JP3735925B2 - Method for purifying polyurethane esterase and method for decomposing ester polyurethane - Google Patents

Description

【００２５】
上記に述べたような培地をオートクレーブなどで滅菌し、コマモナス・アシドボランス ＴＢ−３５株又はその同等菌を植菌し、約３０℃で振とう培養する。培養後５〜８日、好ましくは菌体の生育量が最も大きい６日後に培養を止めて酵素を精製する。
【００２６】
（酵素の精製）
本発明のエステル系ポリウレタンエステラーゼの精製方法は、室温で行うことが可能である。ポリウレタンエステラーゼは、菌体表面に付着しているため、精製に当たって、界面活性剤で抽出する。用いる界面活性剤としては、デオキシＢＩＧＣＨＡＰ及びｎ−ドデシル−β−Ｄ−マルトシドが好適である。
デオキシＢＩＧＣＨＡＰ及びｎ−ドデシル−β−Ｄ−マルトシドはそれぞれ、分子量８６２. ０７及び５１０. ６２の界面活性剤である。デオキシＢＩＧＣＨＡＰ及びｎ−ドデシル−β−Ｄ−マルトシドの構造式をそれぞれ（１）、（２）に示す。
【００２７】
【化１】

【００２８】
【化２】

【００２９】
デオキシＢＩＧＨＣＨＡＰは、臨界ミセル濃度が約０. １２％と低く、溶液を２倍程度に希釈することによって、容易にミセルを破壊できる。また、電荷を持たないため、イオン交換クロマトグラフィーの操作に影響を及ぼさない。さらに、使用濃度では硫安分画時に沈殿しない。これらの理由から、特に、デオキシＢＩＧＣＨＡＰが好適である。デオキシＢＩＧＣＨＡＰの使用濃度は、０. ２〜０. ２５％ｗ／ｗが好ましい。０. ２％より低い濃度だと、抽出効果が悪く、０. ２５％より高い濃度だとその後の精製が困難である。
【００３０】
培養後の菌体を集菌後、上記の界面活性剤を含むリン酸バッファー、トリス・ＨＣｌバッファー（ｐＨ約７. ０）などの一般に用いられる緩衝液に懸濁し、攪拌して抽出する。その後、遠心して上清と菌体に分け、上清を粗抽出液とする。粗抽出液は、硫安等の塩析などによって濃縮後、疎水クロマトグラフィー用担体を添加する。担体吸着分を界面活性剤を含む緩衝液に溶出させ、その溶出液を塩析などによって濃縮する。濃縮した担体吸着液を界面活性剤を含む緩衝液に溶かして、陰イオン交換体を添加し、陰イオン交換体に吸着しない画分を、塩析などによって濃縮後、さらに緩衝液に溶かしたものを精製エステラーゼ標品とする。精製過程での担体の添加は、カラムクロマトグラフィーを行うことで代替することができる。
【００３１】
上記に述べた本発明の精製方法によって、粗抽出液から比活性約８倍以上、収率約２５％でポリウレタンエステラーゼを精製できる。本発明の精製方法は、すべてバッチ処理のみで精製を行うことができるので、高価なクロマト機器やカラム等を必要とせず、攪拌器のみで精製が行える。従って、スケールアップが極めて容易、安価に行える。
本発明の精製方法によって、電気泳動に供して、単一のバンドで確認される程度に高純度のポリウレタンエステラーゼが得られる。
【００３２】
（ポリウレタンエステラーゼの性質）
本発明で得られるポリウレタンエステラーゼは、分子量約６２，０００の単量体で、酵素が働くのに適切なｐＨは４〜８、特に好ましくはｐＨ６. ５である。従って、ｐＨ約６〜７. ５の緩衝液中でポリウレタン分解を行うのが望ましい。本発明のポリウレタンエステラーゼは、エステル系ポリウレタンのエステル結合を切断し、アジピン酸とジエチレングリコールを産出する。分解産物のアジピン酸の産生によって、反応溶液のｐＨの低下が起こりやすい。したがって、至適ｐＨから外れるのを防ぐために、例えば、１％のＣａＣＯ₃ の添加などの処理をしてｐＨ管理をすることが望ましい。分解するポリウレタンの形状は特に限定しないが、フィルム状、球状など表面積の大きな形状にするのが望ましい。
【００３３】
本発明のポリウレタンエステラーゼの活性測定は、エステラーゼによりｐ−ニトロフェニルアセテートを基質として、生成するｐ−ニトロフェノールの量を４０５ｎｍの吸光度で測定することによって行う。本発明のポリウレタンエステラーゼの活性の単位として、１分間に１μｍｏｌのｐ−ニトロフェノールを生成する酵素量を１ユニットとする。
【００３４】
【実施例】
以下に実施例を挙げて、本発明をさらに詳細に説明するが、これらの実施例により本発明の範囲を制限することを意図するものではない。
実施例１（マンニトールを炭素源とした細菌の培養）
実験に用いたポリウレタンは、ポリエチレングリコールアジペート（ニッポラン２２００−Ａ、日本ポリウレタン工業社製）に、２，４−トリレンジイソシアネート（和光純薬工業社製）をＯＨ基：イソシアネート基が１：１になるように加えて合成したものである。
３０ｍｌの上記に述べた表１の組成の培地を３００ｍｌの三角フラスコに入れ、１２１℃で１５分間オートクレーブをかけて滅菌処理を行った。滅菌処理後の培地にＴＢ−３５株を１白金耳植菌し、３０℃で回転振とう培養を行った。２日おきにサンプリングを行い、菌体生育量及びポリウレタン分解活性を測定した。菌体生育量は５８０ｎｍでの濁度によって測定した。
【００３５】
比較例１（ポリウレタンを炭素源とした細菌の培養）
表１の培地のうち、マンニトールの代わりにポリウレタンを１％ｗ／ｗ添加した培地で培養し、同様に培養し、菌体生育量及びポリウレタン分解活性を測定した。
実施例１及び比較例１の結果を図１及び図２に示す。
【００３６】
上記の結果から、菌体は培養６日目に最も大きい生育量を示すことがわかった。マンニトールを炭素源とした場合には、ポリウレタンを炭素源としたときと比べて、生育菌体当たりのポリウレタンエステラーゼ活性は約１／２と低い。しかし、菌体の生育は、マンニトールを炭素源としたときのほうがよい。また、マンニトールは安価で、かつ、入手しやすいため、大量の酵素を迅速に調製するためには、ポリウレタンよりもマンニトールを炭素源としたほうが良いとわかった。
【００３７】
実施例２（酵素の抽出）
ポリウレタンを炭素源として培養した菌体を集菌し、湿重量で約１０％になるように２０ｍＭリン酸バッファー（ｐＨ７. ０）を添加し、懸濁した。この懸濁液にデオキシＢＩＧＣＨＡＰ又はｎ−ドデシル−β−Ｄ−マルトシド（共に同仁化学研究所社製）を界面活性剤として０. ２％ｗ／ｗ加え、ボルテックスミキサーによって室温で２時間攪拌して抽出した。その後、遠心して上清と菌体とにわけ、上清のポリウレタンエステラーゼの活性を測定した。
【００３８】
比較例２
界面活性剤として、ＣＨＡＰＳ、ＣＨＡＰＳＯ、ＢＩＧＣＨＡＰ、ｎ−オクチル−β−Ｄ−グルコシド、ｎ−ヘプチル−β−Ｄ−チオグルコシド、ｎ−オクチル−β−Ｄ−チオグルコシド、ＭＥＧＡ−８、ＭＥＧＡ−９、ＭＥＧＡ−１０、スクロースモノカプレート、スクロースモノラウレート、コール酸ナトリウム、又はジギトニン（すべて同仁化学研究所社製）を用いた以外は、実施例２と同様な方法でポリウレタンエステラーゼの活性を測定した。また、界面活性剤未添加の上清及び未処理の菌体についてもエステラーゼ活性の測定をした。
実施例２及び比較例２の結果を表２に示す。
【００３９】
【表２】

【００４０】
上記の結果から、界面活性剤１５種中、デオキシＢＩＧＣＨＡＰ及びｎ−ドデシル−β−Ｄ−マルトシドに高い抽出能がみられた。特に、デオキシＢＩＧＣＨＡＰに高い抽出能がみられた。また、エタノール、アセトン、ジメチルスルホキシド等の有機溶媒での抽出についても同様に調べたが、抽出効率が極めて悪いか、酵素の失活が起こった。
【００４１】
実施例３（酵素の完全精製）
ポリウレタンを炭素源として培養した菌体（湿重量５４. ８ｇ）を集菌し、湿重量で約１０％になるように、０. ２％ｗ／ｗのデオキシＢＩＧＣＨＡＰを含む５００ｍｌの２０ｍＭリン酸バッファー（ｐＨ７. ０）を加えた。室温にて、マグネチックスターラーで３０分間抽出を行い、その後、遠心して上清と菌体とに分け、上清を粗抽出液とした。
粗抽出液に細粉化した硫酸アンモニウムを４５％飽和になるように添加し、３０分間攪拌した。これを遠心分離して、上清を除き、沈殿に２０ｍＭリン酸バッファーを４００ｍｌを加え、再溶解し、これを硫酸沈殿画分とした。
硫酸沈殿画分にフェニル・トヨパール６５０Ｍ（東ソー社製）１００ｍｌを添加し、攪拌器にて４０分間５００ｒｐｍで穏やかに攪拌して、ポリウレタンエステラーゼを吸着させた。反応後、グラスフィルターで濾過して、非吸着成分を取り除き、２０ｍＭリン酸バッファーで数回洗浄した。担体をビーカーに戻し、０. ２５％のデオキシＢＩＧＣＨＡＰを含むリン酸バッファー３００ｍｌを加え、４０分間穏やかに攪拌してポリウレタンエステラーゼを溶出させた。これをフェニル・トヨパール溶出画分とした。
【００４２】
得られたフェニル・トヨパール溶出画分に硫酸アンモニウムを５０％飽和になるように加えて遠心し、沈殿を０. ２％のデオキシＢＩＧＣＨＡＰを含む２０ｍＭリン酸バッファー１０ｍｌに溶解した。溶け残りを遠心して除き、Ｑ−セファロースＦＦ（ファルマシア社製）カラムクロマトグラフィーに供した。カラムは内径１６ｍｍ長さ１０ｃｍのものを用い、移動相には、０. ２％のデオキシＢＩＧＣＨＡＰを含むリン酸バッファーを用いた。カラムを素通りした画分を集め、これをＱ−セファロース素通り画分とした。Ｑ−セファロース素通り画分に硫酸アンモニウムを５０％飽和になるように加えて遠心し、２０ｍＭリン酸バッファーに再懸濁したものを精製酵素標品とした。
実施例３で得られた粗抽出液、硫安沈殿画分、フェニル・トヨパール溶出画分、及びＱ−セファロース素通り画分それぞれについての容量、全蛋白質量、全活性、比活性を表３に示す。
【００４３】
【表３】

【００４４】
上記の結果から、ＴＢ−３５株のポリウレタンエステラーゼは、比活性で８. ３倍、収率２５％で精製された。実施例３では、最終精製段階でＱ−セファロースＦＦのカラムクロマトグラフィーを用いたが、この精製過程においてもカラムを素通りした画分にポリウレタンエステラーゼが含まれていることから、すべての過程がバッチ処理で行えることがわかった。
【００４５】
実施例４（精製した酵素の分子量測定）
実施例３で得られた精製酵素標品をＳＤＳ−ポリアクリルアミド電気泳動によって分子量を測定した。電気泳動は、通常行われているとおりの方法にて行った。
その結果、分子量約６２，０００の位置に単一のバンドが検出された。なお、０. ２％のデオキシＢＩＧＣＨＡＰ存在下でゲル濾過クロマトグラフィーに供したところ、分子量約６２，０００の位置にピークが認められた。
したがって、実施例３の精製方法によって、ポリウレタンエステラーゼは、完全に精製されたことを示し、このエステラーゼは、約６２，０００の分子量を持つ単量体であることがわかった。
【００４６】
実施例５（精製した酵素の諸性質）
１３μｇの実施例３で得られた精製酵素標品（０. ２ユニット）を含む１００ｍＭリン酸緩衝液（ｐＨ７. ０）とポリウレタン約１５ｍｇ（４×４×１ｍｍのフィルム状のもの）を入れ、３０℃で２４時間反応させた。反応終了後、ポリウレタンを取り出し、水洗して重量を測定した。その結果、４. ９ｍｇのポリウレタンの重量減少がみられ、精製された酵素が確かにポリウレタンを分解することがわかった。また、反応液を高速液体クロマトグラフィー（ＨＰＬＣ）で分析したところ、アジピン酸とジエチレングリコールが検出され、その量は分解されたポリウレタン量から計算した理論値とほぼ一致した。このことから、精製された酵素によって、ポリウレタン中のエステル結合が切断され、分解産物として少なくともアジピン酸とジエチレングリコールが生成することがわかった。
【００４７】
実施例６（精製した酵素の至適ｐＨの測定）
ｐＨを種々に調製した緩衝剤を用いた以外は実施例５と同様な条件でポリウレタンを分解させた。用いた緩衝液は、ｐＨ４〜６は酢酸バッファー、ｐＨ６〜８はリン酸バッファー、ｐＨ８〜９はトリス・塩酸バッファーであり、すべて１００ｍＭの濃度であった。ｐＨ６. ５での酵素活性を１００％としたときのｐＨと酵素活性の関係を図３に示す。
図３から、至適ｐＨ６〜７. ５、特にｐＨ６. ５である。従って、ｐＨ約６〜７. ５の緩衝液中でポリウレタン分解を行うのが望ましいことがわかった。ポリウレタンエステラーゼは、至適ｐＨが比較的狭く、ポリウレタンの分解に用いるときには、分解産物のアジピン酸による反応液のｐＨの低下に注意を要すことがわかった。
【００４８】
【発明の効果】
本発明によれば、コマモナス・アシドボランス ＴＢ−３５株は、エステル系ポリウレタン以外の炭素源を基質としても生育し、ポリウレタンエステラーゼを産生する。よって、一般的な培地を用いて、大量のポリウレタンエステラーゼを迅速、かつ安価に調製することができる。
また、本発明の精製方法によって、ポリウレタンエステラーゼの精製に、界面活性剤を用いたバッチ処理で精製ができる。そのため、高価な機械を使用することなく、容易、かつ安価にポリウレタンエステラーゼを得ることができる。本発明の精製方法によれば、ＴＢ−３５株のポリウレタンエステラーゼは粗抽出液と比べて、比活性で約８倍以上に精製できる。
なお、本発明によれば、デオキシＢＩＧＣＨＡＰ又はｎ−ドデシル−β−Ｄ−マルトシドは、酵素を失活させることなく３０分間で抽出できる界面活性剤であることがわかった。特に、デオキシＢＩＧＣＨＡＰは、硫安沈殿の際も沈殿を生じることがなく、電荷を持たないのでイオン交換クロマトグラフィー操作に影響を及ぼすこともないので好適である。
【００４９】
本発明の精製方法で得られるポリウレタンエステラーゼは、エステル系ポリウレタンを分解し、アジピン酸、ジエチレングリコールを得ることが可能である。この分解反応は、常温の反応であり、高価な薬品を必要としない。したがって、分解が困難であったポリウレタンを容易、かつ安価に分解することができ、ゴミ処理などの環境問題解決の糸口となるうえに、資源の再利用をはかることができる。
【図面の簡単な説明】
【図１】マンニトール又はポリウレタンを炭素源として用いたときの菌体生育量を示すグラフ。
【図２】マンニトール又はポリウレタンを炭素源として用いたときのポリウレタン分解活性を示すグラフ。
【図３】精製したポリウレタンエステラーゼの活性とｐＨとの関係を示すグラフ。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a decomposition treatment of an ester polyurethane by an enzyme derived from a microorganism.
[0002]
[Prior art]
Polyurethane is used in various products such as seat cushions for four-wheeled and two-wheeled vehicles, clothing fibers, shoe cushions, adhesives, and paints. However, polyurethane is a thermosetting resin and is difficult to recycle. The following methods can be used to dispose of polyurethane.
[0003]
As the most common disposal method, a landfill process is known in which used polyurethane and polyurethane pieces produced in the manufacturing process are directly landfilled for disposal. The chemical decomposition treatment is a method in which a raw material polyol is regenerated by decomposing it into gas or petroleum by thermal decomposition or by decomposing glycol. The incineration process is to incinerate as garbage in a furnace. The method of decomposing and treating polyurethane by microorganisms is a basic technology stage and has not yet been put into practical use.
[0004]
When the microbial decomposition method is used for polyurethane, polyurethanes made from ether polyols are resistant to microorganisms and are difficult to decompose. In contrast, it has been reported that ester polyurethanes using ester polyols as raw materials are degraded by enzymes produced by microorganisms. For example, Tokyo, Y. et al. etal: Agric. Biol. Chem. , 52, p1937 (1988) describe that ester polyurethanes are hydrolyzed by lipases from Rhizopus arrizus and R. delemar.
In addition, Toshiaki Nakajima-Kambe et al. : In FEMS Microbiology Letters, 129, 39-42 (1995), a bacterium isolated from soil, Comamonas acidborans TB-35 strain (Commonas acidovorans TB-35) decomposes ester polyurethane, and adipic acid and ethylene glycol. It is described to produce.
[0005]
The method of decomposing ester polyurethane by Comamonas acid boranes TB-35 strain is performed as follows.
First, together with polyurethane synthesized from polyethylene glycol adipate and 2,4-tolylene diisocyanate, the strain was cultured in a liquid medium. The degradation metabolite was mainly diethylene glycol.
[0006]
Regarding the purification of polyurethane-degrading enzymes, there are some reports of mold-derived enzymes, but there is no example of complete purification of bacteria-derived enzymes. In Japanese Patent Application No. 8-9674, esterase adhering to the cell surface of the TB-35 strain has ester-based polyurethane degrading activity, and this esterase crude enzyme solution preparation method and crude enzyme solution were used. A method for decomposing ester polyurethanes is disclosed.
[0007]
The preparation method of this esterase crude enzyme solution is as follows. First, Comamonas acid bolans TB-35 strain is cultured in a medium using ester polyurethane as a carbon source, and the culture solution is centrifuged to separate the supernatant and the cells. The separated cells are treated with a surfactant selected from a sodium cholate solution, a sodium deoxycholate solution, and a sodium dodecyl sulfate solution to release esterase from the cells, and the solution containing the esterase is subjected to ultrafiltration. And the surfactant is removed to obtain a crude polyurethane esterase enzyme solution.
[0008]
The esterase roughly purified by the above method is added to the ester polyurethane by 1.9 to 188.0 mU to decompose the polyurethane.
[0009]
A general method for purifying enzymes produced by bacteria is described below.
Breaks cells in buffer solution by grinding, autolysis, sonication, rapid change in pressure, enzymatic degradation, self-digestion, etc. At this time, what is associated with a buffer-insoluble structure such as a cell membrane is further extracted with various surfactants, dilute acid, alkali, alcohol or the like. The resulting crude extract is subjected to column chromatography (ion exchange, gel filtration, affinity, hydrophobic, etc.), dialysis, ultrafiltration, electrophoresis, fractional salting out with ammonium sulfate, magnesium sulfate, etc., fractional precipitation with alcohol, acetone, etc. It concentrates and refines through the process of. Many enzymes are finally obtained in a crystalline state by fractional salting out or fractional precipitation.
[0010]
[Problems to be solved by the invention]
Polyurethane is a low-density resin used for sponges, cushions and the like. Regarding the disposal of polyurethane, the treatment of polyurethane by landfill does not solidify and stabilize. Moreover, since it is bulky with respect to weight, transportation efficiency is bad. Furthermore, since polyurethane is not easily decomposed by microorganisms and does not rot, it occupies a high ratio in the landfill treatment site, causing a shortage of treatment sites.
[0011]
The chemical decomposition treatment has an advantage that the polyol of the raw material can be regenerated by glycol decomposition, but the quality of the decomposition product is not stable because the quality of the polyurethane to be disposed varies. Furthermore, there is a disadvantage that the cost and labor for processing are enormous and the profit is not suitable.
[0012]
The incineration process cannot withstand the high temperatures generated during the incineration of polyurethane in traditional furnaces. In addition, the hot-cure polyurethane used in seat cushions for automobiles and motorcycles uses a condensed phosphate ester containing 29% chlorine as a flame retardant. Gas is generated and corrodes the furnace. In addition, carbon dioxide and toxic gases are generated during incineration, polluting the air environment.
[0013]
In the method of inoculating microorganisms and degrading polyurethane, it becomes difficult to decompose due to mutations in stored strains and contamination with various bacteria.
In addition, since the crude purified esterase obtained by the method of Japanese Patent Application No. 8-9674 contains many impurities, the specific activity is small and the reaction efficiency is not good. In order to clarify the molecular mechanism and reaction mechanism of enzyme action, a completely purified enzyme is required.
[0014]
In general, the method for purifying enzymes from bacteria requires column chromatography several times in order to purify, so it requires expensive chromatographic equipment and columns, expensive chemicals, and until purification. Takes time. In addition, when an organic solvent such as ethanol, acetone and dimethyl sulfoxide is used, the ester polyurethane esterase is inactivated.
In the method of Japanese Patent Application No. 8-9674, sodium cholate or the like was used as a surfactant to roughly purify esterase. However, extraction requires a high concentration of 2% or more, and subsequent purification is difficult. It was.
[0015]
Therefore, in order to carry out mass processing of polyurethane using polyurethane esterase obtained from Comamonas acid boranes TB-35 in the future, a method for purifying esterase was established, and a method for decomposing ester polyurethane using the purified esterase was established. It is necessary to clarify.
[0016]
Accordingly, an object of the present invention is to purify polyurethane esterase from Comamonas acid borane TB-35 strain or mycologically equivalent Comamonas acid borane. Furthermore, it aims at decomposing | disassembling a polyurethane efficiently using the obtained polyurethane esterase.
[0017]
[Means for Solving the Problems]
The present invention relates to a method for purifying an ester polyurethane esterase, wherein a surface-active activity is obtained by centrifuging a supernatant of a culture of Comamonas acidborans having the same mycological properties as those of Comamonas acidborans TB-35 or TB-35. Adding a reagent to extract the components on the surface of the cells, adding a carrier for hydrophobic chromatography to the extract, and collecting the fraction adsorbed on the carrier as an adsorbed fraction of the carrier for hydrophobic chromatography; And a step of adding an anion exchanger to the adsorbed fraction and collecting the fraction not adsorbing the anion exchanger to obtain a purified polyurethane esterase.
[0018]
In this purification method, the non-anion exchanger non-adsorbed fraction may be subjected to SDS-polyacrylamide electrophoresis to cut out a band having a molecular weight of about 62,000.
Further, the fraction not adsorbing the anion exchanger may be subjected to gel filtration column chromatography to collect a peak with a molecular weight of about 62,000.
The surfactant is preferably deoxy BIGCHAP or n-dodecyl-β-D-maltoside.
Furthermore, the present invention provides a method for decomposing a polyurethane using the polyurethane esterase obtained by the above method, in which the polyurethane is decomposed in a solution maintained at a pH of 6 to 7.5.
[0019]
According to the method for purifying an esterase of the present invention, an ester polyurethane esterase having a high specific activity can be obtained simply, quickly and inexpensively compared to the conventional method, and the polyurethane esterase can be completely purified. In addition, according to the method for decomposing ester polyurethane using the purified esterase of the present invention, it is possible to proceed with the decomposition of polyurethane while maintaining a high enzyme activity.
[0020]
DETAILED DESCRIPTION OF THE INVENTION
(Bacterial properties)
The ester polyurethane in the present invention refers to a polyurethane using a polyester as a polyol.
Comonas acidvorans TB-35 strain (FERM P-15181) used in the present invention is a bacterium isolated from soil in Tsukuba City, Ibaraki Prefecture, and is known to degrade polyurethane. INDUSTRIAL APPLICABILITY According to the present invention, an ester polyurethane esterase can be purified using Comamonas acidborans having bacteriological properties equivalent to those of Comamonas acidborans TB-35 strain or TB-35 strain.
[0021]
The following are known as the mycological properties of Comamonas acid bolans TB-35 strain. That is, the cell morphology is gonococcus and gram-negative bacteria. Performs anaerobic breathing and has mobility. Positive for oxidase, catalase, nitrate reduction. Negative for indole formation, arginine dihydrolase, urease, β-galactosidase. Acid from fructose OF is positive. Negative hydrolysis of esculin and gelatin. Tween 80 hydrolysis is positive. Positive for mannitol availability, gluconate availability, adipate availability, malate availability, citrate availability, and acetate availability. Negative regarding glucose utilization, arabinose utilization, mannose utilization, maltose utilization, caprate utilization, phenylacetate utilization, N-acetylglucosamine utilization. The quinone type is ubiquinone (Q-8), and the molar percentage of G + C is 68.8%.
[0022]
(culture)
In the method for purifying ester polyurethane esterase of the present invention, the esterase can be purified by culturing using a substance having no ester bond as a carbon source and culturing Comamonas acid bolans TB-35 strain or its equivalent.
Any carbon source may be used as long as it can use Comamonas acid boranes, for example, mannitol, gluconate, adipate, malate, citrate, acetate, polyurethane and the like, in particular, mannitol, Polyurethane is preferred. The carbon source may be added at about 1% w / w with respect to the medium.
[0023]
When mannitol is used as a carbon source, the esterase obtained is about ½ as the esterase activity per amount of growing cells, compared to when polyurethane is used as the carbon source. However, since the growth of bacteria is faster than when polyurethane is used as the carbon source, a large amount of enzyme can be prepared quickly. Mannitol is a sugar alcohol present in many plants, mushrooms, algae and the like, and is easily available and inexpensive. Therefore, the cost for enzyme purification is also low, which is particularly suitable for the purification of the esterase of the present invention. As an example, Table 1 shows a culture medium when mannitol is used as a carbon source.
In addition, esterase can be purified using a general natural medium instead of mannitol.
[0024]
[Table 1]

[0025]
The medium as described above is sterilized by an autoclave or the like, inoculated with Commamonas acid bolans TB-35 strain or its equivalent, and cultured with shaking at about 30 ° C. The culture is stopped and the enzyme is purified 5 to 8 days after the culture, preferably 6 days after the largest growth of the bacterial cells.
[0026]
(Enzyme purification)
The method for purifying an ester polyurethane esterase of the present invention can be performed at room temperature. Since polyurethane esterase adheres to the surface of the cells, it is extracted with a surfactant during purification. As the surfactant to be used, deoxy BIGCHAP and n-dodecyl-β-D-maltoside are suitable.
Deoxy BIGCHAP and n-dodecyl-β-D-maltoside are surfactants with molecular weights of 862.07 and 510.62, respectively. Structural formulas of deoxy BIGCHAP and n-dodecyl-β-D-maltoside are shown in (1) and (2), respectively.
[0027]
[Chemical 1]

[0028]
[Chemical formula 2]

[0029]
Deoxy BIGHCHAP has a critical micelle concentration as low as about 0.12%, and the micelles can be easily destroyed by diluting the solution to about 2 times. Moreover, since it has no electric charge, it does not affect the operation of ion exchange chromatography. Furthermore, it does not precipitate during the ammonium sulfate fractionation at the concentration used. For these reasons, deoxy BIGCHAP is particularly preferred. The use concentration of deoxy BIGCHAP is preferably 0.2 to 0.25% w / w. When the concentration is lower than 0.2%, the extraction effect is poor, and when the concentration is higher than 0.25%, the subsequent purification is difficult.
[0030]
The cultured cells are collected, suspended in a commonly used buffer solution such as a phosphate buffer containing the above-mentioned surfactant, Tris / HCl buffer (pH about 7.0), and extracted by stirring. Then, it centrifuges and divides into a supernatant and a microbial cell, and makes a supernatant into a crude extract. The crude extract is concentrated by salting out such as ammonium sulfate, and then a carrier for hydrophobic chromatography is added. The adsorbed carrier is eluted in a buffer containing a surfactant, and the eluate is concentrated by salting out. Dissolved concentrated carrier adsorbent in a buffer containing a surfactant, added an anion exchanger, and concentrated the fraction not adsorbed on the anion exchanger by salting out and then dissolved in the buffer. Is a purified esterase preparation. The addition of the carrier during the purification process can be replaced by performing column chromatography.
[0031]
By the purification method of the present invention described above, polyurethane esterase can be purified from the crude extract with a specific activity of about 8 times or more and a yield of about 25%. Since all the purification methods of the present invention can be purified only by batch processing, expensive chromatographic equipment, columns and the like are not required, and purification can be performed only with a stirrer. Therefore, scale-up is extremely easy and inexpensive.
By the purification method of the present invention, a polyurethane esterase having a high purity to the extent that it is confirmed by a single band by electrophoresis is obtained.
[0032]
(Properties of polyurethane esterase)
The polyurethane esterase obtained in the present invention is a monomer having a molecular weight of about 62,000, and the pH suitable for the enzyme to work is 4 to 8, particularly preferably pH 6.5. Therefore, it is desirable to perform polyurethane degradation in a buffer solution having a pH of about 6 to 7.5. The polyurethane esterase of the present invention cleaves an ester bond of an ester polyurethane to produce adipic acid and diethylene glycol. Due to the production of the degradation product adipic acid, the pH of the reaction solution tends to decrease. Therefore, to prevent deviating from the optimum pH, for example, 1% CaCO_ThreeIt is desirable to control the pH by performing a treatment such as the addition of. The shape of the polyurethane to be decomposed is not particularly limited, but it is desirable that the polyurethane has a large surface area such as a film or a sphere.
[0033]
The activity of the polyurethane esterase of the present invention is measured by measuring the amount of p-nitrophenol produced by esterase using p-nitrophenyl acetate as a substrate at an absorbance of 405 nm. As the unit of activity of the polyurethane esterase of the present invention, the amount of enzyme that produces 1 μmol of p-nitrophenol per minute is defined as 1 unit.
[0034]
【Example】
The present invention will be described in more detail with reference to the following examples. However, it is not intended that the scope of the present invention be limited by these examples.
Example 1 (Bacterial culture using mannitol as a carbon source)
Polyurethane used in the experiment was polyethylene glycol adipate (Nippolan 2200-A, manufactured by Nippon Polyurethane Industry Co., Ltd.), 2,4-tolylene diisocyanate (manufactured by Wako Pure Chemical Industries, Ltd.), OH group: isocyanate group was 1: 1. In addition, it is synthesized.
30 ml of the medium having the composition shown in Table 1 described above was placed in a 300 ml Erlenmeyer flask and sterilized by autoclaving at 121 ° C. for 15 minutes. One platinum ear of the TB-35 strain was inoculated into the sterilized medium and cultured at 30 ° C. with rotary shaking. Sampling was performed every two days, and the cell growth and polyurethane degrading activity were measured. The cell growth was measured by turbidity at 580 nm.
[0035]
Comparative Example 1 (Bacterial culture using polyurethane as a carbon source)
Of the media shown in Table 1, the cells were cultured in a medium supplemented with 1% w / w polyurethane instead of mannitol, cultured in the same manner, and the cell growth and polyurethane degrading activity were measured.
The results of Example 1 and Comparative Example 1 are shown in FIGS.
[0036]
From the above results, it was found that the cells showed the largest growth amount on the sixth day of culture. When mannitol is used as the carbon source, the polyurethane esterase activity per growing cell is as low as about 1/2 compared to when polyurethane is used as the carbon source. However, cell growth is better when mannitol is used as the carbon source. In addition, since mannitol is inexpensive and easy to obtain, it was found that mannitol should be used as a carbon source rather than polyurethane in order to quickly prepare a large amount of enzyme.
[0037]
Example 2 (Enzyme extraction)
Cells cultured using polyurethane as a carbon source were collected, and 20 mM phosphate buffer (pH 7.0) was added and suspended to a wet weight of about 10%. To this suspension, 0.2% w / w of deoxy BIGCHAP or n-dodecyl-β-D-maltoside (both manufactured by Dojindo Laboratories) as a surfactant was added and stirred at room temperature for 2 hours with a vortex mixer. Extracted. Thereafter, the mixture was centrifuged to separate the supernatant and the cells, and the activity of polyurethane esterase in the supernatant was measured.
[0038]
Comparative Example 2
As surfactants, CHAPS, CHAPSO, BIGCHAP, n-octyl-β-D-glucoside, n-heptyl-β-D-thioglucoside, n-octyl-β-D-thioglucoside, MEGA-8, MEGA-9 The activity of polyurethane esterase was measured in the same manner as in Example 2 except that MEGA-10, sucrose monocaprate, sucrose monolaurate, sodium cholate, or digitonin (all manufactured by Dojindo Laboratories) were used. . In addition, the esterase activity was measured for the supernatant not added with the surfactant and the untreated cells.
The results of Example 2 and Comparative Example 2 are shown in Table 2.
[0039]
[Table 2]

[0040]
From the above results, high extractability was observed for deoxy BIGCHAP and n-dodecyl-β-D-maltoside among 15 surfactants. In particular, deoxy BIGCHAP showed high extraction ability. Further, extraction with an organic solvent such as ethanol, acetone, dimethyl sulfoxide, etc. was examined in the same manner, but the extraction efficiency was extremely poor or the enzyme was deactivated.
[0041]
Example 3 (Complete purification of enzyme)
500 ml of 20 mM phosphate buffer containing 0.2% w / w deoxy BIGCHAP so as to collect cells (wet weight 54.8 g) cultured using polyurethane as a carbon source so that the wet weight is about 10% (PH 7.0) was added. Extraction was performed with a magnetic stirrer at room temperature for 30 minutes, and then the mixture was centrifuged to separate the supernatant and cells, and the supernatant was used as a crude extract.
Finely divided ammonium sulfate was added to the crude extract so as to be 45% saturated and stirred for 30 minutes. This was centrifuged, the supernatant was removed, 400 ml of 20 mM phosphate buffer was added to the precipitate and redissolved, and this was used as the sulfate precipitation fraction.
100 ml of phenyl Toyopearl 650M (manufactured by Tosoh Corp.) was added to the sulfuric acid precipitate fraction, and gently stirred at 500 rpm for 40 minutes with a stirrer to adsorb the polyurethane esterase. After the reaction, the mixture was filtered through a glass filter to remove non-adsorbed components and washed several times with 20 mM phosphate buffer. The carrier was returned to the beaker, 300 ml of a phosphate buffer containing 0.25% deoxy BIGCHAP was added, and gently stirred for 40 minutes to elute the polyurethane esterase. This was the phenyl-toyopearl elution fraction.
[0042]
To the obtained phenyl-toyopearl elution fraction, ammonium sulfate was added to 50% saturation and centrifuged, and the precipitate was dissolved in 10 ml of 20 mM phosphate buffer containing 0.2% deoxy BIGCHAP. The undissolved residue was removed by centrifugation and subjected to Q-Sepharose FF (Pharmacia) column chromatography. A column having an inner diameter of 16 mm and a length of 10 cm was used, and a phosphate buffer containing 0.2% deoxy BIGCHAP was used for the mobile phase. Fractions that passed through the column were collected and used as Q-sepharose pass-through fractions. A purified enzyme preparation was prepared by adding ammonium sulfate to the Q-sepharose pass-through fraction to 50% saturation, centrifuging, and resuspending in 20 mM phosphate buffer.
Table 3 shows the capacity, total protein mass, total activity, and specific activity for the crude extract, the ammonium sulfate precipitate fraction, the phenyl-toyopearl elution fraction, and the Q-sepharose pass-through fraction obtained in Example 3, respectively.
[0043]
[Table 3]

[0044]
From the above results, the polyurethane esterase of the TB-35 strain was purified with a specific activity of 8.3 times and a yield of 25%. In Example 3, column chromatography of Q-Sepharose FF was used in the final purification stage. Even in this purification process, since the polyurethane esterase was contained in the fraction passed through the column, all processes were batch processed. I found out that
[0045]
Example 4 (Measurement of molecular weight of purified enzyme)
The purified enzyme preparation obtained in Example 3 was measured for molecular weight by SDS-polyacrylamide electrophoresis. Electrophoresis was performed by the method as usual.
As a result, a single band was detected at a molecular weight of about 62,000. When subjected to gel filtration chromatography in the presence of 0.2% deoxy BIGCHAP, a peak was observed at a molecular weight of about 62,000.
Therefore, it was shown that the polyurethane esterase was completely purified by the purification method of Example 3, and this esterase was found to be a monomer having a molecular weight of about 62,000.
[0046]
Example 5 (Various properties of purified enzyme)
13 μg of 100 mM phosphate buffer (pH 7.0) containing the purified enzyme preparation (0.2 units) obtained in Example 3 and about 15 mg (4 × 4 × 1 mm film-like) of polyurethane, The reaction was carried out at 30 ° C. for 24 hours. After completion of the reaction, the polyurethane was taken out, washed with water and weighed. As a result, a weight loss of 4.9 mg of polyurethane was observed, and it was found that the purified enzyme surely degraded the polyurethane. Further, when the reaction solution was analyzed by high performance liquid chromatography (HPLC), adipic acid and diethylene glycol were detected, and the amounts almost coincided with the theoretical values calculated from the amount of decomposed polyurethane. From this, it was found that the ester bond in the polyurethane was cleaved by the purified enzyme, and at least adipic acid and diethylene glycol were produced as degradation products.
[0047]
Example 6 (Measurement of optimum pH of purified enzyme)
The polyurethane was decomposed under the same conditions as in Example 5 except that buffering agents having various pHs were used. The buffers used were acetate buffer at pH 4-6, phosphate buffer at pH 6-8, and Tris / HCl buffer at pH 8-9, all at a concentration of 100 mM. FIG. 3 shows the relationship between pH and enzyme activity when the enzyme activity at pH 6.5 is taken as 100%.
From FIG. 3, the optimum pH is 6 to 7.5, particularly pH 6.5. Accordingly, it has been found desirable to perform polyurethane degradation in a buffer having a pH of about 6 to 7.5. It has been found that polyurethane esterase has a relatively narrow optimum pH, and when it is used for degradation of polyurethane, it is necessary to pay attention to lowering the pH of the reaction solution due to the degradation product adipic acid.
[0048]
【The invention's effect】
According to the present invention, Comamonas acid boranes TB-35 strain grows using a carbon source other than ester polyurethane as a substrate, and produces polyurethane esterase. Therefore, a large amount of polyurethane esterase can be prepared quickly and inexpensively using a general medium.
Further, by the purification method of the present invention, polyurethane esterase can be purified by batch treatment using a surfactant. Therefore, polyurethane esterase can be obtained easily and inexpensively without using an expensive machine. According to the purification method of the present invention, the polyurethane esterase of the TB-35 strain can be purified about 8 times or more in specific activity as compared with the crude extract.
In addition, according to this invention, it turned out that deoxy BIGCHAP or n-dodecyl- (beta) -D-maltoside is a surfactant which can be extracted in 30 minutes, without deactivating an enzyme. In particular, deoxy BIGCHAP is suitable because it does not cause precipitation even during ammonium sulfate precipitation and does not have an electric charge and therefore does not affect the ion exchange chromatography operation.
[0049]
The polyurethane esterase obtained by the purification method of the present invention can decompose ester polyurethane to obtain adipic acid and diethylene glycol. This decomposition reaction is a normal temperature reaction and does not require expensive chemicals. Therefore, polyurethane that has been difficult to decompose can be easily and inexpensively decomposed, and it can be used as a clue to solving environmental problems such as waste disposal, and resources can be reused.
[Brief description of the drawings]
FIG. 1 is a graph showing the growth of bacterial cells when mannitol or polyurethane is used as a carbon source.
FIG. 2 is a graph showing polyurethane decomposing activity when mannitol or polyurethane is used as a carbon source.
FIG. 3 is a graph showing the relationship between the activity of purified polyurethane esterase and pH.

Claims (5)

In the method for purifying ester polyurethane esterase, a step of adding a surfactant to the supernatant obtained by centrifuging the culture solution of Comonas acidovorans strain TB-35 (FERM P - 15381), and extracting components on the surface of the cells, Adding a carrier for hydrophobic chromatography to the extract and collecting the fraction adsorbed on the carrier as an adsorbed fraction of the carrier for hydrophobic chromatography; adding an anion exchanger to the adsorbed fraction; A method for purifying polyurethane esterase, comprising collecting non-exchanger fractions to obtain purified polyurethane esterase , wherein the polyurethane esterase is a monomer having a molecular weight of about 62,000 and an optimum pH of 4 to 8, the ester bond of the ester polyurethane is cleaved, and adipic acid and polyethylene Characterized in that to produce the glycol method.   The method for purifying polyurethane esterase according to claim 1, further comprising excising a band having a molecular weight of about 62,000 by subjecting the non-ion exchanger fraction to SDS-polyacrylamide electrophoresis.   2. The method for purifying polyurethane esterase according to claim 1, further comprising: subjecting the anion exchanger non-adsorbed fraction to gel filtration column chromatography and collecting a peak with a molecular weight of about 62,000.   The purification method according to any one of claims 1 to 3, wherein the surfactant is deoxy BIGCHAP or n-dodecyl-β-D-maltoside.   A method for decomposing a polyurethane, comprising decomposing the polyurethane in a solution having a pH of 6 to 7.5 using the polyurethane esterase obtained by the purification method according to any one of claims 1 to 3.

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