苯酚消减

Enzyme and Microbial Technology 36 (2005) 147–152 Rapid communication Efficacy of amphiphile-modified laccase and removal of phenolics in aq Yoshitsune Shin-yaa,∗, Hnin Nwe Ayeb, Kyung-Ji duate S okayam ogical U June 2 Abstract This stud d lacca the removal yalkyle The amphip h impr dimethoxyp rease i modified lac ing mo of which usually behave as hazardous pollutants of natural environments. The modified laccase exhibited a remarkably improved efficiency over that of the native laccase in terms of removing various phenolic substances. These results revealed that the enzymatic oxidation by the amphiphile-modified laccase is suitable for the efficient removal of phenolics in aqueous systems. © 2004 Elsevier Inc. All rights reserved. Keywords: A 1. Introdu Contam produced b dying, pho circuit-boa phenolic c receiving w effluent co inated with by various adsorption, Abbreviat dichloropheno HPLC, high zyme; NE, n dride; RA, re trinitrobenzen ∗ Correspo E-mail a 0141-0229/$ doi:10.1016/j mphiphile; Chemical modification; Laccase; Oxidative removal; Phenolic pollutants; Polyalkyleneoxide-co-maleic anhydride; Thermal stability ction inated water containing phenolic compounds is y various industrial processes such as oil refining, to processing, metal plating, coal conversion, and rd manufacturing process [1–3]. As most of the ompounds are toxic and cause coloration of the aters, it is therefore essential to decontaminate ntaining such compounds. The effluents contam- phenolic compounds are conventionally treated methods including solvent extraction, distillation, and chemical oxidation. Although these methods ions: AAP, 4-aminoantipyrine; CP, chlorophenol; DCP, l; DM, degree of modification; DMP, 2,4-dimethoxyphenol; performance liquid chromatography; ME, modified en- ative enzyme; PAOMA, polyalkyleneoxide-co-maleic anhy- lative oxidative activity; TCP, trichlorophenol; TNBS, 2,4,6- esulfonate; WR, weight ratio of modifier to enzyme nding author. Tel.: +81 3 5734 3246; fax: +81 3 5734 3315. ddress: yshin-ya@kuramae.ne.jp (Y. Shin-ya). are useful, they have some drawbacks such as high cost, in- complete purification, formation of other hazardous byprod- ucts, or restricted applicability as regards which pollutants they can effectively remove. Microbial or enzyme-based treatments for the removal of phenolic compounds have offered some distinct advantages over physical and chemical removal methods [4–8]. Thus, using enzymes as decontaminating agents has received great attention because of their potential to remove pollutants from the environment without creating the harsh side effects asso- ciated with other methods. The significant advantages of this enzymatic method include the mild condition of enzymatic treatment, the requirement of only trace amounts of enzymes, the ability to decontaminate low concentrations of contami- nants, and the ability to handle large volumes of effluent as well. Peroxidases, laccases, and tyrosinases catalyze the oxidation of phenolics using either hydrogen peroxide or molecular oxygen, generating phenoxy radicals that react with themselves or other phenolics to form dimers. These re- – see front matter © 2004 Elsevier Inc. All rights reserved. .enzmictec.2004.07.013 a Department of International Development Engineering, Gra Tokyo Institute of Technology, Ishikawadai 4th Bldg., 2-12-1 O b Department of Engineering Chemistry, Yangon Technol Received 17 March 2004; received in revised form 30 y is mainly related to the characterization of a chemically modifie of phenolics in aqueous systems. An amphiphilic copolymer, pol hile-modified laccase displayed higher catalytic activity along wit henol. The chemical modification resulted in a 20- to 10-fold inc case was applied to remove a range of phenolic compounds includ in enzymatic oxidation ueous solution n Honga, Toshio Kajiuchia chool of Science and Engineering, a, Meguru, Tokyo 152-8550, Japan niversity, Yangon, Myanmar 004; accepted 7 July 2004 se from the Trametes sp. for use in the oxidation and neoxide-co-maleic anhydride was used as a modifier. oved thermal stability in the oxidation reaction of 2,6- n thermal stability at 25 and 40 ◦C, respectively. The no-, di-, and tri-chlorophenols, cresol, and xylenol, all 148 Y. Shin-ya et al. / Enzyme and Microbial Technology 36 (2005) 147–152 actions eventually lead to the production of higher oligomers and polymers of low solubility, which then precipitate and can be readily removed by sedimentation or filtration. Laccases, have been Laccases ( utilize mol pounds; in which is ha environmen Since they utilize vari they are v decolorizat Howeve tions, main is still prev bility and activity ha practical ap Numero come these engineering lation from [16], and c low-molec to be succe an amphiph dride (PAO in order to ified enzym The aim modified la and to char idative acti of various 2. Materia 2.1. Mater Laccase sample from weight and 3.0, respec SUNBRIG pon Oil & the modifie shown in Fi 2,6-dimeth 2,4- and 2, (TCP), cre Chemical I directly wi . Struct ndom c degree Chem e che lymer e ami dride e che lymer ed me differ 15 M b laccas of the iluted uffer a modifi fter a ). Degre e deg ge of prote e nativ rotein molecule was reported to be proportional to the rophotometric absorbance at 420 nm obtained from 2,4, itrobenzenesulfonate (TNBS) method [24]. The mea- ent of absorbance of the TNBS reaction mixture was ed out using a UV-3210 spectrophotometer (HITACHI, . Chemical modification of laccase using PAOMA as a modifier. MA ents the maleic anhydride group in PAOMA molecule. Reaction was out at pH 8.5, 2 ◦C. one of the more promising groups of enzymes, used to decontaminate phenol-polluted systems. EC 1.10.3.2), which are multi-copper oxidases, ecular oxygen for the oxidation of phenolic com- contrast, peroxidases require hydrogen peroxide, rmful to the environment. Therefore, laccases are tal-friendly and potentially attractive catalysts. have a wide range of substrate specificity and can ous types of aromatic compounds as substrates, ery useful in the process of detoxification and ion of various phenolic pollutants [9–12]. r, the use of these enzymes for practical applica- ly as agents for environmental cleanup purposes, ented by several limitations. In general, low sta- the potential for drastic reductions in enzymatic ve always been considered as hindrances to the plication of enzymatic systems. us studies have been conducted in order to over- limitations. Several methods based on protein [13], immobilization in solid supports [14], iso- thermophilic organisms [15], the use of additives hemical modification with polymeric [17–19] and ular weight compounds [20,21] have been reported ssful for preparing stable enzymes. In particular, ilic polymer, polyalkyleneoxide-co-maleic anhy- MA), have been widely used as enzyme modifiers overcome the limitations associated with unmod- es [22,23]. of the present study was to prepare a chemically ccase using an amphiphilic polymer, PAOMA, acterize the modified enzyme in terms of its ox- vity, thermal stability, and the removal efficiency phenolics. ls and methods ials obtained from the Trametes sp. was a donated Daiwa Kasei K.K., Osaka, Japan. The molecular isoelectric point of the laccase were 6.2× 104 and tively. Polyalkyleneoxide-co-maleic anhidride, HT AGM-0530®, was kindly supplied from Nip- Fat Co., Japan, and this copolymer was used as r. The chemical structure of the copolymer are g. 1. All phenolic substrates used in this study, i.e., oxyphenol (DMP), o-,m-, p- chlorophenols (CPs), 6-dichlorophenols (DCPs), 2,4,6-trichlorophenol sol, and xylenol, were obtained from Wako Pure ndustries, Ltd., Japan. All chemicals were used thout further purification. Fig. 1 The ra ization 2.2. Th copo a fre anhy Th copo scrib with of 0. The that was d ate b (the herea “NE” 2.3. Th centa ified in th the p spect 6-trin surem carri Fig. 2 repres carried ure of the polyalkyleneoxide-co-maleic anhydride (PAOMA). opolymerization degree (ran) and the alternative copolymer- (alt) are 32 and 30, respectively. The EO/AO is 0.6. ical modification of laccase mical modification of laccase with the PAOMA was based on the condensation reaction between no group of the enzyme surface and a maleic- group of the copolymer (Fig. 2). mical modification of laccase with the PAOMA was performed according to a previously de- thod [22,23]. The native laccase was incubated ent amounts of PAOMA copolymer in a solution orate buffer, pH 8.5, at 2 ◦C with gentle stirring. e concentration in the mixture was 2 wt.%, and modifier was 2–18 wt.%. The resulting solution at the required concentration with the appropri- nd then was used for the following experiments ed laccase obtained by this method is referred to s “ME” and the native laccase is referred to as e of modification ree of modification (DM) was defined as a per- the number of modified amino groups in the mod- in relative to the number of free amino groups e protein. The number of free amino groups in Y. Shin-ya et al. / Enzyme and Microbial Technology 36 (2005) 147–152 149 Japan). DM was calculated by the following equation: DM = (AN − AM) × 100 where AN a tive and mo concentrati 2.4. Lacca The acti rically usin quinone, is ric absorba of 49.6 mM tained lacca acetate buf The reactio amount of was monito defined as quinone dim 2.5. Therm In order 310 mg/L l and 40 ◦C i was withdr surement o The residu to the initi as calculate time t)/(enz 2.6. Efficie The rem systems wa insoluble conducted systems fo catalyzed b ing approp were incub day at 25 ◦ a DISMIC if needed, The concen determined for single liquid chro component In the ca pound reac termediate potassium ferricyanide reagent. The resulting compound was a quinoneimine-type dye that absorbed light at 510 nm. The percent removal of phenolics was calculated by subtracting bsorba f a re esidua ulti-c sis. T m i. Japan Kyoto , Inc., vis de ere fi applie L. Th O-6A n was .1% a mL/m e rem nal c spect val (% esults Chem e mo C wit ) to o n DM ased w pprox ino gr ted ex porte ino gr e N-te 6 ami the PA Oxida order ffect o prelim ent, . As s matic ed the ses, in d the AN nd AM represent the absorbance at 420 nm of na- dified laccases, respectively, at the same enzyme on. se activity assay vity of laccase was determined spectrophotomet- g DMP as a substrate. The DMP oxidized product, a colored compound that shows spectrophotomet- nce at 469 nm and has an extinction coefficient −1 cm−1 [25]. The reaction mixture which con- se (3 mg/L) and DMP (0.9 mM) in 50 mM sodium fer (pH 5.0) was incubated at 25 ◦C for 10 min. n was initiated by the addition of an appropriate enzyme solution, and the increase in absorbance red at 469 nm. One unit of enzyme activity was the amount of enzyme producing 1�mol of the mer per minute from DMP. al stability assay to examine the thermal stability of the enzyme, accase solutions of NE and ME were exposed at 25 n a thermostat shaker. An aliquot of each solution awn at the appropriate time intervals for the mea- f enzyme activity using the above assay method. al activity was expressed as a percentage relative al enzyme activity with respect to each enzyme, d below: residual activity = (enzymatic activity at ymatic activity at time 0). ncy of the removal of phenolic compounds oval of phenolics from contaminated water s achieved by enzymatic conversion into water- substances. In brief, batch experiments were in single-component and multi-components r the oxidative removal of phenolic compounds y the NE and the ME. Reaction mixtures contain- riate amounts of phenolic substrates and enzymes ated with shaking at 100 oscillations/min for 1 C. Thereafter, the samples were filtered using filter unit (0.45 mm, ADVANTEC, Japan) and the resulting filtrates were diluted with buffer. trations of residual phenolics in the filtrate were by two analytical methods, colorimetric method component systems [26] and high performance matography (HPLC) analysis for multi-phenolic s systems. se of the colorimetric method, the phenolic com- ted with 4-aminoantipyrine (4-AAP) yields an in- species, which was oxidized in the presence of the the a that o R the m analy (4.6 m Ltd., tific, dyne UV– ples w were 100 m a CT elutio and 0 of 1.0 Th and fi Cf, re remo 3. R 3.1. Th at 2 ◦ (WR twee incre was a of am repor are re �-am in th 4 or with 3.2. In the e were treatm alone enzy show lacca prove nce of a substrate solution without enzyme from action mixture treated with enzyme. l phenolics in the enzymatic reaction mixture for omponents systems were also detected by HPLC he HPLC system was equipped with Fluofix 120N d.× 250 mm) (Wako Pure Chemical Industries, ), a Shimadzu LC-6A pump (Shimadzu Scien- , Japan) with a 7125 Rheodyne injector (Rheo- Cotati, CA, USA), a SPD-M6A photodiode array tector (Shimadzu Scientific, Kyoto, Japan). Sam- ltered through a 0.45�m filter (ADVANTEC) and d to the system. The injection volume was set at e elution temperature was regulated at 40 ◦C by column oven (Shimadzu Scientific). An isocratic performed using a 50:50 mixture of acetonitrile queous phosphoric acid, maintained at a flow rate in. oval percentage was calculated from the initial oncentrations of phenolic substrate (i.e., Ci and ively) by the following equation: ) = ( Cf Ci ) × 100. and discussion ical modification of laccase dification of laccase was carried out at pH 8.5 and h a varying weight ratio of modifier to enzyme btain different DM values. The relationship be- and WR is shown in Fig. 3 (a). In this case, DM ith increases in the WR, and the maximum DM imately 65% at a WR value of 9. Although number oups in the laccase used in this study has not been actly, similar kinds of laccases from Trametes sp. d to have 6 or 9 amino groups per molecule; 5 or 8 oups in the lysine residues and 1 �-amino group rminal [27]. The results showed that maximally no groups per laccase molecule were maleylated OMA. tive activity of modified laccases to distinguish the effect of modification from f the modifier on enzyme activity, activity assays inarily performed using three types of laccase i.e., NE alone, NE and PAOMA mixture, and ME hown in Fig. 4, the modifier slightly affected the activity of laccase and the PEOMA-modification highest activity among the three treatments by dicating that the PAOMA modification itself im- enzymatic activity of laccase. 150 Y. Shin-ya et al. / Enzyme and Microbial Technology 36 (2005) 147–152 Fig. 3. (a) Degree of modification (DM) and relative activity (RA) as a function of PAOMA-modifier to laccase weight ratio (WR). (b) Relationship between RA and DM. The modification was carried out at pH 8.5, 2 ◦C using 2 wt.% laccase solution. Laccase activity assay was performed at pH 5.0, 25 ◦C using 3 mg/L laccase concentration. Fig. 3 (b) illustrates the plot of the relative activities (RA) of modified laccase versus the WR used in the modification. The RA value was calculated by dividing the activity of mod- ified laccase by the activity of the native laccase. The results Fig. 4. Effect 2, and 3 repr (9:1, w/w), an groups in PAO enzymatic rea laccase. Lacc at pH 5.0, 37 showed that all of the modified laccase trials exhibited much higher activity than those with the native laccase, indicating that this attachment of PAOMA to laccase did not lead to steric hindrance of the enzymatic reaction. The higher activ- ity of modified laccases could be attributed to the alternation of surface hydrophobicity on the laccase. The increased lac- case activity in this study is in agreement with the results obtained from polyethylene glycol-modified laccases [28]. From the kinetic analysis, the enhanced laccase activities of the polyethylene glycol-modified laccases were attributed to the increase in the catalytic efficiency. 3.3. Thermal stability of PAOMA-modifled laccase The thermal stability of the native and modified laccases was examined by measuring the activity of the enzyme re- tained after exposing the enzyme to a temperature of 25 and 40 ◦C for different time intervals. Fig. 5 shows the residual activities of NE and ME retained after incubation at 25 and 40 ◦C. Table 1 shows the first-order deactivation constants (kd) of the NE and ME. Chemical modification of laccase re- sulted in a 20- and 10-fold decrease in the kd value at 25 and 40 ◦C, respectively. The stabilization shown in the ME may have been due to the cross-linking caused by the multipoint attachment of PAOMA to several reactive amino groups on the surface of a polypeptide chain. In addition, protection from self-oxidation of laccase could have been achieved by chemical modification. The stabilization effect of chemical of modifier and modification on oxidative reaction. No. 1, esent RA for NE alone, RA for mixture of modifier and NE d RA for ME alone (WR = 9), respectively. Maleic anhydride MA modifier in the system No. 2 was hydrolyzed prior to the ction in order to avoid undesirable modification reaction with ase concentration was set at 3 mg/L. Reaction was performed ◦C. Fig. 5. Therm solution (30 m assay was car ME and NE r al stability of ME and NE at (a) 25 ◦C and (b) 40 ◦C. Laccase g/L) was incubated at pH 5.0. Then, the remaining activity ried out at pH 5, 25 ◦C using 3.0 mg/L laccase concentration. epresent modified and native laccases, respectively. Y. Shin-ya et al. / Enzyme and Microbial Technology 36 (2005) 147–152 151 Table 1 First-order deactivation constants, kd, of ME and NE at 25 and 40 ◦C Enzyme Temperature (◦C) kd (h−1) ME 25 5.0× 10−3 NE 25 1.1× 10−1 ME 40 1.1× 10−2 NE 40 1.1× 10−1 NE and ME represent native and modified laccases, respectively. The degree of modification for the ME was 63%. modification has been reported in pectinase, protease, and lipase, etc. [29,30]. 3.4. Efficiency of the removal of various phenolic compounds Fig. 6 shows the percent removal of DCP by NE and ME at various enzyme concentrations through the 1-day treatment at 25 ◦C. In this plot, it can be seen that modified laccase was more efficient for the removal of DCP at every concentration than the native laccase. The percent removal of DCP was found to be 75% for the ME and 45% for the NE at 12 mg/L of laccase in the reaction mixture. Table 2 depicts the percent removal of various pheno- lic compounds, namely, DMP, CPs, DCPs, TCP, cresol, and xyleno ardous poll Although i case of bot centage ran tested in sin that the atta the remova Fig. 6. Percen centrations un 12 mg/L; pH, DCP was ana and modified Table 2 Percent removal of various phenolics in single component systems by NE and ME at pH 5.0, 25 ◦C Phenolics DMP o-CP p-CP m-CP 2,4-DCP 2,6-DCP 2,4,6-TCP Cresol Xylenol Phenolics, 0.4 nolics were a and ferricyan spectively. Th Table 3 Percent remov at pH 5.0, 25 Phenolics o-CP 2,4-DCP TCP phenoli tion tim present native and modified laccases, respectively. The ME had 63% of modification. ble 3 compares the removal efficiency of the NE and n a multi-components system containing three pheno- -CP, 2,4-DCP, and 2,4,6-TCP. Native laccase was found crease in removal efficiency when treating a mixture of olics, in comparison with the case of decontamination gle component systems. In multi-component phenolics ms, it was reported that oxidative transformation was licated and that phenolics removal was inefficient due e great decrease of enzymatic activity [10]. The ineffi- removal of NE in this study was similar to the laccase vior reported in the above literature. In contrast, the ME ed its efficient performance with respect to treating a olics mixture, as well as in treating a single substrate. result opens promising perspectives for applying the MA-modified laccase in a practical removal process of olics from polluted water. l, all of which are commonly specified as haz- utants by US Environmental Pr