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1 Bioresource Technology 100 (2009) Contents lists available at ScienceDirect Bioresource Technology journal homepage: Characterization of catechol derivative removal by lignin peroxidase in aqueous mixture Shaul Cohen a,c, Paula A. Belinky a,b, Yitzhak Hadar c, Carlos G. Dosoretz d, * a MIGAL Galilee Technology Center, Kiryat Shmona 11016, Israel b Tel Hai Academic College, Kiryat Shmona 12210, Israel c Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel d Division of Environmental, Water and Agricultural Engineering, Faculty of Civil and Environmental Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel article info abstract Article history: Received 24 July 2008 Received in revised form 8 November 2008 Accepted 11 November 2008 Available online 18 December 2008 Keywords: Lignin peroxidase Catechol removal Phenol removal Product-dependent inactivation Wastewater treatment The use of lignin peroxidase (LIP) as an alternative method for the removal of four catechols (1,2-dihydroxybenzene): catechol (CAT), 4-chlorocatechol (4-CC), 4,5-dichlorocatechol (4,5-DCC) and 4-methylcatechol (4-MC) typical pollutants in wastewater derived from oil and paper industries, was evaluated. The removal of 2 mm catecholic substrates by 1 lm LIP after 1 h was in the following order: 4,5-DCC (95%) > 4-CC(90%) > CAT(55%) > 4-MC(43%). Except for 4-MC, all reactions were accompanied by the formation of insoluble products, leading to LIP precipitation. LIP was exposed to soluble or insoluble product-dependent inactivation, depending on the substrates tested, immediately at the start of the reactions. Despite immediate enzyme inactivation, removal of catecholic substrates continued, resulting in oligomeric product formation. Major oxidation products analyzed were compatible with dimeric, trimeric and tetrameric structures. Ether linkages and a benzoquinone structure were detected in two purified oligochlorocatechols. Catechol derivatives removal initiated by LIP, seems to be different for each catecholic substrate in terms of substrate consumption and transformation, and of enzyme activity. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Phenol-polluted wastewater is commonly produced by a number of industrial and agricultural activities. Phenolic compounds and their derivatives, e.g. catechols, are considered priority pollutants because they are harmful to living organisms even at low concentrations. In humans and mammals, catechols can occur as metabolites in the degradation of estrogens or in endogenous compounds, such as neurotransmitters and their precursors. In nature, catechols are formed as intermediary products of the degradation of aromatic compounds by microorganisms. Catechol is used in a variety of applications: as a reagent for photography, in rubber and plastic production and in the pharmaceutical industry. Substituted catechols, especially chlorinated and methylated ones, are by-products in pulp and oil mills (reviewed in Schweigert et al., 2001). If catechols are released into the environment, they can accumulate in the soil, groundwater, and surface water, and they have therefore become an issue of great environmental concern. * Corresponding author. Tel.: ; fax: addresses: carlosd@tx.technion.ac.il, cgdosoretz@gmail.com (C.G. Dosoretz). Removal of these compounds from wastewater can be addressed by conventional remediation methods (e.g., solvent extraction, chemical oxidation and adsorption to activated supports) (Freeman and Harry, 1995). Although effective, some of these methods present a number of disadvantages, such as high cost, time-consuming procedures and formation of toxic residues. Biological technologies dealing with the use of oxidoreductive enzymes, e.g. laccases, tyrosinases and peroxidases, may offer an efficient alternative means of addressing the clean-up of phenolpolluted wastewater (Shuttleworth and Bollag, 1986; Adam et al., 1999; May, 1999; Regalado et al., 2004). Oxidoreductases can catalyze the transformation of several phenolic compounds through an oxidative-coupling reactions. This results in the formation of less soluble high-molecular-weight compounds that can be easily removed from water by sedimentation or filtration (Gianfreda et al., 2003). The initial oxidation or removal of a wide range of toxic phenols by these enzymes has already been shown. For example, oxidation of phenol by tyrosinase (Ikehata and Nicell, 2000), horseradish peroxidase (HRP) (Wagner and Nicell, 2002) and soybean peroxidase (SBP) (Bassi et al., 2004; Caza et al., 1999; Kinsley and Nicell, 2000; Wilberg et al., 2002; Wright and Nicell, 1999) chlorophenols /$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi: /j.biortech

2 2248 S. Cohen et al. / Bioresource Technology 100 (2009) and catechols by laccase (Aktas et al., 2003; Bollag et al., 2003; Gianfreda et al., 2003; Park et al., 1999), HRP (Park et al., 1999; Wagner and Nicell, 2002; Ward et al., 2004), SBP (Bassi et al., 2004; Dubey et al., 1998; Wright and Nicell, 1999) and tyrosinase (Park et al., 1999; Wada et al, 1993); methylphenols by laccase (Ghosh et al., 2008), HRP (Wagner and Nicell, 2002) SBP (Caza et al., 1999; Wright and Nicell, 1999) and tyrosinase (Wada et al, 1993); methoxyphenols by laccase (Lante et al., 2000) and tyrosinase (Wada et al, 1993), and bromophenols by HRP (Levy et al., 2003). Lignin peroxidase (LIP) is considered one of the most important enzymes of the extracellular lignin degradation system secreted by the white-rot fungus, Phanerochaete chrysosporium (Hatakka, 1994). LIP possesses a higher redox potential than any other peroxidase or oxidase (Hammel et al, 1986; Kersten et al., 1990) and has been reported to oxidize aromatic compounds with calculated ionization potential (IP) values of up to 9.0 ev (Have et al., 1998; Ward et al., 2003a). LIP is of interest in wastewater treatment processes and in catalyzing difficult chemical transformations. It is also of agricultural and environmental importance on account of its role in lignin biodegradation (Tien and Kirk, 1988). Phenol (Chung and Aust, 1995a), chloro- and bromophenols (Ward et al., 2002, 2003b), and guaiacol (Koduri and Tien, 1995) are some of the aromatic substrates that have been studied for their oxidation by LIP for bioremediation purposes. As catechols are widely distributed, and considering the advantages of LIP relative to other peroxidases and oxidases, we set out to perform a study of the in vitro LIP-catalyzed removal of toxic catechol and halogenated and methylated catechols, in order to assess LIP s potential for the treatment of catechol-polluted wastewater. 2. Methods 2.1. Materials Catecholic substrates: catechol (CAT), 4-methylcatechol (4-MC), 4-chlorocatechol (4-CC) and 4,5-dichlorocatechol (4,5-DCC) and hydrogen peroxide (H 2 O 2 ) (30% solution) were all obtained from Sigma Aldrich (Rehovot, Israel). The concentration of stock solutions of H 2 O 2 was determined at 240 nm using an extinction coefficient of 39.4 M 1 cm LIP purification LIP isoenzyme H1 was produced from low nitrogen cultures of P. chrysosporium Burds BKM-F-1767 (ATCC 24725) as previously described (Rothschild et al., 1997). The enzyme was purified by MonoQ (Dosoretz et al., 1990), using a M sodium acetate gradient at ph 6.0. The purified enzyme had an RZ (A409/A280) value >4. LIP concentration was determined at 409 nm using an extinction coefficient of 169 mm 1 cm Substrate consumption studies All reactions were carried out in 1 ml 50 mm sodium tartrate buffer, ph 3.5 at 25 C. Substrate consumption studies were conducted using 1 lm LIP, 2 mm substrate and 0 4 mm H 2 O 2. To prevent H 2 O 2 -dependent enzyme inactivation, optimal H 2 O 2 concentration was added stepwise in aliquots of 250 lm at 1.5 min intervals in the absence or presence of 0.04% gelatin (Ward et al., 2002), known to suppress product-dependent inactivation (Nakamoto and Machida, 1992). One hour after the reactions were initiated an equal volume of acetonitrile (ACN) was added in order to stop the reactions (Ward et al., 2002). To determine the level of oxidation, the remaining substrate was determined by reverse phase high performance liquid chromatography (RP-HPLC). HPLC analyses were conducted in a Hewlet Packard HPLC (HP 1100, Waldbronn, Germany) provided with a diode array detector. A Lichrospher 100 RP-18 column (25 cm 5 mm i.d., 5 lm; Merck, Darmstadt, Germany) was employed. All solvents were of far UV quality HPLC grade purity where available. Elution was performed using a gradient system modified from a previously described method (Waldron et al., 1996). The gradient profile consisted of a solvent A (10% v/v aqueous ACN and 0.1% formic acid) and solvent B (40% v/v aqueous methanol, 40% v/v aqueous ACN and 0.1% formic acid) in the following program: initially, 100% A; linear gradient over 15 min to 100% B; held isocratically at 100% B for a further 5 min, and then linear gradient over 7 min to 100% A. The flow rate was maintained at 1 ml/min. Peak detection was at 210, 280, 400 and 500 nm. Catechols were quantified by integration of peak-areas at 280 nm, with reference to calibration, which were made using known amounts of catechol and derivatives LIP follow-up during substrate consumption reactions Follow up of LIP was done by measuring residual activity and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis Residual activity Residual LIP activity was tested on 100 ll samples taken from reactions that were incubated for 5 min at 25 C. Gelatin, when added was at a concentration of 0.04% (Ward et al., 2002). Residual LIP activity was assayed by monitoring the oxidation of veratryl alcohol (VA) at 310 nm in 1 ml mixtures consisting of 2 mm VA, 0.4 mm H 2 O 2 in 50 mm sodium tartrate buffer, ph 2.5 (Tien and Kirk, 1988). A 500 ll aliquot was also withdrawn from reactions and immediately added to an equal volume of ACN to quench the reaction. Remaining substrate was then determined by RP- HPLC SDS PAGE One and twenty four hours catechols reaction mixtures (1.5 ml) were centrifuged for 5 min at 7000 rpm. Supernatants and sediments were separated, frozen at 80 C, freeze-dried and evaporated to dryness. The dried samples were redissolved in double distillated water to reach 4 lg protein, and loaded. SDS PAGE was performed in gels containing 12% acrylamide according to Laemmli (1970), using Bio-Rad Mini Protean II gel electrophoresis system. Pre-stained protein marker (BioLabs Ò, Petach-Tikva, Israel) was used as molecular weight marker. LIP was stained with Coomassie brilliant blue (Sigma-Aldrich, Rehovot, Israel) Analysis of polymerization products from catechol derivative Reactions containing 6 mm catecholic substrates, 1 lm LIP and 4mMH 2 O 2 (added in aliquots of 250 lm at 1.5 min intervals) were carried out for gel-permeation and mass spectrometry (MS) (3 ml) and fourier transform infrared (FTIR) analyses (25 ml). The reactions mixtures were incubated for 24 h and then harvested as detailed below Molecular weights determination by gel-permeation chromatography Reaction mixtures were centrifuged for 10 min at 12,000 rpm. Supernatants and sediments were separated, frozen at 80 C, freeze-dried and evaporated to dryness. The dried fractions were 3-fold concentrated in tetrahydrofuran (THF), filtered through 0.45 lm nylon filter and injected to gel-permeation column.

3 S. Cohen et al. / Bioresource Technology 100 (2009) Gel-permeation chromatography was performed using a Waters Styragel Ò HR1 (7.8 mm 300 mm i.d., 5 lm; Waters, Milford, USA). TSK polystyrene standards with molecular weights of 300, 500, 1000, 2500 and 5000 were employed (TOSOH Corporation, Tokyo, Japan). Elution was performed using tetrahydrofuran as the mobile phase at a flow rate of 1 ml/min and monitoring was performed at 254 nm (Ward et al., 2003b) Molecular weight determination of major products by MS MS analysis Total reaction mixtures were dried as previously described. Then they were 3-fold concentrated in 50% methanol 50% ACN (v/v), filtered and injected to semi-preparative column for fractionation. Oxidation products were fractionated using a semi-preparative reverse phase Lichrospher 100 reverse phase-18 column (25 cm 10 mm i.d., 10 lm; Lichrocart, Darmstadt, Germany) employing the previously described gradient system. A flow rate of 6 ml/min ensured an elution profile similar to that of the analytical column. Oxidation products were collected and directly injected to the MS. MS analysis was conducted on Quatro Ultima Micromass (Waters Corporation, Manchester, UK). Analysis was performed by using negative ion at a capillary energy of 2.5 kv and cone energy of 80 V in MS scan mode. The source temperature was 150 C and desolvation temperature was 350 C. Nitrogen was used as the desolvation gas at 500 L/h. Micromass Masslynx version 3.5 was used for data analysis Chemical structure characterization by FTIR analysis FTIR analysis was conducted for two oxidation products obtained from chlorocatechols sediments. The dry sediments were extracted with two volumes of dichloromethane. Each extract was then filtered and analyzed by HPLC to verify the presence of only one product. Afterward the solvent was evaporated and the dry products were kept for 24 h in desiccator before analysis. IR spectrophotometry was performed on a Nicolet Magna-IR model 550 spectrophotometer (Madison, WI, USA). The measurements were obtained in cm 1 range for samples in KBr (IR grade, Merck, Germany) pellets. About 2 mg of each dried chlorocatechol monomers and their purified products were separately thoroughly mixed with 98 mg of KBr and pressed into a pellet form, and the spectrum was then recorded. Result analyses were done with the program Omnic (Thermo-Nicolet). Fig. 1. Effect of H 2 O 2 concentration on the removal of catecholic substrates catalyzed by LIP. Reaction mixtures contained 1 lm LIP and 2 mm CAT (j), 4-CC (N), 4,5-DCC (d) or 4-MC () in 50 mm tartrate buffer ph 3.5, and varying concentrations of H 2 O 2. Reactions were stopped after 1 h by addition of an equal volume of acetonitrile and the remaining substrate was measured by HPLC with monitoring at 280 nm. Results are presented as the average of three replicates (bars indicate standard deviation). 55% removal of CAT and 3 mm for 90% removal of 4-CC (Fig. 1). Further increase in H 2 O 2 resulted in progressive decrease in CAT, 4-MC and 4-CC overall oxidation. Removal of 4,5-DCC remained constant from 1 to 3 mm H 2 O 2 and then begun decreasing as well. This trend typifies H 2 O 2 -dependent inactivation (Chung and Aust, 1995a). The overall oxidation of all catecholic substrates, which was maximal after 1 h incubation, remained constant even after 24 h incubation. Significant removal of about 50% of all catecholic substrates was already observed after 2.5 min (Fig. 2). Maximum removal of CAT (67%) and 4-MC (57%) was achieved 5 min after reactions had begun and was about 10% higher than after 1 h. The decrease in CAT and 4-MC removal probably resulted from reduction of the oxidized monomer back to its native form. In contrast, chlorinated catecholic substrates removal reached an asymptotic value after 1 h Elemental analysis Sediments obtained from 25 ml catechol and 4,5-dichlorocatechol oxidation reactions (reactant concentration as mentioned in Section 2.3.) after 24 h were frozen at 80 C, freeze-dried and evaporated to dryness. The dried sediments were weighted and analyzed for their C, H, N content (%w/w) by Elemental analyzer EA-1108 (Fisons, Italy). Yield balance was performed according to initial enzyme and substrate amounts, substrate removal, typical protein composition based on empirical protein formula of C 4 H 6 O 1.2 N(Bailey and Ollis, 1997) and to elemental analysis data. 3. Results 3.1. Effect of H 2 O 2 concentrations on catechol derivative removal by LIP Optimal H 2 O 2 concentrations for maximum removal of 2 mm catecholic substrates by 1 lm LIP after 1 h were: 1 mm for 95% and 43% removal of 4,5-DCC and 4-MC, respectively, 2 mm for Fig. 2. Time course of catechol derivative removal catalyzed by LIP. Reaction mixtures contained 1 lm LIP and 2 mm of CAT (j), 4-CC (N), 4,5-DCC (d) or 4-MC () in 50 mm tartrate buffer, ph 3.5. Hydrogen peroxide concentration was: 3 mm for 4-CC, 1 mm for 4,5-DCC and 4-MC, and 2 mm for CAT. Reactions were stopped at the designated times by addition of an equal volume of acetonitrile and remaining substrate was measured by HPLC with monitoring at 280 nm. Results are presented as the average of three replicates (bars indicate standard deviation).

4 2250 S. Cohen et al. / Bioresource Technology 100 (2009) Table 1 Nitrogen, carbon and hydrogen contents in sediments obtained from CAT and 4,5-DCC reactions. Compound Sediment weight (mg) 3.2. Effect of gelatin and H 2 O 2 addition on CAT and 4-MC removal by LIP The low removal of both CAT and 4-MC by LIP was thought to be caused by H 2 O 2 -dependent (Chung and Aust, 1995a), or productdependent inactivation of LIP (Ward et al., 2001a, 2002). To overcome this and improve CAT and 4-MC removal, optimal H 2 O 2 concentrations were added in a stepwise manner to prevent H 2 O 2 -dependent-inactivation of LIP (Ward et al., 2001a), in the presence of 0.04% gelatin which can suppress product-dependent inactivation by preventing hydrogen bonding between the hydroxyl groups of the phenolic products and the enzyme (Nakamoto and Machida, 1992). Indeed, stepwise addition of H 2 O 2 improved the removal of CAT from 55% to 75% and 4-MC from 44% to 55%, relative to that obtained by single-step addition. On the other hand, gelatin did not significantly improve the removal of either substrates, regardless of how H 2 O 2 was added. Moreover, others additives such as polyethylene glycol (PEG) or Tween 20, which are also known to suppress product-dependent inactivation (Wu et al., 1997), did not improve CAT or 4-MC removal by LIP (data not shown) Characterization of the different reactions N (% w/w) C (% w/w) H (% w/w) Catechol ± ± ± 0.2 4,5-Dichlorocatechol ± ± ± 0.1 Results are presented as the average of three replicates. Except for 4-MC, the different mixtures became turbid during oxidation at different intervals of time and consequently, sediments were formed. The reaction mixture of 4,5-DCC became turbid 3 min after the reaction was initiated, the CAT reaction mixture after 30 min, and the 4-CC reaction mixture after 1 h. The quantity of sediment obtained correlated with the time that turbidity was initiated. Highest quantity of sediments which were obtained from CAT and 4,5-DCC oxidation reactions were analyzed for their C, H and N contents by elemental analyzer. The measured N content indicated the presence of protein in the sediments (Table 1). Yield balance calculations revealed that both sediments were composed of LIP (20%) and oxidation products (80%). Whereas in 4,5-DCC reaction mixture, all of the LIP and 80% of the oxidation products precipitated, in the CAT reaction mixture, only 80% of LIP and 47% of all products precipitated. Inclusion of LIP in CAT and chlorinated-catechols precipitates was confirmed by SDS PAGE. Supernatants and sediments were examined and compared to a mixture containing LIP and CAT in tartrate buffer without the addition of H 2 O 2. After 1 h incubation, LIP was only detected in the sediment of 4,5-DCC reaction mixture while in CAT and 4-CC reactions, part of the enzyme was present in the supernatant and part of it in the sediment (Fig. 3A). Besides 4-MC after 24 h, LIP was only detected in the sediments of all three reactions (Fig. 3B). The degree of LIP precipitation obtained by SDS PAGE analysis correlated with the elemental analysis data and with the time course of turbidity development in each reaction Effect of oxidation products on LIP stability and activity The formation of water-insoluble oxidation products was thought to cause early precipitation of LIP, probably by absorption or binding of those products to the enzyme. To confirm this assumption, the influence of gelatin on LIP precipitation was tested. Inclusion of gelatin in the CAT and 4-CC reaction mixtures totally inhibited sediment formation by preventing turbidity development and LIP precipitation during the first hour of incubation (Fig. 4). In contrast, gelatin added to the 4,5-DCC reaction mixture did not prevent turbidity, i.e., did not prevent the formation of insoluble products and sediment, nevertheless totally prevented LIP precipitation. The influence of oxidation products on LIP precipitation was also evident in reactions performed with increasing ini- Fig. 3. LIP content in supernatants (sup) and sediments (sed) from catecholic substrates reactions. Catecholic substrates reactions were tested after 1 h (A) and 24 h (B). Protein analysis was performed by SDS PAGE. The fluids were separated from the precipitates, lyophilized and solubilized in 400 ll water; 35 ll (4 lg protein) were loaded onto the gel. Results represent one of two replicates.

5 S. Cohen et al. / Bioresource Technology 100 (2009) Products analysis Fig. 4. LIP contents in sediment of 4,5-DCC and supernatant of 4,5-DCC, 4-CC and CAT reactions containing 0.04% gelatin. Catecholic substrates reactions were tested after 1 h. Protein was analyzed by SDS PAGE. The fluids were separated from the precipitates, lyophilized and solubilized in 400 ll water; 35 ll (4 lg protein) were loaded onto the gel. Results represent one of two replicates. tial substrate concentrations (data not shown). In these reactions, more LIP was precipitated, probably due to the adsorption of the enzyme to a higher quantity of oxidation products formed. To check if the low removal of CAT and 4-MC was connected to enzyme inactivation independently to inhibition of precipitation, residual LIP activity was measured in all reaction mixtures. A complete loss of activity was observed within the first 5 min (long before turbidity development) in CAT, 4-CC and 4-MC reactions, even when gelatin was added, and regardless of the manner in which H 2 O 2 was added suggesting enzyme inactivation by water-soluble oxidation products. Although stepwise addition of H 2 O 2 did not prevent loss of LIP activity it continued to improve substrate removal with each addition long after LIP s inactivation (data not shown). In contrast, the 65% decrease in LIP activity after 5 min of 4,5-DCC reaction (with turbidity development) was fully prevented by the addition of gelatin (data not shown) indicating enzyme inactivation by insoluble products. These assumptions were confirmed by testing aliquots from 24 h supernatants (containing only soluble products) of CAT, 4-CC and 4-MC: these aliquots harmed LIP s ability to oxidize veratryl alcohol, while addition of aliquots from the 4,5-DCC supernatant did not (Fig. 5). It should be noticed that even though LIP was inactivated as soon as the reactions began, catecholic substrate removal continued for a long time afterwards (Fig. 2). To characterize the oxidation products, the reaction of LIP with each catecholic substrate was carried out on a large scale. The overall oxidation of all catechols, resulted in oligomeric product formation, as revealed by gel-permeation analysis. In the 4-MC reaction mixture, which contained only soluble products, peaks corresponding to monomer (123 Da), trimer which was probably demethylated (324 Da) and tetramer (530 Da) were noticed. In the CAT reaction, peaks corresponding to monomer (110 Da), dimer (220 Da) and pentamer (550 Da) were detected in the supernatant, while peaks corresponding to tetramer (440 Da) and decamer (1440 Da) were noted in the sediment. In 4-CC, supernatant analysis revealed molecular weights corresponding to monomer (144 Da), dimer which was probably dechlorinated (250 Da) and tetramer (580 Da). In the sediment, peaks corresponding to trimer, probably with additional chlorine atoms (470 Da), and heptamer (1000 Da), were noticed. Another peak with a lowmolecular-weight (130 Da) was also predominant in the sediment. Although located near the monomer, its spectral characteristics were completely different (not shown). This phenomenon repeated itself in the 4,5-DCC sediment analysis, in which a major peak (160 Da) was detected near the monomer (179 Da). These products, exhibiting lower molecular weights than their monomers, were probably high-molecular-weight oligomers that interacted with the column: as a consequence they eluted later and were erroneously detected as low-molecular-weight products. Analysis of the 4,5-DCC supernatant revealed molecular weights corresponding to dimer (300 Da) and tetramer (600 Da) which seemed to be dechlorinated, and dimer (400 Da) which probably had an additional chlorine atom. No correspondence was found between the removal and degree of polymerization. High removal of 4,5-DCC and low removal of 4- MC resulted in similar degrees of polymerization. MS analysis of the major oxidation products indicated the formation of dimers and tetramers in the CAT reactions, dimers and tetramers in the 4-MC reactions, dimers, trimers and tetramers in the 4-CC reactions and dimers, trimers and tetramers in the 4,5-DCC reactions. High-molecular-weights detected in the gelpermeation analysis were not observed in the MS, probably because different solvents were used in each analysis for product extraction, or because of high-mass breakdown in the MS. Analysis of two major products obtained from 4-CC and 4,5-DCC overall oxidation in the MS, suggested trimers formation. IR spectra of the two major products in comparison to those of their monomers were analyzed. In both cases, the intensity of the absorption in the range of cm 1, ascribed to C C and C H bonds of an aromatic ring, and the intensity of the absorption at 1500 cm 1 and 3200 cm 1, ascribed to C@C and OH bonds of an aromatic ring, respectively, in the substrate spectra, significantly decreased in the product spectra. The detection of two new peaks in the corresponding products (1385 cm 1, 1580 cm 1 ), by using Omnic (Thermo-Nicolet) program, indicated the formation of ether (C O C) linkages. The intense absorption at 1650 cm 1 in both products suggested the formation of a benzoquinone structure. 4. Discussion Fig. 5. Effect of soluble oxidation products on VA oxidation by LIP. Supernatants (100 ll) obtained from CAT (j), 4-CC (N), 4,5-DCC (s) and 4-MC () of24hreactions mixtures, were added to 1 ml oxidation reaction mixture containing 1 lm LIP, 2 mm VA, 50 mm tartrate buffer, ph 2.5 and 0.5 mm H 2 O 2, about 20 s after onset of the reaction. Control consisted of VA oxidation reaction supplemented with 100 ll water (}). VA oxidation rate was monitored at 310 nm. LIP was shown to differently catalyze the removal of four catecholic substrates according to the nature of the substitutes. The maximum overall oxidation for each substrate after 1 h reaction was achieved as function of H 2 O 2 concentrations in the following order: 4,5-DCC > 4-CC > CAT > 4-MC. The presence of chlorine, an electron-withdrawing group, on the catechol molecule increased its removal while the presence of methyl, an electron-donating

6 2252 S. Cohen et al. / Bioresource Technology 100 (2009) group, decreased it. Opposite effects of methyl on the CAT removal by laccase (Gianfreda et al., 2003) have been reported. In this case, 4-MC removal after 24 h was higher (100%) than that of CAT (75%). It has been suggested that the molecular structure of the substrate influences its redox potential, thus determining its higher or lower affinity for oxidization by the enzyme (Gianfreda et al., 2003). Ward et al. (2003a) showed that initial enzymatic oxidation of substituted phenols by LIP is in inverse correlation to substrate oxidation potential (OP), which is known to increase in the presence of an electron-withdrawing group, such as halogens. However, when considering the overall oxidation (i.e, enzymatic and radical reaction and precipitation) of each catecholic substrate, the ease of oxidation is inversely correlated to their pka values (pka 4-MC >pka CAT >pka 4-CC >pka 4,5-DCC ) but is not inversely correlated to their OP values. In other words, low pka of substrate with an electron-withdrawing group causes an increase in the ionized form of the catecholic monomer, which, probably, further promotes its removal. Sediments formed during catechol and chlorinated-catechols removal contained not only polymerization products but also enzyme (80 100% of the initial enzyme concentration), as was revealed by elemental and SDS PAGE analyses. These findings demonstrate, for the first time, the ability of peroxidases to undergo incorporation into polymerization products formed during enzymatic removal of phenolic derivatives. The ability of gelatin to suppress LIP precipitation in CAT and chlorinated-catechols reaction mixtures, probably by inhibiting the formation of hydrogen bonds between polymerization products and the enzyme, indicated that the insoluble products formed in these reactions were responsible for LIP precipitation. Nevertheless, the nature of the bond between the enzyme and the products is not yet clear. LIP precipitation caused by insoluble products was not the only reason for LIP s inactivation since no insoluble products were formed in 4-MC reaction and, although in CAT reaction gelatin was able to inhibit LIP precipitation, the removal of CAT was still low. Residual activity results clearly indicated that while in the 4,5-DCC reaction, insoluble products caused LIP inactivation, in the CAT, 4-CC and 4-MC reactions, soluble products were responsible for its inactivation. It could be concluded that two modes of LIP inactivation probably occur during the removal of the different catecholic substrates. LIP inactivation by phenol oxidation products has been reported for 4-bromophenol oxidation (Ward et al., 2002) and for ferulic acid (Ward et al., 2001b). In those reactions, inclusion of gelatin not only minimized LIP s loss activity but also substantially improved substrate consumption. In our work, although addition of gelatin to the reaction mixtures of CAT, 4-CC and 4-MC protected LIP from precipitation, it did not improve substrate removal. Inclusion of other additives, such as polyethylene glycol and Tween 20, which have been reported as well to reduce product inactivation of peroxidases (Wu et al., 1997) during phenol oxidation, neither improved catechol removal by LIP nor protected its activity. Reduction of 25 and 80% in enzyme activity during CAT and 4- MC removal, respectively, have been reported for laccase (Gianfreda et al., 2003). It was suggested that the decrease in laccase activity resulted from its absorption or binding to soluble products (insoluble products have not been formed). Hydrogen peroxide-dependent inactivation did not seem to be the reason for LIP inactivation during catechol derivative removal, since stepwise addition of H 2 O 2, known to reduce H 2 O 2 -dependent inactivation (Conesa et al., 2002), did not suppress loss of LIP activity in CAT, 4-CC and 4-MC reactions. Furthermore, veratryl alcohol, which is known to protect LIP from H 2 O 2 -dependent inactivation due to its ability to revert Cpd III back to the native enzyme (Ward et al., 2001b, 2002), was not able to prevent LIP inactivation, even though it was added at a higher concentration than CAT, 4-CC or 4- MC. Taken together, these findings suggest that the pattern of LIP inactivation by catecholic substrates is different from that for phenols. Although, LIP was inactivated at the beginning of the CAT, 4-CC and 4-MC reactions, their removal continued, independent of LIP. This was confirmed by the fact that addition of acetonitrile, for enzyme inactivation (Ward et al., 2002), to a 10 s 4,5-DCC reaction did not prevent the continuation of substrate removal or product formation (not shown). An additional evidence is the stepwise addition of H 2 O 2 improved CAT and 4-MC removal long after LIP lost its activity. In this context, it should be noted that H 2 O 2,by it self, was not able to remove the tested catechols (data not shown). These findings confirm that LIP is needed only to initiate the oxidative reaction: its continuation depends on radical reactions that are responsible for removal of the remaining catecholic substrates and in consequence polymerization products are formed. This assumption would explain the absence of typical Michaelis Menten behavior during catechol derivative oxidation by LIP (data not shown). The absence of typical Michaelis Menten behavior during catechol oxidation was also reported for laccase (Gianfreda et al., 2003). The differences in the extents of catecholic substrates consumed probably resulted from the different amounts of radicals formed before LIP inactivation. It could be concluded that while addition of chlorine to the catechol molecule facilitates the radical reactions, thus improving substrate removal addition of methyl to the catechol molecule decreases substrate removal. Catecholic reaction mixtures, which were colorless at the beginning, achieved different colors soon after removal begun. Mixture colors changed to light yellow for 4-MC, orange for 4,5- DCC, reddish-brown for 4-CC and to dark brown for CAT, at 24 h incubation. The change in solution colors was in correlation not only with the formation of oxidation products but also with the degree of polymerization. Dark color, as in CAT and 4-CC reaction mixtures, correlated with high degree of polymerization and bright color, as in 4-MC and 4,5-DCC, with lower degree of polymerization. The degree of polymerization of the different substrates seems to be related to their solubility in water. It is reasonable to assume that in CAT and 4-CC reactions, oligomers with high-molecular-weights (1000 Da) were obtained due to the formation of only soluble products at the beginning of the reaction, enabling oligomer lengthening in the soluble phase. In contrast, in the 4,5-DCC reaction, insoluble products were formed as soon as the reaction began (as reflected by the earlier turbidity), preventing lengthening of the catecholic chain. Consequently, lowmolecular-weight oligomers were formed. However, product polarity does not seem to be the cause for the low degree of polymerization of 4-MC, since its overall oxidation reaction was characterized by only soluble products. In this case, the lowmolecular-weight products obtained could be explained by the low removal of 4-MC. Formation of catecholic oligomers has also been reported for CAT and 3-chlorocatechol (3-CC) by HRP (Ward et al., 2004). The reactions with HRP were conducted at ph 7 and only soluble products were formed; nevertheless, the degree of CAT polymerization was similar to its polymerization by LIP. For 3-CC, oligomers with low-molecular-weight (up to 500 Da) were obtained. Benzoquinone structure and ether linkages observed in catechol oxidation products obtained in this work have also been reported for polyguaiacol synthesized by MnP (Iwahara et al., 2000) and for polycatechol synthesized by laccase (Aktas et al., 2003) and SBP (Dubey et al., 1998). To conclude, the removal mechanism of catechol derivatives initiated by LIP, although not yet fully understood, seems to be dif-

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