JP2005081278A - Method for adsorption removing phenolic compound using chitosane bead - Google Patents
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Abstract
Description
本発明は、水系からフェノール系化合物を効率よく除去する方法に関する。 The present invention relates to a method for efficiently removing a phenolic compound from an aqueous system.
フェノール及びその誘導体は、製油精製、化学薬品、繊維、染料、パルプ工場など幅広い工業分野からの排水に含まれ、直接的に、または分解中間体として間接的に環境中に放出され、土壌、河川・湖沼などを汚染する。その中でもアルキル置換体や塩素置換体は毒性が高く、生分解性も低いことから、活性汚泥などの微生物処理によって廃水からのこれら化合物の分解・除去が困難である。 Phenol and its derivatives are contained in wastewater from a wide range of industrial fields such as oil refining, chemicals, textiles, dyes and pulp mills, and are released directly into the environment or indirectly as degradation intermediates to soil, rivers・ Contaminate lakes. Among them, alkyl-substituted products and chlorine-substituted products are highly toxic and have low biodegradability. Therefore, it is difficult to decompose and remove these compounds from wastewater by microbial treatment such as activated sludge.
これまでに、パルス放電処理、活性炭ろ過法、オゾン酸化と活性汚泥処理の併用、液膜法、緑藻・藍藻などの微細藻類などによるフェノール化合物の分解除去に関する報告がなされている。
しかしながら、上記パルス放電処理等のフェノール化合物の分解除去方法は、いずれも設備が大型である、コストが高いなどの問題がある。これに対し、本発明者らは、フェノール系化合物をチロシナーゼによってキノン化合物に酸化させると、このキノン化合物をキトサンフィルムにより効果的に吸着除去できることを見出した。キトサンは、カニやエビの甲殻から精製するキチンを脱アセチル化して得られる安価な物質であり、コストも下げることが可能である。 However, all the methods for decomposing and removing phenolic compounds such as the pulse discharge treatment have problems such as large equipment and high cost. In contrast, the present inventors have found that when a phenolic compound is oxidized to a quinone compound by tyrosinase, the quinone compound can be effectively adsorbed and removed by the chitosan film. Chitosan is an inexpensive substance obtained by deacetylating chitin purified from crab and shrimp shells, and the cost can be reduced.
しかし、工業廃水の十分な浄化のためには、さらに高効率なフェノール系化合物の除去方法が必要とされている。そこで、本発明は、フェノール系化合物を効率的に、かつ低コストで吸着除去する処理技術を提供することを目的とする。 However, in order to sufficiently purify industrial wastewater, a more efficient method for removing phenolic compounds is required. Then, an object of this invention is to provide the processing technique which carries out adsorption removal of a phenolic compound efficiently and at low cost.
本発明者らは、上記課題に鑑みて鋭意研究を重ねた結果、フェノール系化合物をチロシナーゼによって、キノン化合物に酸化したのち、ビーズ状のキトサンを用いることによって非常に効率よく、このキノン化合物を吸着できることを見出した。また、通常チロシナーゼの基質とならないフェノール系化合物も、過酸化水素を加えることによってチロシナーゼによって酸化され、これによってキトサンビーズにより効率よく水系から除去することができることを見出し、本発明を完成するに至った。 As a result of intensive studies in view of the above problems, the present inventors have adsorbed this quinone compound very efficiently by oxidizing the phenolic compound into a quinone compound with tyrosinase and then using bead-shaped chitosan. I found out that I can do it. In addition, phenolic compounds that are not normally substrates for tyrosinase are also oxidized by tyrosinase when hydrogen peroxide is added, and thus can be efficiently removed from the aqueous system by chitosan beads, and the present invention has been completed. .
即ち、本発明は、
［１］フェノール系化合物を含む水系に、チロシナーゼを加えて前記フェノール系化合物をキノン化させ、このキノン化合物をビーズ状キトサンで吸着する工程を含む、水系からフェノール化合物を除去する方法；
［２］前記フェノール系化合物を含む水系に、前記チロシナーゼと前記キトサンを同時に加えることを特徴とする、上記［１］に記載の方法；
［３］前記フェノール系化合物が、フェノール、ｐ−クレゾール、ｍ−クレゾール、アルキルフェノール、ハロゲン化フェノールおよびカテコールからなる群から選択される、上記［１］または［２］に記載の方法
［４］前記接触させる工程を、ｐＨ６．０〜７．０として行う、上記［１］から［３］のいずれか１項に記載の方法；
［５］前記接触させる工程を、４０〜４５℃で行う、上記［１］から［４］のいずれか１項に記載の方法；
［６］前記チロシナーゼの濃度を０．０２０〜０．０３０ｍｇ／ｃｍ3となるように加える、上記［１］から［５］のいずれか１項に記載の方法；
［７］前記接触させる工程で、前記水系に過酸化水素を加える上記［１］から［６］のいずれか１項に記載の方法；
［８］フェノール系化合物を含有する、水系の被処理液を反応槽に供給し、この反応層にチロシナーゼを供給するか、又は、チロシナーゼを支持する手段に前記被処理液を供給して、前記フェノール系化合物をキノン化合物に酸化させ、このキノン化合物を含む前記被処理液が在る前記反応槽にビーズ状のキトサンを供給するか、又はこの処理液をビーズ状のキトサンを支持する手段に供給して、前記被処理液から前記フェノール化合物を分離するシステム；
［９］被処理液のフェノール化合部を除去するために、ビーズ状に形成されたキトサン含有分離材；
［１０］上記［９］に記載の分離材が容器内に充填された分離装置、に関する。
That is, the present invention
[1] A method of removing a phenol compound from an aqueous system, comprising adding tyrosinase to an aqueous system containing a phenolic compound to quinonize the phenolic compound and adsorbing the quinone compound with beaded chitosan;
[2] The method according to [1] above, wherein the tyrosinase and the chitosan are added simultaneously to an aqueous system containing the phenolic compound;
[3] The method according to [1] or [2] above, wherein the phenolic compound is selected from the group consisting of phenol, p-cresol, m-cresol, alkylphenol, halogenated phenol and catechol. The method according to any one of [1] to [3], wherein the contacting step is performed at a pH of 6.0 to 7.0;
[5] The method according to any one of [1] to [4], wherein the contacting step is performed at 40 to 45 ° C.
[6] The method according to any one of [1] to [5] above, wherein the concentration of tyrosinase is added so as to be 0.020 to 0.030 mg / cm 3 ;
[7] The method according to any one of [1] to [6] above, wherein hydrogen peroxide is added to the aqueous system in the contacting step;
[8] An aqueous treatment liquid containing a phenolic compound is supplied to the reaction tank, and tyrosinase is supplied to the reaction layer, or the treatment liquid is supplied to a means for supporting tyrosinase, A phenolic compound is oxidized to a quinone compound, and bead-shaped chitosan is supplied to the reaction vessel containing the liquid to be treated containing the quinone compound, or this treatment liquid is supplied to a means for supporting the bead-shaped chitosan. A system for separating the phenol compound from the liquid to be treated;
[9] A chitosan-containing separating material formed in a bead shape in order to remove the phenol compound part of the liquid to be treated;
[10] The present invention relates to a separation apparatus in which the separation material according to [9] is filled in a container.
以下に、本明細書において記載する記号、用語等の意義、本発明の実施の形態等を示して、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail by showing the meanings of symbols, terms, and the like described in the present specification, embodiments of the present invention, and the like.
本発明にかかる方法では、チロシナーゼ（ＥＣ１．１４．１８．１）を用いて、フェノール系化合物を酵素的に酸化する。チロシナーゼ（ＥＣ１．１４．１８．１）は、酸化還元酵素の一種であり、酸素存在下でジヒドロキシ−Ｌ−フェニルアラニン（ＤＯＰＡ）をＤＯＰＡキノンに酸化することが知られるが、基質特異性が低く、ＤＯＰＡ以外に多くのフェノール化合物に対して酵素活性を示す。従って、複数種類のフェノール系化合物を含む水系に添加すれば、それらの化合物を同時に酸化することも可能であり、一般に酵素活性が高く、単位重量あたりのユニット数が大きいので、コストを下げることもできる。 In the method according to the present invention, the phenolic compound is enzymatically oxidized using tyrosinase (EC 1.14.18.1). Tyrosinase (EC1.14.18.1) is a kind of oxidoreductase and is known to oxidize dihydroxy-L-phenylalanine (DOPA) to DOPAquinone in the presence of oxygen, but has low substrate specificity. It shows enzyme activity against many phenolic compounds other than DOPA. Therefore, if it is added to an aqueous system containing a plurality of types of phenolic compounds, it is possible to oxidize these compounds at the same time. Generally, the enzyme activity is high and the number of units per unit weight is large, so the cost can be reduced. it can.
なお、チロシナーゼに代えて、フェノール系化合物を基質として酸化する他の酵素を用いることもでき、例えば、カテコールオキシダーゼ、ラッカーゼ、ペルオキシダーゼ、ポリフェノールオキシダーゼなどが挙げられる。 Instead of tyrosinase, other enzymes that oxidize using phenolic compounds as substrates can also be used, and examples include catechol oxidase, laccase, peroxidase, polyphenol oxidase, and the like.
ここで、キノン化合物とは、芳香族炭化水素中のベンゼン核の水素原子２個を酸素原子２個で置換した構造のジカルボニル化合物を意味する。 Here, the quinone compound means a dicarbonyl compound having a structure in which two hydrogen atoms of a benzene nucleus in an aromatic hydrocarbon are substituted with two oxygen atoms.
本発明にかかる方法では、フェノールの酵素的酸化により得られたキノン化合物がビーズ状のキトサンに吸着する。キトサンは、カニやエビなどの甲殻から単離されるキチンを脱アセチル化することに生成され、安価に入手することができる。 In the method according to the present invention, a quinone compound obtained by enzymatic oxidation of phenol is adsorbed to bead-shaped chitosan. Chitosan is produced by deacetylating chitin isolated from shells such as crabs and shrimps, and can be obtained at low cost.
キノン化合物は、キトサンに含まれるアミノ基との反応性が高く、効率よくキトサンに吸着される。アミノ基がプロトン化（−ＮＨ2基→−ＮＨ3 +基）していると、キノン化合物との反応効率が著しく低下することが知られるが、キトサン中のアミノ基はｐＫ値が高く、プロトン化が起こりにくい。また、キトサンは、ｐＨ６．５以上で水に不溶であり、吸着担体として好適である。キトサンは分子量が小さすぎると溶解性が高くなり、分子量が大きすぎると粘性が高く取り扱いにくいため、分子量１０4〜１０5程度のものを用いることが好ましい。 The quinone compound has high reactivity with the amino group contained in chitosan and is efficiently adsorbed on chitosan. It is known that when the amino group is protonated (-NH 2 group → -NH 3 + group), the reaction efficiency with the quinone compound is remarkably lowered, but the amino group in chitosan has a high pK value, Is unlikely to occur. Chitosan has a pH of 6.5 or higher and is insoluble in water, and is suitable as an adsorption carrier. Chitosan has a molecular weight of about 10 4 to 10 5 because the solubility is high when the molecular weight is too small, and the viscosity is high and difficult to handle when the molecular weight is too large.
本発明にかかる方法には、特にビーズ状のキトサンが用いられる。これにより、キノン化合物と反応する表面積が増大するので、より好適にキノン化合物を吸着させることができる。本発明にかかる方法は、工業廃水の浄化に用いられることが期待されるが、かかる場合に高効率な吸着除去が非常に重要となる。ビーズ状のキトサンは、例えば、キトサンを酸性溶液に分散溶解後、凝固材と接触させてキトサンを再生することによって製造することができる。 In particular, bead-shaped chitosan is used in the method according to the present invention. Thereby, since the surface area which reacts with a quinone compound increases, a quinone compound can be adsorb | sucked more suitably. The method according to the present invention is expected to be used for purification of industrial wastewater. In such a case, highly efficient adsorption removal is very important. The bead-shaped chitosan can be produced, for example, by dispersing chitosan in an acidic solution and then regenerating chitosan by bringing it into contact with a coagulant.
なお、キトサンに代えて、キノン化合物を吸着する他のアミノ基を有する高分子化合物を用いることもでき、例えば、ポリアリルアミン、ポリビニルアミン、直鎖型ポリエチレンイミン、分岐型ポリエチレンイミンなどが挙げられる。 In addition, it can replace with chitosan and the high molecular compound which has another amino group which adsorb | sucks a quinone compound can also be used, for example, polyallylamine, polyvinylamine, linear polyethyleneimine, branched polyethyleneimine etc. are mentioned.
フェノール化合物を含む水系と、チロシナーゼおよびビーズ状キトサンとは、同時に接触させることが好ましい。チロシナーゼとフェノール化合物を混合して放置すると、フェノール化合物はキノン化合物に変化した後、さらに別の化合物へと変化し、キトサンと反応しない物質となってしまう。従って、キノン化合物に変化した直後にキトサンと反応させる必要があり、フェノール化合物とチロシナーゼとを接触させるのと同時に、この水系にビーズ状キトサンを接触させることが望ましい。 The aqueous system containing the phenol compound, tyrosinase and beaded chitosan are preferably contacted simultaneously. When tyrosinase and a phenol compound are mixed and allowed to stand, the phenol compound changes to a quinone compound, and then changes to another compound, resulting in a substance that does not react with chitosan. Therefore, it is necessary to react with chitosan immediately after changing to a quinone compound, and it is desirable to contact beaded chitosan with this aqueous system simultaneously with contacting the phenol compound and tyrosinase.
本発明にかかる方法で除去するフェノール化合物は、チロシナーゼの基質となりうるものである限り特に限定されない。例えば、フェノール、ｐ−クレゾール、ｍ−クレゾール、アルキルフェノール、ハロゲン化フェノール、カテコールなどが挙げられる。 The phenol compound removed by the method according to the present invention is not particularly limited as long as it can be a substrate for tyrosinase. Examples thereof include phenol, p-cresol, m-cresol, alkylphenol, halogenated phenol, catechol and the like.
本発明において、フェノール系化合物にチロシナーゼとビーズ状キトサンとを接触させる工程は、チロシナーゼの至適ｐＨ、および至適温度下、すなわちｐＨ５．０〜８．０、２０〜５０℃で行い、特にｐＨ６．０〜７．０、４０〜４５℃で行うと、酵素活性が最大となって好ましい。また、この際、チロシナーゼ濃度を０．０２０〜０．０３０ｍｇ／ｃｍ3とすることによって、最も好適に酸化反応を進めることが可能となる。 In the present invention, the step of bringing the phenolic compound into contact with tyrosinase and beaded chitosan is carried out at an optimum pH of tyrosinase and at an optimum temperature, that is, pH 5.0 to 8.0, 20 to 50 ° C., particularly pH 6 When carried out at 0.0 to 7.0 and 40 to 45 ° C., the enzyme activity is maximized, which is preferable. At this time, the oxidation reaction can be most suitably advanced by setting the tyrosinase concentration to 0.020 to 0.030 mg / cm 3 .
本発明にかかる方法は、また、チロシナーゼの添加と同時に過酸化水素を加えることを含む。例えば、４−ｔｅｒｔ−ブチルフェノール（４ＴＢＰ）は、通常チロシナーゼの基質とならないが、過酸化水素を添加することによって、チロシナーゼに酸化されることが知られる（非特許文献１）。従って、過酸化水素を加えることによって、４ＴＢＰ等もキトサンに吸着させることができ、本発明にかかる方法によって水系から除去することができる。過酸化水素は、添加量が多すぎると、キノン化合物をさらに別の物質に変化させ、キトサンによって吸着させることができなくなる。 The method according to the invention also includes adding hydrogen peroxide simultaneously with the addition of tyrosinase. For example, 4-tert-butylphenol (4TBP) is not normally a substrate for tyrosinase, but is known to be oxidized to tyrosinase by adding hydrogen peroxide (Non-patent Document 1). Therefore, by adding hydrogen peroxide, 4TBP and the like can also be adsorbed on chitosan and can be removed from the aqueous system by the method according to the present invention. If the amount of hydrogen peroxide added is too large, the quinone compound is further changed to another substance and cannot be adsorbed by chitosan.
また、本発明は、フェノール系化合物を含有する、水系の被処理液を反応槽に供給し、この反応層にチロシナーゼを供給するか、又は、チロシナーゼを支持する手段に前記被処理液を供給して、前記フェノール系化合物をキノン化合物に酸化させ、このキノン化合物を含む前記被処理液が在る前記反応槽にビーズ状のキトサンを供給するか、又はこの処理液をビーズ状のキトサンを支持する手段に供給して、前記被処理液から前記フェノール化合物を分離するシステム、被処理液のフェノール化合部を除去するために、ビーズ状に形成されたキトサン含有分離材および、この分離材が容器内に充填された分離装置も提供する。 The present invention also provides an aqueous treatment liquid containing a phenolic compound to a reaction tank, and supplies tyrosinase to the reaction layer, or supplies the treatment liquid to a means for supporting tyrosinase. Then, the phenolic compound is oxidized to a quinone compound, and bead-shaped chitosan is supplied to the reaction vessel in which the liquid to be treated containing the quinone compound is present, or the treatment liquid is supported by bead-shaped chitosan. A system for separating the phenol compound from the liquid to be treated, a chitosan-containing separating material formed in a bead shape to remove the phenol compound portion of the liquid to be treated, and the separating material in the container Also provided is a separation device packed in the container.
かかるシステム、分離材および分離装置は、工業廃液等からのフェノール系化合物の吸着除去に供され、大規模な浄化処理に用いることができる。 Such a system, a separating material, and a separating apparatus are used for adsorption removal of phenolic compounds from industrial waste liquids and the like, and can be used for large-scale purification treatment.
本発明にかかる方法によれば、水系に含まれるフェノール化合物を、大型の設備を必要とせずに、低コストで効率よく除去することができる。本発明にかかる方法を用いて、工業廃水中に含まれるフェノール化合物を処理し、その排出を防止することが可能である。 According to the method concerning this invention, the phenolic compound contained in an aqueous system can be efficiently removed at low cost, without requiring a large sized installation. By using the method according to the present invention, it is possible to treat a phenol compound contained in industrial wastewater and prevent its discharge.
酵素反応のｐＨ及び温度依存性(至適ｐＨと温度の決定)は初速度より評価した。結果を図１に示す。 The pH and temperature dependence of enzyme reaction (determination of optimum pH and temperature) was evaluated from the initial rate. The results are shown in FIG.
45℃でpHを5〜8でチロシナーゼ緩衝溶液20cm3と基質(m-クレゾール、p-クレゾール)緩衝溶液20cm3を混合し(混合時の両溶液のpHは同じ)、酵素濃度が0.04mg/cm3、基質濃度が0.5mMとなるようにした。酵素濃度0.04mg/cm3、基質(m-クレゾール、p-クレゾール)、温度45℃でチロシナーゼはリン酸緩衝溶液pH4〜8(イオン強度0.1M)で酵素反応を示し、最も高い酵素反応を示したpHは、いずれの基質の場合も７となり、pH5〜7で高い活性が得られ、この範囲での使用が好ましい。 Tyrosinase buffer at 5-8 pH with 45 ° C. solution 20 cm 3 and the substrate (m-cresol, p- cresol) buffer solution 20 cm 3 were mixed (pH of both solutions during mixing is the same), the enzyme concentration is 0.04 mg / cm 3 , and the substrate concentration was 0.5 mM. At an enzyme concentration of 0.04 mg / cm 3 , substrates (m-cresol, p-cresol) and a temperature of 45 ° C, tyrosinase shows enzyme reaction with phosphate buffer solution pH 4-8 (ionic strength 0.1M), showing the highest enzyme reaction The pH is 7 for any substrate, and high activity is obtained at pH 5 to 7, and use within this range is preferred.
酵素濃度0.04mg/cm3で基質をp-クレゾールとして、pH6で酵素反応を行うと、60℃以下で酵素反応を示した(60℃では、熱による変性により酵素活性は短時間で失活する)。図２に示されるように、20〜50℃程度の範囲での使用が好ましく、酵素反応が最大となった45℃での使用が特に好ましい。 When the enzyme reaction was carried out at pH 6 with the substrate concentration of 0.04 mg / cm 3 and p-cresol as the substrate, the enzyme reaction was observed at 60 ° C or lower (at 60 ° C, the enzyme activity was deactivated in a short time due to heat denaturation. ). As shown in FIG. 2, use in the range of about 20 to 50 ° C. is preferable, and use at 45 ° C. at which the enzymatic reaction is maximized is particularly preferable.
キトサンは和光純薬(株)製のキトサン１０００を用い、pH2〜3の塩酸にキトサン試料を溶解させ、濃度1g/100cm3とした。キトサンの分子量にも依るが、濃度3g/100cm3程度までは調製可能である。濃度1g/100cm3のキトサン溶液約４ｇを直径約３cmのシャーレに展開し、50℃で蒸発乾固させ、キトサンフィルムを作成した。キトサンフィルムをシャーレからはく離し、0.1〜1ＭのNaOH水溶液で洗浄し、溶解時にプロトン化したアミノ基を脱プロトン化させた。続いて純水で洗浄後、減圧乾燥した。 As chitosan, chitosan 1000 manufactured by Wako Pure Chemical Industries, Ltd. was used, and the chitosan sample was dissolved in hydrochloric acid having a pH of 2 to 3 to a concentration of 1 g / 100 cm 3 . Depending on the molecular weight of chitosan, it can be prepared up to a concentration of about 3 g / 100 cm 3 . About 4 g of a chitosan solution having a concentration of 1 g / 100 cm 3 was developed on a petri dish having a diameter of about 3 cm and evaporated to dryness at 50 ° C. to prepare a chitosan film. The chitosan film was peeled from the petri dish and washed with a 0.1 to 1 M NaOH aqueous solution to deprotonate the amino groups that were protonated during dissolution. Subsequently, it was washed with pure water and then dried under reduced pressure.
45℃でpHを５〜８で混合溶液の酵素濃度が0.04mg/cm3、基質(m-クレゾール、p-クレゾール)濃度が0.5mMとなるようにし、更に上述で作成したキトサンフィルムを加え、所定時間ごとに60分間溶液の波長300〜600nmでのスペクトルを測定した(チロシナーゼはo-クレゾールに対しては活性を示さない、ただし、過酸化水素を加えた際については現在実験を行ってないので、不詳)。また、同条件で酵素・基質混合溶液に10〜60分間(10分間隔)キトサンフィルムを浸漬させた。そのキトサンフィルムを純水で洗浄後減圧乾燥させ、波長300〜600nmでのスペクトルを測定した。 At 45 ° C, the pH is 5-8, the enzyme concentration of the mixed solution is 0.04 mg / cm 3 , the substrate (m-cresol, p-cresol) concentration is 0.5 mM, and the chitosan film prepared above is added, The spectrum of the solution at a wavelength of 300 to 600 nm was measured every predetermined time for 60 minutes (tyrosinase showed no activity against o-cresol, but no experiment was conducted when hydrogen peroxide was added. So unknown.) Moreover, the chitosan film was immersed in the enzyme / substrate mixed solution for 10 to 60 minutes (10 minute intervals) under the same conditions. The chitosan film was washed with pure water and dried under reduced pressure, and the spectrum at a wavelength of 300 to 600 nm was measured.
キトサンフィルムには、波長460nm付近にアミノ基にMichael's付加反応によって化学吸着したと考えられるピークが見られ、浸漬時間とともにそのピークは増大した。このピークの増大はキトサンフィルムによるキノン化合物の吸着を意味し、pH5〜8の範囲で見られた。pH5、6では混合溶液中でキトサンフィルム中のアミノ基の一部がプロトン化するため、キトサンフィルムが溶解または劣化するため、使用条件として好ましくない。また、pH8では酵素活性の低下によりキノン生成が少なくなるため、キトサンフィルムの吸着が下がる。従って、pH7前後での行うと、酵素反応によるキノン化合物の生成とキトサンフィルムによる吸着が首尾よく起こると言える。 In the chitosan film, a peak thought to have been chemically adsorbed to the amino group by Michael's addition reaction was observed near the wavelength of 460 nm, and the peak increased with the immersion time. This increase in peak meant adsorption of the quinone compound by the chitosan film, and was observed in the pH range of 5-8. When the pH is 5 or 6, some of the amino groups in the chitosan film are protonated in the mixed solution, so that the chitosan film is dissolved or deteriorated. In addition, at pH 8, the quinone production is reduced due to a decrease in enzyme activity, so that the adsorption of the chitosan film is lowered. Therefore, it can be said that when it is carried out at around pH 7, the production of quinone compounds by the enzymatic reaction and the adsorption by the chitosan film occur successfully.
図３にp-クレゾールでのpH７でのキトサンフィルム添加と無添加の混合溶液の波長400nmのピーク強度の反応時間変化を示す。 FIG. 3 shows the reaction time change of the peak intensity at a wavelength of 400 nm of the mixed solution with and without addition of chitosan film at pH 7 in p-cresol.
また、図４（ａ）にp-クレゾールでのpH７でのキトサンフィルムのスペクトル変化を、図４(b)にp-クレゾールでのpH５〜８でのキトサンフィルムの波長460nmでの吸光度の浸漬時間に対する変化を示す。図４（ａ）は、下から０分、１０分、２０分、３０分、４０分、５０分、６０分におけるスペクトルを示す。 Fig. 4 (a) shows the spectral change of chitosan film at pH 7 with p-cresol, and Fig. 4 (b) shows the immersion time of the absorbance of chitosan film at pH 5 to 8 with p-cresol at pH 460 nm. The change with respect to is shown. FIG. 4A shows spectra at 0 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes from the bottom.
45℃、pH７で基質をm-クレゾールまたはp-クレゾールとしてチロシナーゼと基質からなる混合溶液中の酵素濃度を0.005〜0.05mg/cm3となるようにして酵素濃度依存性を検討した。いずれの基質においても酵素濃度0.02mg/cm3以下で、比初速度(酵素1mg当たりの初速度)が一定となり、効率よく酵素反応を行うことができる。この濃度以上においても酵素反応は起こるが、比初速度は低下するため、その効率は低下する。 The enzyme concentration dependency was examined by changing the enzyme concentration in a mixed solution composed of tyrosinase and the substrate at 45 ° C. and pH 7 with m-cresol or p-cresol as the substrate, to 0.005 to 0.05 mg / cm 3 . In any substrate, the specific initial rate (initial rate per 1 mg of enzyme) becomes constant at an enzyme concentration of 0.02 mg / cm 3 or less, and an enzyme reaction can be carried out efficiently. Enzymatic reactions occur even above this concentration, but the efficiency is reduced because the specific rate is reduced.
図５(a)にp-クレゾールでのpH７での酵素濃度依存性を、図５(b)にm-クレゾールでのpH７での酵素濃度依存性を波長400nmでの吸光度の反応時間に対する変化として測定した結果を示す。また図５（ｃ）に、p-クレゾールとm-クレゾールでのpH７での酵素濃度依存性を酵素濃度に対する比初速度の変化として測定した結果を示す。 Fig. 5 (a) shows the enzyme concentration dependence at pH 7 with p-cresol, and Fig. 5 (b) shows the enzyme concentration dependence at pH 7 with m-cresol as the change in absorbance at 400 nm with respect to the reaction time. The measurement results are shown. FIG. 5 (c) shows the results of measuring the enzyme concentration dependence of p-cresol and m-cresol at pH 7 as the change in the specific rate relative to the enzyme concentration.
また、キトサンフィルムの入った酵素溶液に基質溶液を加えて、キトサンフィルムのキノン吸着能に及ぼす酵素濃度依存性を評価した結果、いずれの濃度においてもキノン化合物の吸着が確認できたが、m-クレゾールでは0.03mg/cm3以上で、p-クレゾールでは0.02 mg/cm3以上で、キノン吸着を示す波長460nmでのピークの浸漬時間に対する上昇がほぼ類似したため、45℃、pH７、基質濃度0.5mMの条件でキノン吸着に適した酵素濃度をm-クレゾールでは0.03mg/cm3、p-クレゾールでは0.02mg/cm3とした。 In addition, the substrate solution was added to the enzyme solution containing the chitosan film, and the enzyme concentration dependency on the quinone adsorption ability of the chitosan film was evaluated. As a result, the adsorption of the quinone compound was confirmed at any concentration. The increase in cresol at 0.03 mg / cm 3 or more and p-cresol at 0.02 mg / cm 3 or more and the increase in the immersion time of the peak at a wavelength of 460 nm showing quinone adsorption was almost similar, so that the temperature was 45 ° C., pH 7, and the substrate concentration was 0.5 mM. the enzyme concentration suitable quinone adsorbed condition m- cresol 0.03 mg / cm 3, the p- cresol was 0.02 mg / cm 3.
図６（ａ）に、p-クレゾールでのpH７での酵素濃度依存性を、図６（ｂ）にm-クレゾールでのpH７での酵素濃度依存性を、それぞれキトサンフィルムの波長460nmでの吸光度の浸漬時間に対する変化として測定した結果を示す。 Fig. 6 (a) shows the enzyme concentration dependence at pH 7 with p-cresol, and Fig. 6 (b) shows the enzyme concentration dependence at pH 7 with m-cresol. The absorbance of the chitosan film at a wavelength of 460 nm. The result measured as change with respect to immersion time of is shown.
チロシナーゼは4-tert-ブチルフェノール(４ＴＢＰ)を基質とすると、酵素反応を示さないが、過酸化水素の存在下では酵素反応を示す。45℃、pH７、酵素濃度0.02mg/cm3、基質濃度0.5mMとして過酸化水素濃度を0.5〜2.0mMとしてキノン生成の経時変化を混合溶液のスペクトル測定から評価した結果、反応時間に対してキノン化合物の生成を示す400nmのピークは増大するが、それに併せて480nm付近に別の化合物を示すピークが現れた。このピークも反応時間とともに増大し、キノン化合物が更に溶液中で他の化合物へと変化したと考えられる。この480nm付近のピークの増大は過酸化水素濃度が高いほど顕著であり、過酸化水素濃度0.5mMではこのピークはほとんど出現しなかった。これはキノン化合物の別の化合物への変化が起きていないことを示し、キトサンフィルムによるキノン吸着において好ましい条件である。酵素濃度0.04及び0.06mg/cm3においても同様な結果が得られた。しかし、４ＴＢＰにおいてはキノン生成を示す波長400nmでのピーク強度はm-クレゾールやp-クレゾールに比べて低いことがわかった。 Tyrosinase does not show an enzymatic reaction when 4-tert-butylphenol (4TBP) is used as a substrate, but shows an enzymatic reaction in the presence of hydrogen peroxide. 45 ° C, pH 7, enzyme concentration 0.02mg / cm 3 , substrate concentration 0.5mM, hydrogen peroxide concentration 0.5-2.0mM, quinone formation over time was evaluated from spectral measurements of the mixed solution. Although the peak at 400 nm indicating the formation of the compound increased, a peak indicating another compound appeared at around 480 nm. This peak also increases with the reaction time, and it is considered that the quinone compound further changed to another compound in the solution. The increase in the peak near 480 nm is more remarkable as the hydrogen peroxide concentration is higher, and this peak hardly appears at a hydrogen peroxide concentration of 0.5 mM. This indicates that the quinone compound has not changed to another compound, which is a preferable condition for quinone adsorption by the chitosan film. Similar results were obtained at enzyme concentrations of 0.04 and 0.06 mg / cm 3 . However, in 4TBP, the peak intensity at a wavelength of 400 nm indicating quinone formation was found to be lower than that of m-cresol or p-cresol.
図７(a)〜(d)に、４ＴＢＰでの過酸化水素濃度依存性(スペクトルの時間変化)を、図８に４ＴＢＰでの過酸化水素濃度依存性を、波長400nmと480nmでの吸光度の反応時間に対する変化として測定した結果を示す。(a)は0.5mM、(b)2.0mMである。 FIGS. 7 (a) to (d) show the dependence of hydrogen peroxide concentration on 4TBP (change in spectrum over time) in FIG. 8. FIG. 8 shows the dependence of hydrogen peroxide concentration on 4TBP on the absorbance at wavelengths of 400nm and 480nm. The result measured as change with respect to reaction time is shown. (a) is 0.5 mM and (b) is 2.0 mM.
45℃、pH７、酵素濃度0.02mg/cm3、基質濃度0.5mMとして過酸化水素濃度を0.5〜2.0mMの範囲で、キトサンのキノン吸着能を調べた結果、過酸化水素濃度1.0mM以下でそれ以上の濃度に比べて高いキノン吸着を示した。過酸化水素濃度が高いと、溶液中でキノン化合物が更に他の化合物に変化し、キトサンフィルムへの吸着が低下したことから、溶液中でのキノン化合物の存在がキトサンフィルムの吸着能の向上に重要であるといえる。 As a result of examining the quinone adsorption ability of chitosan at 45 ° C, pH 7, enzyme concentration 0.02mg / cm 3 , substrate concentration 0.5mM and hydrogen peroxide concentration in the range of 0.5-2.0mM, Compared with the above concentration, quinone adsorption was high. When the hydrogen peroxide concentration is high, the quinone compound changes to another compound in the solution and the adsorption to the chitosan film decreases, so the presence of the quinone compound in the solution improves the adsorption capacity of the chitosan film. It can be said that it is important.
図９に、４ＴＢＰでの過酸化水素濃度依存性を、波長420nmでのキトサンフィルムの吸光度の浸漬時間に対する変化として測定した結果を示す。 FIG. 9 shows the result of measuring the dependence of hydrogen peroxide concentration on 4TBP as the change in the absorbance of the chitosan film at a wavelength of 420 nm with respect to the immersion time.
上記の結果からチロシナーゼによるキノン生成とキトサンフィルムによるキノン吸着によって水溶液中のフェノール化合物の除去ができることが見いだせたので、更に高いキノン化合物の吸着を行うため、キトサンフィルムの代わりに市販のキトサンビーズを用いて同様の実験を行った。本研究で用いたキトサンビーズは、富士紡績(株)製のキトパールAL-01である。キトパールAL-01は水膨潤した状態で製品化されているため、pH７の緩衝溶液で外部溶液を洗浄、置換し、冷所に保存した。実験では所定重量のキトパールを添加することができないので、所定体積を加えて行った。使用したキトパールは、粒径分布70〜200μm、比表面積70〜100m2/gであり（市販品はこの一種のみで公称値）、1cm3当たりの乾燥キトパール量は、0.070g/cm3（実験値）、水に膨潤させたキトパール1g中に含まれる乾燥キトパールは0.075g、水に膨潤させたキトパール1g中の含水量は0.925g、つまり含水率92.5％（実験値）である。10μm〜数mmまでの粒径が使用可能である。 From the above results, we found that phenol compounds in the aqueous solution can be removed by quinone production by tyrosinase and quinone adsorption by chitosan film. Therefore, in order to adsorb higher quinone compounds, commercially available chitosan beads were used instead of chitosan film. The same experiment was conducted. The chitosan beads used in this study are Chito Pearl AL-01 manufactured by Fuji Boseki Co., Ltd. Since Chitopearl AL-01 was commercialized in a swollen state with water, the external solution was washed and replaced with a pH 7 buffer solution, and stored in a cold place. In the experiment, a predetermined weight of chitopearl could not be added, so a predetermined volume was added. The used chitopearl has a particle size distribution of 70 to 200 μm and a specific surface area of 70 to 100 m 2 / g (the commercial product is only this kind, nominal value), and the dry chitopearl amount per 1 cm 3 is 0.070 g / cm 3 (experimental Value), dry chitopearl contained in 1 g of chitopearl swollen in water is 0.075 g, and the water content in 1 g of chitopearl swollen in water is 0.925 g, that is, the water content is 92.5% (experimental value). Particle sizes from 10 μm to several mm can be used.
45℃、pH７、酵素濃度0.02mg/cm3、基質濃度0.5mMの条件で、キトパール0.2〜2.0cm3を加えたチロシナーゼ・p-クレゾール混合溶液の波長400nmでのピークを測定した。波長400nmでのピークは攪拌時間を長くすると、徐々に低下した。この傾向はキトパール添加量が多いほど顕著であり、キトサンフィルムを加えたときに比べて波長400nmでのピーク強度は著しく小さくなった。これはキトサンフィルムを浸漬するよりもキトパールを用いた方が、より効率よくキノン化合物を吸着できることを示し、キトパール添加量1.0cm3以上ではほぼ同様の結果が得られたので、最適添加量を1.0cm3とした。また、基質をm-クレゾールとした場合も同様にキトサンフィルムを浸漬するよりもキトパールを用いた方が、より効率よくキノン化合物を吸着できた。 45 ° C., pH 7, enzyme concentration 0.02 mg / cm 3, at a substrate concentration 0.5mM conditions were measured peak at the wavelength 400nm tyrosinase · p-cresol mixture solution was added Chitopearl 0.2~2.0cm 3. The peak at a wavelength of 400 nm gradually decreased as the stirring time was increased. This tendency becomes more prominent as the amount of chitopearl added increases, and the peak intensity at a wavelength of 400 nm is significantly smaller than when chitosan film is added. This shows that quinone compounds can be adsorbed more efficiently when chitopearl is used than when dipping the chitosan film, and almost the same result was obtained when the chitopearl addition amount was 1.0 cm 3 or more. It was cm 3. Similarly, when the substrate was m-cresol, the quinone compound could be adsorbed more efficiently when chitopearl was used than when the chitosan film was immersed.
図10に、p-クレゾールでのpH７でのキトパール添加量依存性を、波長400nmでの混合溶液の吸光度の攪拌時間に対する変化として測定した結果を示す。 FIG. 10 shows the results of measuring the dependency of p-cresol on the amount of chitopearl added at pH 7 as the change in the absorbance of the mixed solution at a wavelength of 400 nm with respect to the stirring time.
更に、p-クレゾールを基質としてキトパール添加量を1.0cm3として、反応温度25と35℃で行うと、温度が高いほど、混合溶液の波長400nmでのピーク強度が低下したので、45℃でキトパールを用いて吸着させることが好ましいが、いずれの温度においても攪拌時間60分で、キトパールを添加した溶液の波長400nmでのピーク強度はキトパール不在な溶液の10分の１程度となった。また、p-クレゾールとm-クレゾールを基質とした場合、混合溶液中に60分間浸漬したキトパールは、こげ茶色に変色し、キノン化合物の吸着が確認できた。 Furthermore, when p-cresol was used as the substrate and the amount of chitopearl added was 1.0 cm 3 and the reaction temperature was 25 and 35 ° C., the higher the temperature, the lower the peak intensity at a wavelength of 400 nm of the mixed solution. However, at any temperature, the stirring intensity was 60 minutes, and the peak intensity at a wavelength of 400 nm of the solution to which chitopearl was added was about one-tenth that of the solution without chitopearl. In addition, when p-cresol and m-cresol were used as substrates, chitopearl soaked in the mixed solution for 60 minutes turned dark brown, confirming the adsorption of the quinone compound.
酵素反応を起こすために過酸化水素を必要とする４ＴＢＰにおいても45℃、pH７、酵素濃度0.02mg/cm3、基質濃度0.5mM、過酸化水素濃度0.5mMで、キトパール添加量を1.0〜5.0cm3としてキトパールの吸着能を調べた結果、キトパールを添加した混合溶液の波長400cmでの吸光度は無添加の混合溶液に比べて低下し、キトパールへのキノン化合物の吸着を示した。また、60分間浸漬したキトパールは薄茶色を呈した。 Even in 4TBP that requires hydrogen peroxide to cause enzyme reaction, 45 ° C, pH 7, enzyme concentration 0.02mg / cm 3 , substrate concentration 0.5mM, hydrogen peroxide concentration 0.5mM, and chitopearl addition amount 1.0-5.0cm As a result of examining the adsorption ability of chitopearl as 3 , the absorbance at a wavelength of 400 cm of the mixed solution containing chitopearl was lower than that of the non-added mixed solution, indicating that the quinone compound was adsorbed to chitopearl. Moreover, the chitopearl immersed for 60 minutes was light brown.
図11に、４ＴＢＰでのpH７でのキトパール添加量依存性を、波長400nmでの混合溶液の吸光度の攪拌時間に対する変化として測定した結果を示す。 FIG. 11 shows the result of measuring the dependency of chitopearl addition amount at pH 7 with 4TBP as the change in the absorbance of the mixed solution at a wavelength of 400 nm with respect to the stirring time.
Claims (10)
A separation device in which the separation material according to claim 9 is filled in a container.
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| CN103073104A (en) * | 2013-01-30 | 2013-05-01 | 中国天辰工程有限公司 | Treatment method of caprolactam production waste water |
| CN104692522A (en) * | 2015-02-13 | 2015-06-10 | 中南大学 | Composite chemical for degrading pollutant benzoquinone in wastewater and method for degrading pollutant benzoquinone in wastewater |
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