JP6347315B2 - 酸素添加酵素含有組成物の活性化方法及びこれに基づく汚染物質の無害化方法 - Google Patents
酸素添加酵素含有組成物の活性化方法及びこれに基づく汚染物質の無害化方法 Download PDFInfo
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- JP6347315B2 JP6347315B2 JP2013141383A JP2013141383A JP6347315B2 JP 6347315 B2 JP6347315 B2 JP 6347315B2 JP 2013141383 A JP2013141383 A JP 2013141383A JP 2013141383 A JP2013141383 A JP 2013141383A JP 6347315 B2 JP6347315 B2 JP 6347315B2
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Description
本発明の方法に使用しうる酸素添加酵素は、汚染物質を酸化的に分解しうる酵素であれば特に限定されるものではなく、多種多様な汚染物質を分解するものとして多くの動物又は微生物由来の酸素添加酵素を用いることができる。これらの中でも、特に、芳香環水酸化ジオキシゲナーゼ、又はRieske non-heme iron oxygenaseと称される一群の酵素が好ましく、これらは多くの芳香族化合物分解経路での最初の反応であるcis型二水酸化反応を触媒する。トルエンやナフタレンに加えてベンゼン、クメン、フェナントレン、ピレン等の単環・多環の芳香族炭化水素に限らず、ダイオキシン類、ジベンゾチオフェン、カルバゾール等のヘテロ環式芳香族化合物、PCBs等のビフェニル環化合物等の種々の芳香族化合物の好気的代謝経路において、これらの酵素は初発酸化酵素として一連の分解反応の進行の有無を左右する重要な役割を果たしている。芳香環水酸化ジオキシゲナーゼは、基質を認識し酸化反応を行う酸化酵素(TO:terminal oxygenase)と、電子をNAD(P)HからTOに伝える電子伝達系から構成される多成分酵素である。電子伝達系は、NAD(P)Hから電子を受け取るレダクターゼ(Red)単独で構成される場合と、Redとフェレドキシン(Fdx)の二つで構成される場合がある。
例えば、汚染物質としてのPCBsを分解するためには、コマモナス属、シュードモナス属、アクロモバクター属、ロドコッカス属、及びステノトロフォモナス属からなる群より選択される少なくとも1種、あるいはそれ以上のPCBs分解菌を用いることができる。これらの菌株は、2,3-ビフェニルジオキシゲナーゼ(2,3-biphenyl dioxygenase)が共通に選択する基質特異性を示し、PCBs異性体に対しては更に狭い範囲の基質特異性を示す。具体的には、2,2’,3,4−テトラクロロビフェニルや、3,3’,4,4’−テトラクロロビフェニル、2,3,3’,6−テトラクロロビフェニル,3,4,4’−トリクロロビフェニル、2,2’,3,4’−テトラクロロビフェニル、2,3,3’,4’,テトラクロロビフェニル、2,3,4,4’,テトラクロロビフェニル、2,2’,3,5,5’−ペンタクロロビフェニル、2,2’,4,4’−テトラクロロビフェニル、2,2’,4,5−テトラクロロビフェニル、2,2’,3,5’−テトラクロロビフェニルを選択的に分解する複数の細菌を混合して複合化させることが好ましい。
このような酸素添加酵素含有組成物を、常温、常圧の大気環境下での飽和溶存酸素濃度を超える量の酸素を含有した水性媒体中に溶解又は分散させることにより、酸素添加酵素含有組成物を活性化することができる。特に、酸素添加酵素含有組成物として上述した微生物製剤を用いた場合に、顕著な活性化効果を得ることができる。水性媒体中の溶存酸素濃度の上昇によって、酸素添加酵素含有組成物が活性化されること、例えば、あらかじめ芳香環水酸化ジオキシゲナーゼを高発現させた微生物製剤による基質の分解反応が向上するメカニズムについては現在のところ明らかではないが、考えられる要因は、まず、水性媒体中の微生物細胞外の酸素濃度が高まることで、受動輸送により、極めて短時間に、微生物細胞内の酸素濃度が水性媒体中の酸素濃度近くまで上昇する。次に、微生物細胞内と細胞外の酸素濃度の平衡化現象は、微生物細胞内で行われる芳香環水酸化ジオキシゲナーゼの基質に対する酸素添加反応の速度向上へ寄与していることである。さらに、この酸素添加反応の生成物は、高酸素下で誘導された酸化ストレスによって活性化した酵素組成物中に含まれるその他の代謝系酵素類や補酵素類が、より低分子にまで物質変換を進めるため、基質を完全に分解することができると考えられる。
本明細書において、用語「マイクロバブル」とは、概ね直径1mm以下、好ましくは直径100μm以下の気泡をいう。外部から酸素や空気等の気体を供給して気泡を形成してもよいし、水性媒体中に溶存している酸素や空気等を用いてもよいが、水性媒体の溶存酸素濃度を高めるためには、酸素ガスを外部から供給しつつマイクロバブルを発生させることが好ましい。マイクロバブルは体積当たりの表面積が広く浮上速度も極めて遅いため、効果的に酸素等の気体を液体に溶解させることができる。また、電荷を帯びることで液中に均一に分散し、水性媒体中で油性成分のエマルジョン化を促進する。マイクロバブルは負の表面電荷を有するため、一般に正の表面電荷を持つ微生物菌体等との相互作用を介して、これらを水性媒体中で均一に分散させることが可能になる。
本発明の分解又は無害化方法の対象となる汚染物質は、酸素添加酵素によって酸化的に分解されるものであれば特に限定されるものではないが、単環若しくは多環芳香族化合物であることが好ましく、これらの中にはトルエン及び/又はベンゼン若しくはダイオキシン類及び/又はポリ塩化ビフェニル類を含む。本明細書において、ダイオキシン類とは、ポリ塩素化ジベンゾ−p−ダイオキシン、ポリ塩素化ジベンゾフラン、及びコプラナーPCB(特に、オルト位以外に塩素原子が置換したポリ塩化ビフェニル)の全ての総称である。本発明では、「ダイオキシン類」は、特に断らない限りこれらの化合物の一部又は全部を表す。
次に本発明の1つの実施形態に係る汚染物質の無害化装置を、図面を参照しながら説明する。図3において、本実施形態に係る汚染物質の無害化装置1は、通気手段を兼ねた攪拌翼18を供えた攪拌槽10と、当該攪拌槽と連通し水性媒体を供給するためのバッファータンク20と、汚染物質を供給するタンク30と、酵素製剤投入口40と、これらに酸素ガスを供給又は排出するための通気管19及び排気管23とを備えている。この実施形態においてPCBs等の汚染物質は専用のタンクから攪拌槽に供給されるが、特にこれに限定されるものではなく、バッファータンクと兼用しても良い。攪拌槽には、さらに超音波の振動伝達体50超音波振動子51とからなるマイクロバブル発生部が備えられている。
さらに、マイクロバブル発生装置としては、基質を含む気体を加圧溶解した水を減圧して、マイクロバブルを発生させてバッファータンク20内の水性媒体に供給する装置も用いることができる。あるいは、気液二相流を突起物や衝突体に衝突させて、気泡を剪断してマイクロバブルとして攪拌槽10内の混合液中に供給する装置等、いずれの装置も用いることができる。
コマモナス・テストステロニ(C. testosteroni) YAZ2株及びYU14-111株の培養には、以下の表1に示した組成のミネラル塩合成培地(W培地)を使用した。
既知のPCBs分解細菌のBphA1(ビフェニル−2,3−ジオキシゲナーゼ αサブユニット)のアミノ酸配列の比較から、保存性の高い領域を数カ所選び(Asn-Gln/Ser-Cys-Arg/Ser-His-Arg-Gly-Met(配列番号1)並びにGlu-Gln-Asp-Asp-Gly/Thr-Glu-Asn(配列番号2)など)、縮重プライマーを作製した。
次に、コマモナス・テストステロニ(C. testosteroni) YAZ2株及びYU14-111株の細菌を適当量のTE緩衝液(10mM Tris−HCl,1mM EDTA,pH8.0)に懸濁し、加熱処理して得たゲノムDNAを含む抽出物を鋳型として使用した。作製した縮重プライマーを用いて、94℃、3分→[94℃、30秒→58〜60℃、30秒→72℃、1分(30サイクル)]→72℃、2分、の反応条件でPCRを行った。また、BphA1遺伝子を持たないネガティブコントロールとして、大腸菌K−12株のゲノムを含む熱抽出物を用いて同様の反応を行った。
上記の参考例1に倣い、ビフェニルジオキシゲナーゼ遺伝子を発現するコマモナス・テストステロニ(C. testosteroni)YAZ2株の細菌製剤を作製し、この細菌製剤を波長660nmの濁度で10又は60(湿菌重量で約15mg又は90mg)に調整して、ビフェニル及びPCBs異性体である2,2’−ジクロロビフェニル、4,4’−ジクロロビフェニル、および3,3’,4,4’−テトラクロロビフェニルの標準品を、それぞれ0.5ppmの濃度で混合したPCBs混合溶液と、30℃で加温しながら24時間にわたり接触反応させた。試験は2回繰り返して行った。
ガスクロマトグラフィーの温度プログラムは、初期温度80℃から20℃/分の昇温速度で130℃まで昇温し、引き続き8℃/分で300℃まで昇温した。分析カラムは非極性のキャピラリーカラム(アジレント社製、HP-5ms、0.25mm×15m、0.25μm)を使用した。
両者のTICチャートを比較すると、試験サンプルではビフェニルをはじめ、PCBs異性体である2,2’−ジクロロビフェニルや4,4’−ジクロロビフェニル、3,3’,4,4’−テトラクロロビフェニルのピークは消失した。
すなわち、ビフェニルジオキシゲナーゼ遺伝子を発現する細菌は、ビフェニル及びPCBs異性体を分解することを示した。この結果は、細菌製剤がPCBs分解酵素であるビフェニルジオキシゲナーゼを含有していることを示唆した。
氷冷した20mMリン酸ナトリウム緩衝液(pH7.5)に図4に示すような超音波ホモジナイザー(SMT社製、UH-50)に内部がガス通過可能な中空ホーン(出口内径−外径:φ2.6-φ6.0)を取り付けた装置を用いて酸素マイクロバブルを10分間連続充填した。この装置からは、直径20μm以下のマイクロバブルが発生し、10分間のバブリングにより、20mMリン酸ナトリウム緩衝液中の溶存酸素濃度は約28ppmまで上昇した。
このマイクロバブル充填溶液0.5mLあたりに、界面活性剤トリトンX−100を最終濃度0.01%又は0.005%、ビフェニルジオキシゲナーゼを発現する細菌製剤を波長660nmの濁度で10又は60(湿菌重量で約15mg又は90mg)、PCBs汚染廃油又は商用PCBsであるカネクロールKC−300(GLサイエンス社製)を10ppm又は100ppmとなるように加えてPCBs分解反応溶液とし、30℃で加温しながら転倒撹拌した。このとき、20mMリン酸ナトリウム緩衝液に対するPCBs含有油の混合比率は、容量比で約99:1である。試験は3回繰り返して行った。
定量解析は内部標準法で行い、また各定点での分析はすべて3回繰り返し行って、定量値の平均と標準偏差を求めた。
以上の結果から、通常の酸素分圧下での反応と比較して、酸素マイクロバブルを充填するとコマモナス・テストステロニ(C. testosteroni)YU14-111製剤によるPCBsの分解効率が約5〜8%向上することが分かった。
実施例1と同様の方法にて、酸素マイクロバブル充填による高酸素溶存下(初期濃度約28ppm)の20mMリン酸ナトリウム緩衝液中で、ビフェニルジオキシゲナーゼを高発現させたコマモナス・テストステロニ(C. testosteroni)YAZ2細菌製剤と、商用PCBsのカネクロールKC−300(初期濃度100ppm)とを反応させ、48時間後までの残存PCBs濃度の経時的変化を追い、その結果を図6に示した。
2 マイクロバブル発生装置
10 攪拌槽
11 スイッチバルブ
12 レギュレーター
13 メインバルブ
14 フィルター・レギュレーター
15 エアーポンプ(コンプレッサー)
16 逆止弁
17 ロータリージョイント
18 攪拌翼
19 通気管
20 バッファータンク
30 PCBタンク
40 微生物製剤投入口
50 振動伝達体
51 超音波振動子
52 気体供給口
53 気体流路
54 気体放出口
55 超音波放射面
56 ケーブル
57 振動制御器
Claims (6)
- 酸素添加酵素を含有する組成物を、常温、常圧の大気環境下での飽和溶存酸素濃度を超える量の酸素を含有した水性媒体中に溶解又は分散させること、及び前記水性媒体中へマイクロバブルを供給することを含み、前記マイクロバブルが、前記水性媒体及び/又は前記水性媒体と油性成分の混合物へ、酸素を流しながら超音波処理を行うことにより発生することを特徴とする、酸素添加酵素含有組成物の活性化方法。
- 飽和溶存酸素濃度を超える量の酸素を含有した水性媒体中で、酸素添加酵素含有組成物と汚染物質を含む油性成分との混合物を攪拌すること、及び前記水性媒体中へマイクロバブルを供給することを含み、前記マイクロバブルが、前記水性媒体及び/又は前記水性媒体と油性成分の混合物へ、酸素を流しながら超音波処理を行うことにより発生することを含む、汚染物質の分解又は無害化方法。
- 前記マイクロバブルが、前記水性媒体及び/又は前記水性媒体と油性成分の混合物を、加圧処理することにより発生する請求項1または2に記載の方法。
- 前記汚染物質が、トルエン、ベンゼン、ダイオキシン類及び/又はポリ塩化ビフェニル類を含む、単環若しくは多環芳香族化合物からなる請求項2または3に記載の方法。
- 前記酸素添加酵素含有組成物が、芳香環水酸化ジオキシゲナーゼを微生物細胞内で発現させた微生物製剤である請求項1〜4の何れか一項に記載の方法。
- 前記混合物が、界面活性剤又はアルコールを含み、前記水性媒体と油性成分とのエマルジョンである請求項2〜5の何れか一項に記載の方法。
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JP2010046595A (ja) * | 2008-08-20 | 2010-03-04 | Arthur:Kk | 汚染土壌の微生物分級浄化方法及び汚染土壌の微生物分級浄化装置 |
JP5037479B2 (ja) * | 2008-11-18 | 2012-09-26 | シャープ株式会社 | 浄化処理装置及び浄化処理方法 |
JP5297221B2 (ja) * | 2009-02-16 | 2013-09-25 | 株式会社テクノ菱和 | 水溶性有機化合物の除去システム |
JP2011000567A (ja) * | 2009-06-22 | 2011-01-06 | Arthur:Kk | 土壌・地下水汚染物質の分解浄化方法及びそのシステム |
JP2012035181A (ja) * | 2010-08-05 | 2012-02-23 | Shimizu Corp | 汚染土壌及び汚染地下水の原位置浄化処理方法 |
JP2012040476A (ja) * | 2010-08-16 | 2012-03-01 | Shimizu Corp | 汚染土壌または地下水の浄化方法および装置 |
JP6099569B2 (ja) * | 2011-10-28 | 2017-03-22 | サンスター技研株式会社 | 組成物およびその製造方法 |
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JP2015012830A (ja) | 2015-01-22 |
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