nitrogen sources to the environment itself is an environmental issue. In terms of inducing eutrophication, it is not necessarily preferred in practice. In addition, there is no solution regarding the point that tryptophan becomes a competitive inhibitor in TCE degradation.
[0011]
Therefore, the search, creation, and development of degrading bacteria that do not require toxic inducers such as phenol and toluene for the expression of degrading activity were also advanced. For example, Shields et al., A P4 Pseudomonas cepacia that does not require an inducer (in this case phenol or toluene) and has TCE resolution (the classification has been changed to Pseudomonas sp.) Acquired by a technique using a transposon (Appl. Environ. Microbiol., 58, 3977 (1992), PCT Publication No. WO 92/19738). It has also been reported that a methylocinastricospolium OB3b strain, which is a methane-utilizing TCE-degrading bacterium, has obtained a TCE-degrading mutant strain that does not require the inducer methane (US Pat. No. 5,316,940). Furthermore, in Japanese Patent Application Laid-Open No. 8-294387, a strain JM1 (international deposit based on the Budapest Treaty), which performs a mutation induction operation with nitrosoguanidine and can decompose volatile organochlorine compounds and aromatic compounds without requiring an inducer. Number: FERM BP-5352) has been reported.
[0012]
On the other hand, as one method to avoid the outflow of inducer to the environment, TCE resolution is expressed using the inducer during preculture, and the bacteria grown in the preculture are administered to the repair site as resting cells. Attempts have also been made (Environ. Sci. Technol., 30, 1982 (1996)).
[0013]
In fact, the above-mentioned inducer is used in the purification treatment that does not require the addition of an inducer at the repair site, using a mutant strain that does not require the addition of the inducer as described above, or a bacterium that has been proliferated in a pre-culture and that does not require the inducer to be added at the repair site. It has been reported that the control of the purification process becomes easier and the purification efficiency is also increased as compared with the case of using. However, regarding the expression of degradation activity as needed and the continuation of degradation, the growth control of degrading bacteria is a major issue that should be solved in the future. In addition, when resting cells are used, if the amount of TCE degradation that can be decomposed or the capacity of the degradation duration exceeds the capacity of the resting cells that have been put in the first place, subsequent TCE degradation is impossible. In addition, when it is carried out on a larger scale, it takes time for the quiescence treatment itself, so that some degradation of degradation activity is inevitable during that time. Since these restrictions are included, the processing apparatus becomes large-scale, the processing work becomes complicated, and this is accompanied by a practical disadvantage that hinders cost reduction.
[0014]
Therefore, currently, a plasmid incorporating a DNA fragment containing the gene region encoding oxygenase or hydroxylase, which is a TCE-degrading enzyme, is introduced into the host microorganism, and the TCE is constitutively constructed by a harmless inducer or in the absence of the inducer. Attempts have been made to express degradation activity. For example, Pseudomonas Mendsina KR-1 (Japanese Patent Laid-Open No. 2-503866), Pseudomonas putida KWI-9 (Japanese Patent Laid-open No. 6-106991), Pseudomonas putida BH (Study meeting on groundwater / soil contamination and prevention measures) 3rd Lecture Collection, 213 (1994)), a transformant carrying a hybrid gene of a Pseudomonas putida F1 derived toluene-degrading enzyme gene and a Pseudomonas pseudomonage biphenyl-degrading enzyme gene (Japanese Patent Laid-Open No. 7-143882) ) And the like.
[0015]
Thus, various problems associated with addition of inducers when decomposing volatile organic chlorine compounds such as chlorinated ethylene compounds and aromatic compounds such as phenol and toluene, which are difficult to decompose, using microorganisms, Alternatively, many of the problems related to optimization of degrading enzyme activity expression are being avoided. As a result of such improvements, the environmental remediation process by the decomposition of microorganisms has reached a level that can be technically sufficiently practical for a wide range.
[0016]
On the other hand, there are not a few cases where long-term purification is required even in the repair processing of an organic compound-contaminated environment by microbial decomposition. When applied to this long-term purification, there is a need to maintain the microbial degradation activity over a long period of time. However, in the current technology, considering the date required for long-term purification, the problem that the microbial degradation activity is deactivated in a very short time still remains unsolved.
[0017]
In addition, in the repair treatment of the environment contaminated with organic compounds by microbial decomposition, it takes time and cost to culture microorganisms to be used, and it is strongly desired to make maximum use of the ability of the cultured microorganisms. Therefore, a means for solving the problem that the above-mentioned microorganism degrading activity is deactivated in a very short time is desired.
[0018]
The cause of the phenomenon that the degradation activity of this microorganism is deactivated in a short period of time is the self-digestion of the degradation enzyme in the cell, lack of energy necessary for degradation, toxicity derived from the degradation enzyme expression itself, degradation intermediate product Toxicity due to degradation is considered, but among these, toxicity due to degradation intermediate products, toxicity problems derived from degradation enzyme expression itself involve damage and death of microorganisms due to toxicity, inactivation of degradation enzyme, etc. This is a very important problem in the sense of the essential disappearance of the biodegradation activity for the organic compounds intended for treatment. Solving the problems of toxicity due to the degradation intermediate products and the degradation enzyme expression itself is necessary for maximizing effective use of the ability of the cultured microorganisms.
[0019]
[Problems to be solved by the invention]
The present invention solves the above-mentioned problems. The object of the present invention is to determine the toxicity derived from the expression of the degrading enzyme itself against microorganisms exhibiting degrading activity such as volatile organochlorine compounds which are hardly degradable. It is an object of the present invention to provide a method for removing the influence of toxicity caused by degradation intermediate products produced by an enzymatic reaction. More specifically, an enzyme that further decomposes a degradation intermediate product that exhibits toxicity, or an enzyme that inhibits or suppresses a secondary enzyme reaction that produces a cytotoxic substance derived from the degradation enzyme expression itself. Is produced by microorganisms that exhibit degradation activity such as volatile organochlorine compounds that are hardly degradable, and a method for sequentially removing these toxic substances is provided. In addition, the present invention uses a method for removing the effects of toxicity derived from the above-described degradation enzyme expression itself and the degradation intermediate product produced by the enzymatic reaction, and is a volatile property that is hardly degradable using microorganisms. Another object of the present invention is to provide a method for improving the efficiency of decomposition of organic chlorine compounds. Furthermore, the true object of the present invention is to use the above-described method for improving the efficiency of the decomposition treatment, and to decompose and treat volatile organochlorine compounds that are difficult to decompose using microorganisms that can maintain high efficiency over a long period of time. It is another object of the present invention to provide a method of using the same decomposition / treatment method for repairing environmental pollution.
[0020]
[Means for Solving the Invention]
In order to solve the above-mentioned problems, the present inventors have intensively studied and searched for an enzyme having a function of suppressing degradation of degradation activity due to toxicity due to degradation intermediate product or toxicity derived from degradation enzyme expression itself, When microorganisms are brought into contact with these refractory organic compounds to cause degradation, co-expression or overexpression of alkyl hydroperoxide reductase together with the enzymes and proteins of the degradation system itself can lead to toxicity due to degradation intermediate products or It was found that the toxicity derived from the degradation enzyme expression itself can be effectively suppressed and detoxified. The present invention has been completed based on such knowledge.
[0021]
That is, in the method for improving the efficiency of the organic compound decomposition treatment using the microorganism of the present invention, when the organic compound targeted for the treatment is brought into contact with a microorganism having a resolution of the organic compound, the decomposition is performed. In addition to the expression of an enzyme protein system having a resolution of the organic compound itself, the microorganism is co-expressed with an alkyl hydroperoxide reductase. In addition, the organic compound decomposition treatment method using the microorganism of the present invention is a microorganism having the resolution of the organic compound targeted for the treatment, and in addition to the expression of the enzyme protein system having the resolution of the organic compound itself. The method is characterized in that the microorganism co-expressing alkyl hydroperoxide reductase is brought into contact with the organic compound to cause degradation.
[0022]
Alternatively, the method for improving the efficiency of environmental restoration contaminated with organic matter using the microorganism of the present invention is a method for improving the efficiency of restoration when an environment contaminated with an organic compound is restored using microorganisms. When an organic compound causing contamination is brought into contact with a microorganism capable of decomposing the organic compound for decomposition, in addition to the expression of an enzyme protein system having the decomposability of the organic compound itself, It is a method characterized by co-expressing alkyl hydroperoxide reductase. In addition, the method for repairing an environment contaminated with an organic substance using the microorganism of the present invention is a method for repairing an environment contaminated with an organic compound using a microorganism, the microorganism having a resolution of the organic compound causing the contamination. In addition to the expression of an enzyme protein system having the resolution of the organic compound itself, the microorganism that co-expresses alkyl hydroperoxide reductase is contacted with the organic compound to cause degradation. Is the method.
[0023]
In the method of the present invention, the action of the co-expressed alkyl hydroperoxide reductase suppresses a decrease in degradation activity due to toxicity due to degradation intermediate products or toxicity derived from degradation enzyme expression itself by the mechanism described below. .
[0024]
In the method of the present invention, the enzyme group used for microbial decomposition of the chlorinated ethylene compound or aromatic compound to be treated is various oxygenase or hydroxylase enzyme groups expressed by the microorganism. As the reaction mechanism of these enzymes, it is known that the enzyme system receives electrons from NADH, the enzyme active center is activated by these electrons and oxygen, and oxygen is added to the organic compound as a substrate at this active center. Yes. In an enzyme system that exchanges electrons, it is known that leakage of electrons occurs with a certain probability. In other words, an undesirable side reaction occurs at a certain frequency rather than activation of the enzyme active center. For example, when the leaked electron reaches oxygen, active oxygen such as superoxide anion, hydrogen peroxide, hydroxy radical, etc. It is known that seeds arise. The above-mentioned enzymes used for microbial degradation of chlorinated ethylene compounds and aromatic compounds are also enzyme systems that exchange electrons, and toxicity due to the expression of degrading enzymes is also caused by such secondary reactions. Various active oxygen species and peroxides generated by the active oxygen species are likely to be the cause. Similarly, as for the toxicity due to the decomposition intermediate product, for example, the intermediate product itself is a peroxide, and many of them show cytotoxicity. Alternatively, the degradation intermediate product itself is toxic, but many of them are rendered harmless when reduced.
[0025]
In the method of the present invention, the alkyl hydroperoxide reductase used by co-expression is an enzyme having a function of catalyzing the reduction of the alkyl hydroperoxide, which is a peroxide. It can participate in the reduction detoxification. In other words, alkyl hydroperoxide reductase directly plays a role in biological defense functions such as reduction detoxification of peroxides caused by various oxidative stresses, conjugation and excretion of various accompanying foreign compounds. .
[0026]
Microorganisms have glutathione transferase as one of the enzymes that play a role in the defense function of the cells themselves. This glutathione transferase is an enzyme widely found not only in microorganisms but also in higher animal cells, and is an enzyme that catalyzes the rearrangement reaction of glutathione as a substrate. Specifically, using the above-mentioned catalytic action, for example, the peroxide generated by oxidative stress is reduced and detoxified, or conjugated with various exogenous compounds that enter and take up into the cell, to the outside of the cell. Involved in biological defense functions such as excretion. It is known that the above-mentioned reduction detoxification of peroxides, conjugation of foreign compounds, extracellular discharge, etc. are achieved by the function of glutathione and glutathione transferase in concert.
[0027]
Glutathione is a peptide (L-γ-glutamyl-L-cysteinylglycine) compound composed of glutamic acid, cysteine and glycine, and is present in almost all cells and biosynthesized by microorganisms themselves. Glutathione is found to have various physiological activities, and its function is based on the γ-glutamyl group and the SH group of the cysteine residue. Glutathione has a reducing ability and causes other molecules (small molecules, enzymes, proteins) to newly generate or maintain SH groups. Certain enzyme proteins may form mixed disulfides with glutathione and regulate enzyme activity. It also plays a very important role in terms of biological defense such as reduction detoxification of peroxides caused by oxidative stress, conjugation and excretion of various foreign compounds. Another important function of glutathione is maintaining a redox system. In vivo, approximately 95% or more is usually present in reduced form (GSH). The redox reaction between the reduced form (GSH) and the oxidized form (GSSG) is coupled to the pentose phosphate cycle, and is performed by an oxidase and a reductase, that is, both glutathione reductase and glutathione oxidase.
[0028]
In the method of the present invention, various oxygenase or hydroxylase enzyme groups are highly expressed as enzyme groups used for microbial degradation of chlorinated ethylene compounds and aromatic compounds to be treated. Along with this, a series of enzymatic reactions with the oxygenase or hydroxylase enzymes produce toxic peroxides as decomposition intermediate products, or they exhibit toxicity due to reactive oxygen species produced by the secondary reactions described above. Peroxides may be formed. In many cases, these harmful peroxides are rendered harmless and discharged to the outside by a biological defense function in which glutathione and glutathione transferase function together. However, with the high expression of oxygenase or hydroxylase enzyme group, the production amount of undesired peroxides and the like increases, and when the biological defense capability inherent to the microorganism itself is exceeded, the cell itself is damaged. As a result, the efficiency of the enzymatic reaction by the oxygenase or hydroxylase enzyme group is rapidly reduced.
[0029]
In the method of the present invention, the alkyl hydroperoxide reductase is co-expressed, so that it is derived mainly from the peroxide generated as an intermediate product or reactive oxygen species in the enzyme reaction of oxygenase or hydroxylase enzymes. A substantial part of various peroxides is reduced and detoxified by an enzyme reaction of co-expressed alkyl hydroperoxide reductase. As a result, the peroxide produced beyond the ability of biological defense functions such as glutathione transferase possessed by the microorganism itself is reduced and detoxified by the co-expressed alkyl hydroperoxide reductase. It is possible to prevent / suppress the damage to itself and the accompanying decrease in the efficiency of the enzyme reaction by the oxygenase or hydroxylase enzymes.
[0030]
In other words, in the method of the present invention, glutathione originally possessed by the microorganism itself has the ability to remove and detoxify the toxicity due to the degradation intermediate product produced by the enzyme reaction of the microorganism itself, or the toxic byproduct derived from the degradation enzyme expression itself. By artificially co-expressing or over-expressing alkylhydroperoxide reductase that plays a role in complementing the biological defense function such as transferase and the biological defense function, it is possible to function both in cooperation with each other. It is a thing. As described above, the biological defense function can be made more efficient or enhanced, so that damage to cells and the like that lead to a decrease in degrading enzyme activity are reduced and suppressed. Furthermore, even when the alkyl hydroperoxide reductase is artificially co-expressed or overexpressed, it does not adversely affect the expression of the degradation enzyme system of the microorganism itself, thus solving the problem of degradation of degradation activity. Was able to. In particular, when the decomposition process is repeated, the degradation activity decreases or progresses with the lapse of time related to the decomposition process. The product can be reduced, detoxified or discharged out of the cell sequentially. Therefore, the inhibitory effect on degradation of degradation activity is essentially independent of the passage of time and continues for a long period as long as the microorganisms survive.
[0031]
In addition, these effects are particularly useful in the restoration of the environment by microorganisms, which are often carried out outdoors, because they use alkyl hydroperoxide reductase co-expressed by the microorganisms themselves. That is, in the method for improving the efficiency of decomposition processing of pollutants according to the present invention, if there are no pollutants, it can be sustained even when the pollutants are continuously supplied as long as the conditions under which the microorganisms can survive are satisfied. It shows a typical effect.
[0032]
In such microorganisms used for environmental repair, conventionally provided genetic engineering techniques can be used as a method of co-expressing or overexpressing alkyl hydroperoxide reductase together with a target enzyme decomposing enzyme. That is, it is possible to apply a method in which an alkyl hydroperoxide reductase gene is recombinantly introduced into a microorganism and expressed using a genetic engineering technique. Furthermore, there are no particular restrictions on the origin of the alkyl hydroperoxide reductase gene used for this genetic recombination. For example, it is preferable to use an alkyl hydroperoxide reductase gene derived from Escherichia coli, specifically, an alkyl hydroperoxide reductase gene derived from Escherichia coli K-12 (Takara Shuzo) as the alkyl hydroperoxide reductase gene. Many organisms each have an alkyl hydroperoxide reductase, and many amino acid sequences or gene base sequences are specified. Therefore, genes encoding these known alkyl hydroperoxide reductases can be utilized. In addition, when a method in which an alkyl hydroperoxide reductase gene is recombinantly introduced and expressed is applied, the microorganism itself is overexpressed when the alkyl hydroperoxide reductase is originally expressed.
[0033]
The decomposing microorganism used in the method of the present invention may be classified into any genus as long as it is a microorganism capable of decomposing aromatic compounds and / or organochlorine compounds. It can be selected from the following genera from which microorganisms capable of decomposing organochlorine compounds such as chlorinated ethylene compounds are collected. Therefore, the genus Esherichia, Pseudomonas, Burkholderia, Acinetobacter, Moraxella, Alcaligenes, Vibrio, Nocardia Genus, Bacillus genus, Lactobacillus genus, Achromobacter genus, Arthrobacter genus, Micrococcus genus, Mycobacterium genus, Methylosinus Microbes belonging to the genus, Methylomonas, Welchia, Methylocystis, Nitrosomonas, Saccharomyces, Candida, Torulopsis, etc. It is done.
[0034]
Pollutants as environmental purification targets using these microorganisms are persistent organic compounds, and specific examples include chlorinated ethylene compounds such as trichlorethylene (TCE) and dichloroethylene (DCE). . In addition, the method of the present invention can be equally used for purification of the environment contaminated with the above-mentioned pollutants, whether it is liquid, soil, or air. That is, as long as the contaminant can be brought into contact with the microorganism to be used, it does not depend on the form of the contaminant.
[0035]
DETAILED DESCRIPTION OF THE INVENTION
In the method for improving the efficiency of environmental repair of the present invention, as long as the contaminant can be brought into contact with the microorganism to be used as described above, it does not depend on the form of the contaminant. Therefore, when the environment polluted by the organic compound is a liquid such as ground water or waste liquid, it can be most easily implemented. For example, the present invention can be carried out in such a manner that a microorganism is supported on a carrier, and a liquid containing an organic compound to be treated, that is, contaminated ground water or waste water is brought into contact with the microorganism supported on the carrier. Specifically, the microorganisms supported on the carrier are stored in a container, a liquid containing an organic compound to be treated is introduced from one end of the container, and the treated liquid is taken out from the other end of the container. It is preferable to take a form. If necessary, the above-described decomposition treatment can be repeated a plurality of stages, and a method of reducing the concentration of the organic compound to be treated stepwise can be adopted.
[0036]
The method of the present invention can also be applied when the object to be treated is soil contaminated with an organic compound to be treated. For example, a liquid containing microorganisms, a suspension of bacteria, a liquid that immerses microorganisms carried on a carrier, and a liquid that contains soil and microorganisms are brought into contact with microorganisms to contaminate the organic compounds that contaminate the soil. Decomposition treatment can be performed by transferring to a liquid containing sucrose. More specifically, it can also be carried out by introducing it into the soil, and in the introduction into the soil, a mode in which a liquid containing microorganisms is introduced into an injection well provided in the introduced soil is preferable.
[0037]
On the contrary, it is possible to adopt a system in which soil contaminated with an organic compound to be treated is introduced into a liquid containing microorganisms. It is good also as an aspect which mixes the liquid containing microorganisms, and soil. Moreover, it is good also as an aspect which contacts the support | carrier which carry | supported microorganisms, and soil. In these embodiments, the organic compound that contaminates the soil gradually moves to the liquid that covers the microorganisms or the liquid that soaks the carrier, and is decomposed by contact and mixing. It is desirable to keep minutes.
[0038]
The method of the present invention, particularly the method for improving the efficiency of environmental restoration, can also be applied to the purification of air contaminated with organic compounds to be treated. For example, air that is contaminated with the organic compound to be treated can be washed with water, and an indirect means for treating the organic compound to be treated that has been taken into the washing water can be employed. Alternatively, a method may be employed in which water or the like is aerated with air contaminated with the organic compound to be treated, and the organic compound to be treated is taken into a liquid such as water. More specifically, a method of directly introducing contaminated air into a liquid containing microorganisms, or a mode in which a carrier carrying microorganisms and air are brought into contact with each other can be employed. Further, when contacting the carrier carrying microorganisms with air, the carrier carrying microorganisms is housed in a container, contaminated air is introduced from one end of the container, and the treated air is fed from the other end to the container. It is preferable that the discharge is performed outside. If necessary, the above-described decomposition treatment can be repeated a plurality of stages, and a method of reducing the concentration of the organic compound to be treated stepwise can be adopted.
[0039]
In the method of the present invention, in particular, the environmental pollution that is highly demanded to apply the method for improving the efficiency of environmental remediation is when the organic compound that pollutes the environment is an organic compound that is hardly decomposable and highly volatile. It is. Among them, among refractory and highly volatile organic compounds, chlorinated aliphatic compounds that are used as solvents, more specifically, trichlorethylene or dichloroethylene, that is, trans-1,2- When dichloroethylene, cis-1,2-dichloroethylene, or 1,1-dichloroethylene is an object to be treated, it is preferable to employ the method of the present invention. In particular, in contamination with trichlorethylene or dichloroethylene, soil contamination, groundwater and other water contamination, air contamination due to volatility thereof, and the above three types of contamination are both present, and it is preferable to apply the method of the present invention. .
[0040]
In carrying out the method of the present invention, one of the microorganisms that can be used is Barkholderia cepacia KK01 strain (International deposit number under the Budapest Treaty: FERM BP-4235). In the publication, it is a strain that has been announced as a fungus that degrades aromatic compounds such as phenol and cresol, and in Japanese Patent Application Laid-Open No. 6-296711, an organochlorine compound such as TCE is used as an inducer. It is disclosed to decompose.
[0041]
In carrying out the method of the present invention, one of the microorganisms that can be used, JM1 strain (International Deposit No. FERM BP-5352 under the Budapest Treaty) JP-A-8-294387 discloses that a mutant strain obtained by mutating by the above-mentioned method can decompose organochlorine compounds such as TCE without using an inducer. This strain: JM1 strain was initially labeled as “Corynebacterium sp. JM1 strain” as belonging to the genus Corynebacterium. Because it was recognized as “No”, the indication of the microorganism identification was changed to “JM1 strain”.
[0042]
The above-mentioned bulk holderia cepacia KK01 strain (FERM BP-4235), JM1 strain (FERM BP-5352), etc. have a degrading enzyme system capable of decomposing organochlorine compounds such as TCE. E. coli-derived alkyl hydroperoxide reductase gene, specifically, an alkyl hydroperoxide reductase gene derived from Escherichia coli K-12 (Takara Shuzo), etc. The body is preferably used in the present invention. In addition to these strains, for example, by utilizing microorganisms capable of degrading aromatic compounds and / or organochlorine compounds belonging to various genera described above, for example, alkyl hydroperoxide reductase genes derived from E. coli, specifically, The present invention can also be used in the present invention as a transformant that is genetically recombined using an alkyl hydroperoxide reductase gene derived from E. coli K-12 strain (Takara Shuzo Co., Ltd.) to co-express alkyl hydroperoxide reductase.
[0043]
In carrying out the method of the present invention, recombinant Escherichia coli transformed with an organochlorine-degrading gene derived from Barkholderia cepacia KK01, which is one of the microorganisms that can be used, includes, for example, E. coli TMOKK01 (FERM BP-6916) stock. This transformed Escherichia coli retains an organochlorine compound-degrading gene derived from the bulk Chorderia cepacia KK01 strain. In addition, for example, an alkyl hydroperoxide reductase gene derived from Escherichia coli, specifically, an Escherichia coli K-12 strain ( It is preferably used in the present invention as a transformant which is genetically modified using an alkyl hydroperoxide reductase gene derived from Takara Shuzo Co., Ltd. and co-expresses the alkyl hydroperoxide reductase. In addition, for transformed Escherichia coli that similarly holds exogenous organochlorine compound-degrading genes derived from various strains and has been given organochlorine-degrading activity, for example, alkyl hydroperoxide reductase genes derived from Escherichia coli, specifically Can be used in the present invention as a transformant that undergoes genetic recombination using an alkyl hydroperoxide reductase gene or the like derived from Escherichia coli K-12 (Takara Shuzo) and co-expresses the alkyl hydroperoxide reductase.
[0044]
As a medium used for culturing these microorganisms, basically, it only needs to contain a carbon source, nitrogen source, phosphorus source, inorganic salts, etc. necessary for growth. It is preferable to use a liquid medium, or a liquid medium described in a document in which each strain is disclosed. Accordingly, the basic medium used for culturing the microorganism is not particularly limited as long as it contains an appropriate amount of components necessary for the growth of the strain. For example, a commonly used LB medium or M9 medium Or a basic salt medium such as MSB medium, and can be prepared by adding an appropriate amount of a carbon source or the like necessary for growth.
[0045]
The composition of M9 medium is shown below.
[0046]
Na₂HPO_Four: 6.2g
KH₂PO_Four: 3.0g
NaCl: 0.5g
NH_FourCl: 1.0 g (in 1 liter of medium; pH 7.0)
The culture can be performed under aerobic conditions, and may be performed in a liquid medium or a solid medium. The culture temperature is appropriately selected depending on the microorganism to be used, but is usually preferably selected between 15 ° C and 37 ° C.
Hereinafter, with reference to specific examples, the procedure for carrying out the present invention and the degree of efficiency obtained by the present invention will be described in more detail.
[0047]
【Example】
(Example 1)
E. coli HB101 Of alkyl hydroperoxide reductase gene from strainE. coli strain HB101 was cultured overnight at 37 ° C. in 50 ml of LB medium. After culture, the cells were collected and total DNA was prepared by a conventional method. Using this total DNA as a template, PCR was performed using Primer 1 and Primer 2 shown below to amplify the alkyl hydroperoxide reductase gene.
Primer 1
5'-AACTTATCGATAAGCTTATGGAAACGC-3 '
Primer 2
5'-GCAATTGCAGGTGAAGCTTACTTC-3 '
For the PCR reaction, a commercially available LA-PCR kit (Takara Shuzo) was used, and the composition of the reaction solution and the reaction conditions were in accordance with the instructions (standard conditions, procedures) attached to the commercially available kit. By this PCR reaction, an amplified product of about 2500 bp could be obtained. This approximately 2500 bp amplification product is completely digested with the restriction enzyme HindIII, and then ligated using the commercially available ligation kit (Takara Shuzo) to the complete digestion product of the restriction enzyme HindIII of the wide host range plasmid vector pBBR122 (Mo Bi Tec). Reaction was performed. The resulting vector was used to transform the host E. coli strain HB101 (Takara Shuzo).
[0048]
For selection of transformants, an agar LB medium containing chloramphenicol at a concentration of 50 μg / ml was used using a selection marker possessed by the broad host range plasmid vector pBBR122 itself. The presence or absence of the alkyl hydroperoxide reductase gene in the plasmid vector was confirmed for multiple transformant strains selected as colonies on the agar LB medium containing the chloramphenicol. Whether the alkyl hydroperoxide reductase gene is inserted into the broad host range plasmid vector pBBR122 as designed is checked by completely digesting the recombinant plasmid recovered by the alkaline method with the restriction enzyme HindIII. Only transformants carrying the inserted recombinant plasmid were selected. This selected transformant was cultured, the cultured cells were disrupted, and the plasmid was separated and recovered, whereby an alkyl hydroperoxide reductase gene recombinant plasmid could be obtained. The transformed recombinant Escherichia coli carrying the recombinant plasmid into which the alkyl hydroperoxide reductase gene has been inserted is labeled with the identification for identification of Escherichia coli TMOAHP, and deposited by the Institute of Industrial Technology, Ministry of International Trade and Industry. The deposit number is FERM P-17656. This recombinant Escherichia coli had the resolution of chlorinated aliphatic hydrocarbon compounds, and the other mycological properties were the same as the host Escherichia coli HB101 strain.
[0049]
(Example 2)
Effect of co-expressed alkyl hydroperoxide reductase on TCE degradation of JM1 strain (liquid system)
Using an alkyl hydroperoxide reductase gene recombinant plasmid prepared by the method described in Example 1, the plasmid was introduced into the host strain JM1 by electroporation (Bio-Rad). The resulting transformant has the same TCE resolution as the host strain JM1 (FERM BP-5352), and alkyl hydroperoxide reductase is expressed from the newly introduced E. coli-derived alkyl hydroperoxide reductase gene. .
[0050]
The culture conditions, collection conditions, etc. of the bacteria to be subjected to electroporation were in accordance with instructions attached to a commercially available electroporation apparatus. The apparatus was set to have a capacitance of 25 μF, a resistance value of 200Ω, and the amount of DNA to be donated was 1 μg. The suspension of the introduced strain is OD₆₀₀100 μl of a solution in which the cell density was adjusted so as to be 10 was used. A cuvette with a 0.2 mm gap was used (Bio-Rad), and the applied voltage was 2.5 kV. Here, the cuvette used was previously cooled to 4 ° C. The time constant during electroporation was 4.8 ms.
[0051]
Under the above conditions, the transformant of JM1 strain into which the alkylhydroperoxide reductase gene recombinant plasmid was introduced was selected using the chloramphenicol resistance selection marker possessed by the broad host range plasmid vector pBBR122 itself, as in Example 1 above. Agar M9 medium (containing 0.5% sodium glutamate) containing chloramphenicol at a concentration of 100 μg / ml was used.
[0052]
A strain forming a colony on the agar M9 medium (containing 0.5% sodium glutamate) containing chloramphenicol was selected as an alkyl hydroperoxide reductase gene recombinant JM1 strain. The collected alkyl hydroperoxide reductase gene recombinant JM1 strain was inoculated into 200 ml of a liquid M9 medium containing 0.5% sodium glutamate, and shake culture was performed at 25 ° C. in a 500 ml shake flask. At the time when 20 hours had elapsed from the start of culture, which was in the late logarithmic growth phase, the cells were collected by centrifugation. The collected cells were resuspended in M9 medium containing no equivalent amount of carbon source, and 10 ml of the cell suspension was injected into a 27.5 ml vial. Separately, the JM1 strain that has not undergone recombination of the alkyl hydroperoxide reductase gene, that is, the host JM1 strain itself, is cultured, collected and suspended in the same manner, and 10 ml of the suspension is stored in a 27.5 ml vial. Injected into.
[0053]
2 vials injected with these bacterial suspensions and 1 vial containing 10 ml of M9 medium only as a control, so that the total concentration in each vial is about 12 ppm. A predetermined amount of a saturated aqueous solution of TCE was added, and sealed with a butyl rubber stopper with a Teflon liner and an aluminum seal.
[0054]
These sealed vials were shaken at 30 ° C., and the TCE concentration in the vial gas phase was measured by gas chromatography after a certain time. After the measurement, the stopper was removed and once opened, and a predetermined amount of a saturated aqueous solution of TCE having a concentration in the liquid similar to the initial concentration was added again, and then the stopper was sealed again. However, the control vial was left in its initial state. Subsequently, the re-sealed vial was shaken at 30 ° C., and when a certain time had elapsed, the TCE concentration in the gas phase portion of the vial was measured by gas chromatography.
[0055]
The above measurement and resealing procedure were repeated to evaluate the effect of co-expressed alkyl hydroperoxide reductase on the repeated degradation of TCE. Specifically, 1st time: 1 hour later, 2nd time: 1 hour later (total 2 hours later), 3rd time: 1.5 hours later (total 3.5 hours later), 4th time: 1.5 hours later (total 5 hours later) , 5th: 1.5 hours later (total 6.5 hours later), 6th: 2 hours later (total 8.5 hours later), 7th: 2 hours later (total 10.5 hours later), 8th time: 2 hours later (total 12.5 hours later) After), 9th: A total of 9 measurements were performed 10 hours later (22.5 hours after total). During this time, in order to maintain the bacteria, when performing the third, fifth, and seventh measurements described above, a total of three times, formaldehyde as an energy source in each bacterial suspension so that the final concentration is 1 mM. In a vial. Table 1 shows the measurement results.
[0056]
As shown in Table 1, in bacteria that did not co-express alkyl hydroperoxide reductase, undegraded TCE was measured at the sixth time (after a total of 8.5 hours), but alkyl hydroperoxide reductase was co-expressed. In fungi, undegraded TCE has not been measured. From the 7th time (after 10.5 hours in total), undegraded TCE is measured even in bacteria co-expressing alkyl hydroperoxide reductase, but undegraded TCE measured in bacteria not co-expressing alkyl hydroperoxide reductase The amount is significantly lower than the amount. From this comparison, it is judged that the co-expressed alkyl hydroperoxide reductase is clearly improving the efficiency of the degradation treatment or suppressing the degradation of degradation efficiency. Therefore, it was shown that by co-expressing alkyl hydroperoxide reductase, the degradation efficiency of TCE by microorganisms can be increased and the high efficiency can be maintained for a long time.
[0057]
[Table 1]

Unit is ppm, detector detection limit is 0.1ppm,
ND: Below detection limit (≦ 0.1ppm)
Ahp: Alkyl hydroperoxide reductase
(Example 3)
Effect of co-expressed alkyl hydroperoxide reductase on DCE degradation of JM1 strain (liquid system)
As in the case of the above-mentioned TCE decomposition, it was verified that the decomposition efficiency could be achieved by co-expressed alkyl hydroperoxide reductase when decomposing other chlorine-substituted ethylene. The fungus used was the alkyl hydroperoxide reductase gene recombinant JM1 strain described in Example 2, and the degradation rate of the host JM1 strain (FERM BP-5352) was compared.
[0058]
In the test of this example, the decomposition target substances are cis-1,2-dichloroethylene (cis-1,2-DCE), trans-1,2-dichloroethylene, and 1,1-dichloroethylene (1,1-DCE). It is. For each dichloroethylene, as in Example 2, a suspension of alkyl hydroperoxide reductase transgenic JM1 strain, a suspension of host JM1 strain, and M9 medium alone as a control were injected into the vial 3 Each book was prepared. Each DCE was added so that the concentration in the liquid in each vial was 5.0 ppm. After adding DCE, each vial was sealed with a butyl rubber stopper with a Teflon liner and an aluminum seal.
[0059]
These sealed vials were shaken at 30 ° C., and the DCE concentration in the vial gas phase was measured by gas chromatography after a certain time. After the measurement, the stopper was removed and once opened, and a predetermined amount of DCE saturated aqueous solution having a concentration in the liquid similar to the initial concentration was added again, and then the stopper was sealed again. However, the control vial was left in its initial state. Next, the re-sealed vial was shaken at 30 ° C., and when a certain time had elapsed, the DCE concentration in the gas phase portion of the vial was measured by gas chromatography.
[0060]
The above measurement and resealing operation were repeated to evaluate the effect of the co-expressed alkyl hydroperoxide reductase on the repeated degradation of DCE. The measurement is basically performed in the same manner as in Example 2. Specifically, the first time (after 1 hour), the second time (after 1.5 hours, a total of 2.5 hours), the third time (after 2 hours, a total of 4.5 hours later) ), 4th time (after 2.5 hours, after 7 hours in total), 5th time (after 3 hours, after 10 hours in total), and 6th time (after 12 hours, after a total of 22 hours), a total of 6 measurements were performed. During this time, in order to maintain the bacteria, when performing the first, third, and fifth measurements described above, a total of three times, formaldehyde as the energy source in each bacterial suspension so that the final concentration is 1 mM. In a vial. Table 2 shows the measurement results for cis-1,2-DCE, Table 3 shows the measurement results for cis-1,2-DCE, and Table 4 shows the measurement results for 1,1-DCE.
[0061]
As shown in Table 2, in bacteria that did not co-express alkyl hydroperoxide reductase, undegraded cis-1,2-DCE was measured at the third time (after 4.5 hours in total). In bacteria that co-express reductase, undegraded cis-1,2-DCE has not been measured. From the fourth time (after 7 hours in total), undegraded cis-1,2-DCE is measured in bacteria that co-express alkyl hydroperoxide reductase, but in bacteria that do not co-express alkyl hydroperoxide reductase The amount is significantly lower than the amount of undecomposed cis-1,2-DCE measured. From this comparison, it is judged that even in the degradation of cis-1,2-DCE, the co-expressed alkyl hydroperoxide reductase clearly improves the efficiency of the degradation process or suppresses the degradation of degradation efficiency. .
[0062]
As shown in Table 3, in bacteria not co-expressing alkyl hydroperoxide reductase, undegraded trans-1,2-DCE is measured after the third time (after 4.5 hours in total). In bacteria that co-express peroxide reductase, undegraded trans-1,2-DCE has not been measured. From the 4th time (after 7 hours in total), undegraded trans-1,2-DCE is measured in bacteria that co-express alkyl hydroperoxide reductase, The amount is significantly lower than the amount of undegraded trans-1,2-DCE measured. From this comparison, it is judged that even in the degradation of trans-1,2-DCE, the co-expressed alkyl hydroperoxide reductase is clearly improving the efficiency of the degradation process or suppressing the degradation efficiency. .
[0063]
As shown in Table 4, in bacteria that did not co-express alkyl hydroperoxide reductase, undegraded 1,1-DCE was measured after the third round (after 4.5 hours in total), but alkyl hydroperoxide reductase Undegraded 1,1-DCE has not been measured in bacteria that co-express. From the 4th time (after 7 hours in total), undegraded 1,1-DCE is measured in bacteria co-expressing alkyl hydroperoxide reductase, but in bacteria not co-expressing alkyl hydroperoxide reductase. The amount is significantly lower than the amount of undegraded 1,1-DCE. From this comparison, it is judged that even in the degradation of 1,1-DCE, the co-expressed alkyl hydroperoxide reductase clearly promotes the efficiency of the degradation process or suppresses the degradation of the degradation efficiency.
[0064]
Therefore, it was shown that by co-expressing alkyl hydroperoxide reductase, the degradation efficiency of each DCE by microorganisms can be increased, and the high efficiency can be maintained for a long time.
[0065]
[Table 2]

Unit is ppm, detector detection limit is 0.1ppm,
ND: Below detection limit (≦ 0.1ppm)
Ahp: Alkyl hydroperoxide reductase
[0066]
[Table 3]

Unit is ppm, detector detection limit is 0.1ppm,
ND: Below detection limit (≦ 0.1ppm)
Ahp: Alkyl hydroperoxide reductase
[0067]
[Table 4]

Unit is ppm, detector detection limit is 0.1ppm,
ND: Below detection limit (≦ 0.1ppm)
Ahp: Alkyl hydroperoxide reductase
From the results, in addition to the TCE degradation treatment, the effect of co-expressing alkyl hydroperoxide reductase was also demonstrated in the liquid DCE degradation treatment using the JM1 strain (FERM BP-5352). It can also be seen that the effect of co-expressing the alkyl hydroperoxide reductase is not substantially dependent on the type of DCE and the position of chlorine substitution.
[0068]
(Example 4)
Effect of co-expressed alkyl hydroperoxide reductase on TCE degradation of KK01 strain (liquid system)
The alkyl hydroperoxide reductase gene recombinant plasmid prepared by the method described in Example 1 was introduced into bulk Holderia cepacia KK01 strain (FERM BP-4235) by electroporation (Bio-Rad) method. The resulting transformant has the same TCE resolution as that of the host strain KK01 (FERM BP-4235), and alkyl hydroperoxide reductase is expressed from the newly introduced E. coli-derived alkyl hydroperoxide reductase gene. .
[0069]
The culture conditions, collection conditions, etc. of the bacteria to be subjected to electroporation were in accordance with instructions attached to a commercially available electroporation apparatus. The apparatus was set to have a capacitance of 25 μF, a resistance value of 200Ω, and the amount of DNA to be donated was 1 μg. The suspension of the introduced strain is OD₆₀₀100 μl of a solution in which the cell density was adjusted so as to be 10 was used. A cuvette with a 0.2 mm gap was used (Bio-Rad), and the applied voltage was 2.5 kV. Here, the cuvette used was previously cooled to 4 ° C. The time constant during electroporation was 4.7 ms.
[0070]
Under the conditions described above, the transformant of the KK01 strain into which the alkylhydroperoxide reductase gene recombinant plasmid was introduced was selected using the selection marker of the broad host range plasmid vector pBBR122 itself, as in Example 2 above, and 100 μg / ml Agar M9 medium (containing 0.5% sodium glutamate) containing a concentration of chloramphenicol was used.
[0071]
Strains that formed colonies on agar M9 medium (containing 0.1% yeast extract and 2 mM phenol) were selected as alkyl hydroperoxide reductase gene recombinant KK01 strain. The collected alkyl hydroperoxide reductase gene recombinant KK01 strain was inoculated into 200 ml of a liquid M9 medium having the same composition as that of the agar M9 medium except for agar, and cultured at 25 ° C. in a 500 ml shake flask. At the time when 24 hours had elapsed from the start of culture, which was in the late logarithmic growth phase, the cells were collected by centrifugation. The collected cells were resuspended in M9 medium containing no equivalent amount of carbon source, and 10 ml of the cell suspension was injected into a 27.5 ml vial.
[0072]
Separately, the KK01 strain that has not been subjected to alkyl hydroperoxide reductase gene recombination, that is, the host KK01 strain (FERM BP-4235) itself is cultured, collected and suspended in the same manner. The solution was poured into a 27.5 ml vial.
[0073]
2 vials injected with these bacterial suspensions and 1 vial containing 10 ml of M9 medium only as a control, so that the total concentration in each vial is about 12 ppm. A predetermined amount of a saturated aqueous solution of TCE was added, and sealed with a butyl rubber stopper with a Teflon liner and an aluminum seal.
[0074]
These sealed vials were shaken at 30 ° C., and the TCE concentration in the vial gas phase was measured by gas chromatography after a certain time. After the measurement, the stopper was removed and once opened, and a predetermined amount of a saturated aqueous solution of TCE having a concentration in the liquid similar to the initial concentration was added again, and then the stopper was sealed again. However, the control vial was left in its initial state. Subsequently, the re-sealed vial was shaken at 30 ° C., and when a certain time had elapsed, the TCE concentration in the gas phase portion of the vial was measured by gas chromatography.
[0075]
The above measurement and resealing procedure were repeated to evaluate the effect of co-expressed alkyl hydroperoxide reductase on the repeated degradation of TCE. Specifically, 1st time: 1 hour later, 2nd time: 1 hour later (total 2 hours later), 3rd time: 1.5 hours later (total 3.5 hours later), 4th time: 1.5 hours later (total 5 hours later) , 5th: 1.5 hours later (total 6.5 hours later), 6th: 2 hours later (total 8.5 hours later), 7th: 2 hours later (total 10.5 hours later), 8th time: 2 hours later (total 12.5 hours later) After), 9th: A total of 9 measurements were performed 10 hours later (22.5 hours after total). Other operations were in accordance with the procedure described in Example 2 above. Table 5 shows the measurement results.
[0076]
[Table 5]

Unit is ppm, detector detection limit is 0.1ppm,
ND: Below detection limit (≦ 0.1ppm)
Ahp: Alkyl hydroperoxide reductase
As shown in Table 5, in bacteria that did not co-express alkyl hydroperoxide reductase, undegraded TCE was measured at the seventh time (after a total of 8.5 hours), but co-expressed alkyl hydroperoxide reductase. In fungi, undegraded TCE has not been measured. From the 8th time (after 12.5 hours in total), undegraded TCE is measured in bacteria co-expressing alkyl hydroperoxide reductase, but undegraded TCE measured in bacteria not co-expressing alkyl hydroperoxide reductase The amount is significantly lower than the amount. From this comparison, it is judged that the co-expressed alkyl hydroperoxide reductase is clearly improving the efficiency of the degradation treatment or suppressing the degradation of degradation efficiency. Therefore, in the KK01 strain (FERM BP-4235) as well as the above-mentioned JM1 strain, by co-expressing alkyl hydroperoxide reductase, the degradation efficiency of TCE by microorganisms can be increased and the high efficiency can be maintained for a long time. It was shown that an effect can be obtained. From this result, also in KK01 strain (FERM BP-4235), the effect of alkyl hydroperoxide reductase in the degradation treatment of liquid TCE was shown.
[0077]
(Example 5)
Acquisition of alkyl hydroperoxide reductase gene (2)
Construction of plasmid incorporating alkyl hydroperoxide reductase gene and TCE degrading enzyme gene
The Escherichia coli HB101 strain was cultured overnight at 37 ° C. in 50 ml of LB medium, and then total DNA was prepared by a conventional method. Using this total DNA as a template, PCR was performed using primer 3 and primer 4 shown below to amplify the alkyl hydroperoxide reductase gene.
Primer 3
5'-AACTTCTAGATTTGATAATGGAAACGC-3 '
Primer 4(Same base sequence as Primer 2)
5'-GCAATTGCAGGTGAAGCTTACTTC-3 '
In accordance with the PCR procedure described in Example 1 above, an LA-PCR kit (Takara Shuzo) was used, and the reaction solution composition and reaction conditions followed the instructions attached to the kit. An amplification product of about 2500 bp could be obtained by PCR. Restriction of the expression vector pSE280 (Invitrogen) containing the TCE-degrading enzyme gene derived from the KK01 strain (FERM BP-4235) disclosed in JP-A-6-296711 is completely digested with the restriction enzymes XbaI and HindIII. A ligation reaction was performed on the complete degradation product of the enzymes XbaI and HindIII using a ligation kit (Takara Shuzo). The resulting plasmid contains an alkyl hydroperoxide reductase gene derived from Escherichia coli HB101 and a TCE degrading enzyme gene derived from KK01 (FERM BP-4235). This plasmid was used to transform the host strain HB101 (Takara Shuzo). For selection of the transformant, an ampicillin resistance selection marker of the expression vector pSE280 itself was used, and an agar LB medium containing ampicillin at a concentration of 100 μg / ml was used.
[0078]
In the collected transformants, it was checked whether the alkyl hydroperoxide reductase gene was inserted as designed into the expression vector pSE280 containing the TCE-degrading enzyme gene derived from the KK01 strain (FERM BP-4235). The plasmid was digested with restriction enzymes XbaI and HindIII. Through the confirmation, a transformant carrying a recombinant plasmid into which an alkyl hydroperoxide reductase gene was inserted could be obtained. A recombinant plasmid containing the alkyl hydroperoxide reductase gene and the TCE-degrading enzyme gene derived from the KK01 strain (FERM BP-4235) by culturing the selected transformant, crushing the cultured cells, and separating and recovering the plasmid. Could get.
[0079]
(Example 6)
Effect of co-expressed alkyl hydroperoxide reductase on TCE degradation of TCE-degrading recombinant E. coli (liquid system)
Using the transformant prepared by the method of Example 5 and introducing the TCE-degrading enzyme gene derived from the KK01 strain (FERM BP-4235) and the alkyl hydroperoxide reductase gene recombinant plasmid into Escherichia coli HB101 strain (Takara Shuzo), The effect of co-expressed alkyl hydroperoxide reductase in was evaluated.
[0080]
The TCE-degrading enzyme gene and alkyl hydroperoxide reductase gene recombinant E. coli HB101 strain were inoculated on agar LB medium (containing 100 μg / ml ampicillin), and colonies were selected again. The bacterial strain that formed the colony was inoculated into 200 ml of liquid LB medium having the same composition as the agar LB medium (containing 100 μg / ml ampicillin) except for agar, and cultured at 37 ° C. in a 500 ml shake flask. At the time when 12 hours have elapsed from the start of the culture, which is the late phase of logarithmic growth, the cells are collected by centrifugation, resuspended in an M9 medium containing no equivalent carbon source, and 27.5 ml of 10 ml of the bacterial suspension. Into a vial.
In addition, the TCE-degrading genetically modified HB101 strain not recombined with the alkyl hydroperoxide reductase gene was similarly cultured, collected and suspended, and 10 ml of the bacterial suspension was injected into a 27.5 ml vial. TCE saturated aqueous solution was added to these vials and a total of 3 vials containing only 10 ml of M9 medium as a control so that the concentration in the vial was about 12 ppm, and a butyl rubber stopper with a Teflon liner and an aluminum seal were used. Sealed.
[0081]
These sealed vials were shaken at 30 ° C., and the TCE concentration in the vial gas phase was measured by gas chromatography after a certain time. After the measurement, the stopper was removed and once opened, and a predetermined amount of a saturated aqueous solution of TCE having a concentration in the liquid similar to the initial concentration was added again, and then the stopper was sealed again. However, the control vial was left in its initial state. Subsequently, the re-sealed vial was shaken at 30 ° C., and when a certain time had elapsed, the TCE concentration in the gas phase portion of the vial was measured by gas chromatography.
[0082]
The above measurement and resealing procedure were repeated to evaluate the effect of co-expressed alkyl hydroperoxide reductase on repeated degradation of TCE. Specifically, 1st time: 1 hour later, 2nd time: 1 hour later (total 2 hours later), 3rd time: 1.5 hours later (total 3.5 hours later), 4th time: 1.5 hours later (total 5 hours later) , 5th: 1.5 hours later (total 6.5 hours later), 6th: 2 hours later (total 8.5 hours later), 7th: 2 hours later (total 10.5 hours later), 8th time: 2 hours later (total 12.5 hours later) After), 9th: A total of 9 measurements were performed 10 hours later (22.5 hours after total). Other operations were in accordance with the procedure described in Example 2 above. Table 6 shows the measurement results.
[0083]
[Table 6]

Unit is ppm, detector detection limit is 0.1ppm,
ND: Below detection limit (≦ 0.1ppm)
Ahp: Alkyl hydroperoxide reductase
As shown in Table 6, in bacteria that did not co-express alkyl hydroperoxide reductase, undegraded TCE was measured in the sixth time (after a total of 8.5 hours), but co-expressed alkyl hydroperoxide reductase. In fungi, undegraded TCE has not been measured. Since the seventh round (after 10.5 hours in total), bacteria that co-express alkyl hydroperoxide reductase also measure slightly undegraded TCE, but they do not degrade in bacteria that do not co-express alkyl hydroperoxide reductase The amount is much lower than the TCE amount. From this comparison, it is judged that the co-expressed alkyl hydroperoxide reductase is clearly improving the efficiency of the degradation treatment or suppressing the degradation of degradation efficiency. Therefore, co-expression of alkyl hydroperoxide reductase in E. coli HB101 strain transformed with TCE-degrading enzyme gene recombinant plasmid derived from KK01 strain (FERM BP-4235) as well as KK01 strain (FERM BP-4235) above As a result, it was shown that the TCE degradation efficiency by microorganisms can be increased, and that the high efficiency can be maintained for a long time.
[0084]
From these results, the effect of alkyl hydroperoxide reductase in the degradation of liquid TCE was also demonstrated in TCE-degrading enzyme E. coli.
[0085]
(Example 7)
Effect of co-expressed alkyl hydroperoxide reductase on TCE degradation of JM1 strain (soil system)
Similar to the liquid system shown in Example 2 above, it was evaluated that the co-expressed alkyl hydroperoxide reductase was effective in the TCE degradation of the JM1 strain for the purification of TCE-contaminated soil as follows.
[0086]
Sahara through sand (water content 13%, non-sterilized) 50g each in 15 68ml vials, add TCE saturated aqueous solution so that the concentration in soil is 5ppm, seal tightly and leave at 15 ° C for 3 days A simulated TCE-contaminated soil sample was prepared.
[0087]
Then, 5 ml of the prepared suspension of alkyl hydroperoxide reductase gene recombinant JM1 strain was injected into the vial according to the procedure of Example 2. A bacterial suspension of the host JM1 strain (FERM BP-5352) not recombined with the alkyl hydroperoxide reductase gene was prepared in the same manner, and 5 ml was injected into a vial. Further, as a control, 5 ml of M9 medium alone was added to the other. The cell suspension and TCE decomposition experiments were conducted at 20 ° C., which is close to the actual soil temperature.
[0088]
In the TCE decomposition experiment, after injecting the bacterial suspension into the vial, add a TCE saturated aqueous solution so that the concentration when the TCE is completely dissolved in the liquid part of the soil is 10 ppm, and the Teflon liner butyl rubber stopper and aluminum cap And completely sealed. These sealed vials were allowed to stand at 20 ° C., and the TCE concentration in the vial gas phase was measured by gas chromatography after a certain time. After the measurement, the stopper was removed and the tube was opened once, and again, TCE gas having a liquid concentration similar to the initial concentration was added, followed by sealing again. However, the control vial was left in its initial state. Subsequently, the resealed vial was allowed to stand at 20 ° C., and when a certain time had elapsed, the TCE concentration in the gas phase portion of the vial was measured by gas chromatography.
[0089]
The above measurement and resealing procedure were repeated to evaluate the effect of co-expressed alkyl hydroperoxide reductase on repeated degradation of TCE. Specifically: 1st time: 2 hours later, 2nd time: 3 hours later (total 5 hours later), 3rd time: 4 hours later (total 9 hours later), 4th time: 5 hours later (total 14 hours later) 5th time: Measurement was performed 5 times in total, 10 hours later (total 24 hours later). In addition, after adding the 1st, 3rd, and 5th TCE, when sealing, formaldehyde as an energy source was added so as to have a final concentration of 1 mM. Other operations were in accordance with the procedure described in Example 2 above. Table 7 shows the measurement results.
[0090]
[Table 7]

The unit is ppm, the detection limit of the detector is 0.01 ppm,
ND: Below detection limit (≦ 0.01ppm)
Ahp: Alkyl hydroperoxide reductase
As shown in Table 7, in bacteria that did not co-express alkyl hydroperoxide reductase, undegraded TCE was measured at the third time (after 9 hours in total), but co-expressed alkyl hydroperoxide reductase. In fungi, undegraded TCE has not been measured. From the 4th time (after 14 hours in total), some undegraded TCE is measured in bacteria co-expressing alkyl hydroperoxide reductase, but undegraded in bacteria not co-expressing alkyl hydroperoxide reductase This is much lower than the TCE amount. From this comparison, it is judged that the co-expressed alkyl hydroperoxide reductase is clearly improving the efficiency of the degradation treatment or suppressing the degradation of degradation efficiency. From these results, the effect of co-expressed alkyl hydroperoxide reductase was also demonstrated in the degradation treatment of soil-based TCE using JM1 strain. That is, even in contaminated soil remediation, it is possible to effectively suppress the degradation and deactivation of the degradation activity of microorganisms that often occur when the remediation treatment is performed over a long period of time by co-expressing alkyl hydroperoxide reductase. Indicated.
[0091]
(Example 8)
Effect of co-expressed alkyl hydroperoxide reductase on TCE degradation of JM1 strain (gas phase system)
A porous cellulose carrier (average pore size of about 500 mm, diameter of about 5 mm) was sterilized in an autoclave and cooled to room temperature or lower, and packed into three vials. According to the procedure described in Example 2, 15 ml of the prepared suspension of alkyl hydroperoxide reductase gene recombinant JM1 strain was injected into a vial. Further, 15 ml of a bacterial suspension of the host JM1 strain (FERM BP-5352) not recombined with the alkyl hydroperoxide reductase gene was injected into another vial. Further, as a control, 15 ml of M9 medium alone was added to the other vial.
[0092]
In each vial, TCE-contaminated air (prepared with a permeator, gas concentration 100 ppm) was added with a 1 ml gas tight syringe and sealed. TCE decomposition was performed at 25 ° C., and after 4 hours, the TCE concentration in the gas phase was measured by gas chromatography. After the measurement, the stopper was removed and the tube was once opened, and again TCE gas having a gas phase concentration comparable to the initial value was added, followed by sealing again. However, the control vial was left in its initial state. Subsequently, TCE decomposition was performed at 25 ° C. in the resealed vial, and when 4 hours had elapsed, the TCE concentration in the gas phase portion of the vial was measured by gas chromatography.
[0093]
Every 4 hours, the above measurement and reseal operation were repeated to evaluate the effect of the co-expressed alkyl hydroperoxide reductase on the repeated degradation of TCE. Specifically: 1st time: 4 hours later, 2nd time: 4 hours later (total 8 hours later), 3rd time: 4 hours later (total 12 hours later), 4th time: 4 hours later (total 16 hours later) 5th time: Measurement was performed 5 times in total, 4 hours later (20 hours later in total). In addition, when the first, third, and fifth times of TCE gas were added and sealed, formaldehyde as an energy source was added to a final concentration of 1 mM. Other operations were in accordance with the procedure described in Example 2 above. Table 8 shows the measurement results. The TCE concentrations shown in Table 8 are gas concentrations.
[0094]
[Table 8]

The unit is ppm (gas concentration), the detection limit of the detector is 10ppm,
ND: Below detection limit (≦ 10ppm)
Ahp: Alkyl hydroperoxide reductase
As shown in Table 8, undegraded TCE was measured after the 4th time (after 12 hours in total) in both the bacteria not co-expressing alkyl hydroperoxide reductase and the bacteria co-expressing alkyl hydroperoxide reductase. However, the amount of undegraded TCE measured in bacteria co-expressing alkyl hydroperoxide reductase is significantly lower than the amount of undegraded TCE measured in bacteria not co-expressing alkyl hydroperoxide reductase Is. From this comparison, it is judged that the co-expressed alkyl hydroperoxide reductase is clearly improving the efficiency of the degradation treatment or suppressing the degradation of degradation efficiency. From these results, the effect of co-expressed alkyl hydroperoxide reductase was also demonstrated in the decomposition treatment of gas phase TCE using JM1 strain, that is, in the purification of contaminated air. That is, even in the purification of contaminated air, there is an effect of effectively suppressing the degradation and deactivation of the degradation activity of microorganisms, which often occurs when the purification treatment takes a long time, by co-expressing alkyl hydroperoxide reductase. Indicated.
[0095]
【The invention's effect】
In the method for repairing a contaminated environment according to the present invention, by co-expressing alkyl hydroperoxide reductase in a microorganism exhibiting degrading activity, for example, contamination with persistent volatile organic compounds such as the above-mentioned chlorinated ethylene compounds. An efficient biodegradation and purification treatment of the liquid, soil, and air thus made becomes possible. In particular, when purification treatment takes a long time, co-expression of alkyl hydroperoxide reductase has the effect of effectively suppressing degradation and deactivation of the degradation activity of microorganisms over time. Accordingly, there is an advantage that the efficiency of the biodegradation purification process can be achieved by preventing the degradation and deactivation of the degradation activity over time.

Claims (21)

In repairing the environment polluted by the organic compound using a microorganism having the ability to decompose organic compounds,
The organic compound decomposed by the microorganism is a chlorinated aliphatic compound,
Co-expressing the genetically modified alkyl hydroperoxide reductase together with the organic enzyme degrading enzyme system obtained by genetic recombination using the alkyl hydroperoxide reductase gene for microorganisms capable of degrading organic compounds Using a transformant having the ability to
Using the microorganism characterized in that the transformant microorganism is allowed to co-express a recombinant alkyl hydroperoxide reductase together with the organic enzyme degrading enzyme system, and the microorganism is brought into contact with the organic compound for degradation. A method for improving the efficiency of environmental remediation contaminated with organic compounds. The method according to claim 1 , wherein the organic compound decomposed by the microorganism is trichloroethylene or dichloroethylene. 3. The method according to claim 1 , wherein the contaminated environment to be repaired is a liquid such as ground water or waste liquid. 4. The method according to claim 3 , wherein the organic compound is brought into contact with the microorganism by bringing the liquid into contact with a carrier carrying the microorganism. The contact of the liquid with the carrier carrying the microorganism is performed by introducing the liquid from one end of a container containing the carrier carrying the microorganism and discharging the repaired liquid from the other end to the outside of the container. 5. The method of claim 4 , wherein the method is performed. 3. A method according to claim 1 or 2 , characterized in that the contaminated environment to be repaired is soil. The method according to claim 6 , wherein when the organic compound is brought into contact with the microorganism, a liquid containing the microorganism is introduced into the soil. The method according to claim 7 , wherein the introduction of the liquid containing microorganisms into the soil is performed from an injection well provided in the soil. The method according to claim 6 , wherein the organic compound is brought into contact with the microorganism by introducing the soil into a liquid containing the microorganism. 7. The method according to claim 6 , wherein the organic compound is brought into contact with the microorganism by mixing a liquid containing the microorganism and contaminated soil. The method according to claim 6 , wherein the organic compound is brought into contact with the microorganism by bringing the carrier carrying the microorganism into contact with contaminated soil. 3. A method according to claim 1 or 2 , characterized in that the contaminated environment to be repaired is air. The method according to claim 12 , wherein the organic compound is brought into contact with the microorganism by introducing contaminated air into a liquid containing the microorganism. 13. The method according to claim 12 , wherein the organic compound is brought into contact with the microorganism by bringing a carrier carrying the microorganism into contact with contaminated air. When the organic compound is brought into contact with the microorganism, contaminated air is introduced from one end of a container that stores the carrier carrying the microorganism, and the repaired air is discharged from the other end to the outside of the container. 15. The method of claim 14 , wherein: Of subjects subjected to genetic recombination using an alkyl hydroperoxide reductase gene, a microorganism having the organic compound decomposing ability, any one of claims 1 to 15, characterized in that the strain JM1 (FERMBP-5352) The method described in 1. The microorganism having the ability to degrade organic compounds, which is subjected to genetic recombination using an alkyl hydroperoxide reductase gene, is a bulk hordelia cepacia KK01 strain (FERM BP-4235) . 16. The method according to any one of 15 . Of subjects subjected to genetic recombination using an alkyl hydroperoxide reductase gene, claim microorganism having the organic compound decomposing ability, wherein the a microorganism recombined by enzyme gene of the organic compound 1-15 The method as described in any one of . Microorganism recombined by enzyme gene of the organic compound, according to claim, wherein the bulk Burkholderia cepacia KK01 strain (FERM BP-4235) is a microorganism recombined by enzyme gene of organic compounds from 18 The method described in 1. 19. The method according to claim 18 , wherein the microorganism recombined with the enzyme degrading enzyme gene is a transformant having E. coli as a host. The method according to any one of claims 1 to 20, wherein the alkyl hydroperoxide reductase gene is an alkyl hydroperoxide reductase gene derived from Escherichia coli K-12.