Bioresource Technology 102 (2011) 3911–3917 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech On hexenuronic acid (HexA) removal and mediator coupling to pulp fiber in the laccase/mediator treatment Edith M. Cadena a, Xueyu Du b, Göran Gellerstedt b, Jiebing Li b,⇑, Amanda Fillat a, Jordi García-Ubasart a, Teresa Vidal a, Josep F. Colom a a b Textile and Paper Engineering Department, Universitat Politècnica de Catalunya, ETSEIAT, Colom 11, E-08222 Terrassa, Spain Department of Fibre and Polymer Technology, Royal Institute of Technology, KTH, Teknikringen 56-58, SE-10044 Stockholm, Sweden a r t i c l e i n f o Article history: Received 4 October 2010 Received in revised form 28 November 2010 Accepted 30 November 2010 Available online 5 December 2010 Keywords: Biobleaching Coupling Flax Functionalization Hexenuronic acid a b s t r a c t Flax soda/AQ pulps were treated with different fungal laccase–mediator combinations followed by physical and chemical characterization of the pulps to obtain a thorough understanding of the laccase/mediator effects on hexenuronic acid (HexA) removal and the coupling of mediator onto pulps for fiber functionalization. Large differences were found and the presence of lauryl gallate (LG) during Trametes villosa laccase (TvL) treatment (TvL + LG) resulted in a much larger reduction of pulp-linked HexA than the combination of p-coumaric acid (PCA) and Pycnoporus cinnabarinus laccase (PcL). A major portion of LG became attached to the pulp as revealed by an increase in the kappa number and further confirmed by thioacidolysis and 1H NMR analysis of solubilized pulp fractions. Additional experiments with other chemical pulps and isolated pulp xylan and lignin revealed that HexA seems to be the sole pulp component attacked by TvL + LG. As a substrate for TvL, the reaction preference order is PCA > HexA > LG. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Laccases (EC 1.10.3.2) are copper-containing oxidase enzymes found in many plants, fungi, and microorganisms. Together with oxygen, they play an important role in nature in the formation and decomposition of lignin through the generation of phenoxy radicals formed by the abstraction of a hydrogen atom followed by radical polymerization or depolymerization, respectively (Yaropolov et al., 1994). The initiation of lignin oxidation could be the result of a direct attack of lignin by laccase or of an indirect attack with the assistance of redox mediators that are laccase substrates. After laccase oxidation, the oxidized form of the mediator attacks and oxidizes lignin while the mediator returns to its reduced original form. The combination of a fungal laccase and a redox mediator is termed a laccase/mediator-system. Generally, enzymes, including laccases, have high application potential in the modern chemical industry by virtue of their high specificity and environmental friendliness. Within the pulp industry, a major step was taken when it was shown that laccase in the presence of a low molecular mass mediator and oxygen was able to significantly delignify kraft pulp under mild conditions (Bourbonnais and Paice, 1992). Thereafter, many studies using laccase–mediator treatment were conducted to improve and further develop traditional ⇑ Corresponding author. Tel.: +46 8 790 6165; fax: +46 8 790 6166. E-mail address: jbing@kth.se (J. Li). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.127 pulp manufacturing processes. The major interest has focused on the development of stable, recyclable and enviromentally friendly mediators to substitute the currently used chemicals, e.g., chlorinated reagents in pulp bleaching (Bourbonnais et al., 2001). Because the laccase oxidation mechanism involves an initial oxidation of phenolic structures, different phenols, both synthetic and natural, have been used as a mediator together with different types of laccases for pulp delignification (Camarero et al., 2007; Aracri et al., 2009; Fillat et al., 2010). Positive effects were found, such as reduction in kappa number, improvement in brightness, and structural modification of the residual fiber lignin. In addition, the laccase–mediator treatment was shown to positively affect the removal of extractives in pulps (Gutiérrez et al., 2006; Valls et al., 2009). It has been reported that the application of laccase/mediator resulted in a decreased content of hexenuronic acid (4-deoxy-Lthreo-hex-4-enopyranosyluronic acid, HexA) in kraft pulps (Cadena et al., 2010; Valls et al., 2010), which is beneficial since HexA is a problematic component in kraft pulp and in the pulping process. In addition to lignin, it contributes to the kappa number, and this contribution can be substantial for certain types of pulps (Li and Gellerstedt, 1997). The presence of HexA also results in an increase in the consumption of bleaching chemicals such as chlorine dioxide, ozone or peracetic acid, thus increasing the costs of bleaching (Forsström et al., 2005). Because HexA is reactive against bleaching agents, chloro-organic products result from chlorine dioxide-based sequences, thus contributing to the load of AOX in the bleaching effluent. The presence of HexA in the final bleached pulp may also 3912 E.M. Cadena et al. / Bioresource Technology 102 (2011) 3911–3917 cause quality problems such as brightness reversion and increased retention of metal ions (Sevastyanova et al., 2006). The possibility of generating phenoxy radicals in the pulp fiber lignin by a laccase–mediator treatment has also opened new fields of application, i.e., modification of the fiber by addition of functional structures through radical coupling reactions (Chandra and Ragauskas, 2002; Aracri et al., 2009). Thereby, novel properties such as hydrophobicity, antimicrobial or antioxidant effects can be obtained. Direct coupling of the mediator itself to the pulp resulting in increased kappa number, higher klason lignin content and increased surface charge has been reported (Aracri et al. 2009, 2010). Flax is an economically important fiber source that is predominantly used in the textile industry and in the production of strong specialty papers such as bank notes. In the latter application, the material is pulped in alkali (soda–anthraquinone, AQ) to give fibers that are low in residual lignin content with cellulose and a minor amount of xylan as dominating carbohydrate constituents (Karakus et al., 1999). Application of laccase–mediator delignification on such pulps, therefore, seems attractive because expensive bleaching stages involving chlorine dioxide and/or hydrogen peroxide could be substituted with an environmentally benign enzyme stage under mild conditions. In the present work, unbleached as well as TCF-bleached soda– AQ pulp from flax was treated with different fungal laccases in combination with two mediators, p-coumaric acid (PCA) and lauryl gallate (LG), followed by physical and chemical characterization of the fibers. Reference and model experiments with several other chemical pulps and with isolated pulp xylan and flax lignin respectively were also conducted to further elucidate the interactions between pulp fibers and the laccase/mediator system in terms of HexA removal and mediator coupling onto pulp fibers. The hydrophobic mediator LG was chosen based on previous work where it was shown that a high level of internal sizing of paper could be achieved using this compound (Garcia-Ubasart et al., 2010). Ref. NS-51002) and INRA (Marseille, France), respectively. Laccase activity was defined as the amount of enzyme needed to convert 1 lmol/min of 2,20 -azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) to its cation radical. The oxidation of ABTS in 0.1 M sodium acetate buffer (pH 5) at 25 °C was followed by the absorbance increase at 436 nm (e436 = 29,300 M 1 cm 1) on a UV–vis Shimadzu 1603 spectrophotometer. 2.3. Mediators The mediators used were lauryl gallate (LG) and p-coumaric acid (PCA) from Sigma–Aldrich. 2.4. Laccase–mediator treatment Treatment of soda–AQ pulps with laccase–mediator was done with two different enzymatic systems. The TvL–LG combination aimed at obtaining internal sizing of paper and was applied as described elsewhere (Garcia-Ubasart et al., 2010); 40 U of TvL/g of oven dry pulp (odp) together with 4.0% of LG in an EasydyeÒ reactor without oxygen pressure (Ahiba Easydye from Datacolor International) for 1 h. The PcL–PCA treatment was performed with 40 U of PcL/g odp together with 3.5% PCA in a pressurized reactor at 590 kPa oxygen pressure for 4 h according to Fillat et al. (2010). All treatments were done at 4–5% pulp consistency, 50 °C, and in 50 mM sodium tartrate buffer at pH 4. Control experiments were done with addition of either TvL or PcL but without mediator present. After treatment, the pulp was thoroughly washed with deionized water. In model experiments, xylan and lignin were treated with 40 U/ g of laccase (TvL) together with 4.0% of LG or PCA in 50 mM tartrate buffer at pH 4. After 4 h of treatment at 50 °C the sample was filtered and washed thoroughly before drying under vacuum at room temperature. 2.5. Acetone extraction 2. Methods 2.1. Samples Unbleached and TCF-bleached pulp from soda–AQ pulping of flax (Linum usitatissimun) were supplied by CELESA pulp mill (Tortosa, Spain). Before laccase–mediator treatment, the unbleached pulp was subjected to a weak acid washing at room temperature for a complete removal of all residual alkali as well as metal ions. The kappa number and brightness of the unbleached (acid washed) and TCF-bleached pulps were 9.2 and 38.8%ISO and 1.8 and 77.0%ISO, respectively. For reference purposes, two further pulps were used: TCFbleached dissolving pulp from spruce with 5.5% hemicelluloses (obtained from Domsjö Sulfite Mill, Sweden) but without any lignin, hexenuronic acid (HexA), or extractives (after extraction with acetone) (Sevastyanova et al., 2006) and TCF-bleached eucalypt ( Eucalypus grandis) pulp without any lignin but with 68 lmol/g of HexA obtained from a Brazilian pulp mill (Sevastyanova et al., 2005). In model studies, pulp xylan, isolated from the TCF-bleached eucalypt pulp by extraction with DMSO (Sjöström and Enström, 1967), and flax pulp lignin (a gift from Granit AS, Lausanne, Switzerland) were used. 2.2. Laccases Laccases (EC 1.10.3.2) from Trametes villosa (TvL) and Pycnoporus cinnabarinus (PcL) were supplied by Novozymes (Denmark, Before and after laccase–mediator treatment, the pulps were extracted with acetone in a Soxhlet apparatus for 24 h. All acetone filtrates were evaporated and dried under vacuum at 40 °C to obtain the content of extractives. The extracted pulps were further used or analyzed as extractives-free pulps. 2.6. Xylanase treatment Xylanase hydrolysis was conducted on flax soda–AQ pulp samples using 36 U/g odp of Pulpzyme HC (commercial xylanase from Novo Nordisk) at 10% consistency, sodium phosphate buffer at pH 7 and 60 °C for 3 h. The spent filtrate was collected for xylose content determination as described below for carbohydrate composition quantification, and the resulting pulp was thoroughly washed with deionized water before subsequent analysis. 2.7. Lignin–carbohydrate complex (LCC) fractionation LCC fractionation was performed following the procedure depicted in Fig. 1 (Li et al., 2009, 2011). After ball milling for 12 h, the pulp sample was dispersed in DMSO (470 mg/5 mL) under stirring followed by addition of 5 mL/470 mg of 40% aqueous tetrabutylammonium hydroxide (TBAH) (from Fluka International). From the resulting clear solution, glucan–lignin and xylan–lignin fractions were obtained after drop-wise addition of water, which resulted in the precipitation of the glucan–lignin fraction, whereas the xylan–lignin fraction remained in solution. After centrifugation, the glucan–lignin fraction was collected and further purified by washing with water until neutral followed by freeze drying. 3913 E.M. Cadena et al. / Bioresource Technology 102 (2011) 3911–3917 was silylated for 30 min at room temperature with 100 lL of pyridine and N, O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) (containing 1% trimethylchlorosilane (TMCS) (1:1) before analysis by gas chromatography (GC) to quantify the monomeric degradation products, i.e., the uncondensed syringyl (S) and guaiacyl (G) units present in the sample. 3. Results and discussion Fig. 1. Scheme for the fractionation of lignin–carbohydrate complexes from flax soda–AQ pulps after a complete pulp dissolution using DMSO and tetrabutylammonium hydroxide (TBAH). The remaining solution containing the xylan–lignin fraction was dialyzed (Mw cut-off = 1000 Da) against water and isolated by freeze drying. 2.8. Analyses For all pulp samples, kappa number (ISO 302), brightness (ISO 2470) and content of HexA (Gellerstedt and Li, 1996) was determined. On the initial unbleached flax soda–AQ pulp, the klason lignin, carbohydrate composition and ash content were determined according to TAPPI Standards T 222 om-06, T 249 cm-00, and T 211 om-02, respectively. The contribution to kappa number by the mediators was determined by dissolution of LG or PCA in a small amount of acetone before measuring the kappa number according to the standard procedure as described by Li and Gellerstedt (1998). The extractives obtained after acetone evaporation were silylated and analyzed by gas chromatography in combination with mass spectroscopy (GC–MS). GC analysis was carried out using a DB5MS column with helium as carrier gas. The initial temperature was 150 °C for 2 min, and it was then increased by 5 °C/min to 170 °C, followed by an increase by 25 °C/min to 330 °C. Injector temperature was 230 °C, and mass spectra were obtained at 70 eV. The xylan–lignin LCC was dissolved in 0.1 M sodium hydroxide and analyzed by size exclusion chromatography (SEC) using three TSK gel columns (3000PW, 4000PW, 3000PW) coupled in series with 0.01 M sodium hydroxide as the eluent. The flow rate was 1 mL/min, and detection was done by UV using a PDA detector. 1 H NMR spectra of the xylan–lignin LCC and the isolated xylan before and after laccase–mediator treatment were recorded after dissolution in 10% NaOD in D2O on a Bruker Avance 400 MHz instrument using the standard Bruker pulse program at room temperature. 13 C–1H 2-dimensional heteronuclear single quantum coherence (2D HSQC) NMR spectra of the flax lignin dissolved in acetone-d6D2O (5:1) before and after laccase–mediator (TvL + LG) treatment were recorded using standard Bruker pulse program (Zhang and Gellerstedt, 2007). The thioacidolysis reaction was carried out according to Rolando et al. (1992). The thioacidolysis product mixture (about 6 mg) In order to better understand and utilize chemical pulps from flax (Linum usitatissimun) and evaluate the effects of the laccase/ mediator system on the degree of delignification and other possible fiber changes, especially of HexA-removal and fiber functionalization, industrial unbleached (Table 1) and TCF-bleached flax soda–AQ pulp was treated with the Trametes villosa laccase in the presence of lauryl gallate (TvL + LG) (Garcia-Ubasart et al., 2009). In control experiments, treatment with TvL alone was also carried out. For comparison, a second laccase/mediator system consisting of Pycnoporus cinnabarinus together with p-coumaric acid (PcL + PCA) was also investigated since previous work had shown that this combination could promote the formation of covalent linkages between PCA and flax fibers (Aracri et al., 2010). From the results, shown in Table 2, it is obvious that treatment with the combination of laccase/mediator resulted in a large increase in kappa number irrespective of pulp. Simultaneously, the pulps turned dark, as shown by the large drop in brightness. Contrary to the combination of PcL + PCA, the TvL + LG-system was also found to give a substantial reduction in the content of HexA, amounting to around 80% for both the unbleached and the bleached pulp. For the latter pulp, it can be assumed that the original level of HexA is of the same order of magnitude as for the unbleached pulp because it has been shown before that TCFbleaching utilizing oxygen and hydrogen peroxide does not result in any significant reduction of HexA (Vuorinen et al., 1996; Li and Gellerstedt, 1997). Obviously, a laccase/mediator treatment is capable of efficiently eliminating HexA, although there are large differences between various laccases and mediators. It should also be noted that the Trametes villosa laccase when used alone gave a substantial reduction in the amount of HexA present in the flax pulps (Table 2). In an earlier report, it was found that the combination of Trametes villosa and hydroxybenzotriazole (HBT) only led to a HexA reduction of about 20% on eucalyptus pulp (Valls et al., 2010). It was also shown that the combination of xylanase with the laccase Trametes villosa and HBT only removed HexA to an extent of about 35% (Valls et al., 2010). The high HexA removal efficiency of the TvL + LG-system is stronger than the effect of acidic hydrolysis (A-stage) (Vuorinen et al., 1996; Forsström et al., 2005) and comparable to a hot chlorine dioxide bleaching stage (Forsström et al., 2005). In a Dhot-stage, the removal of HexA is around 80–90%. In the absence of a mediator, the two laccases TvL and PcL were both found to affect the content of HexA in the pulps albeit with largely different efficiencies apparently due to the divergences in the laccase species and experimental conditions applied (Table 2). Since it is generally accepted that laccase itself cannot penetrate deep into the pulp fiber wall, these results indicate that the decrease in HexA concentration observed should be due to a direct oxidative elimination of the easily accessible HexA located on the Table 1 Chemical compositions of initial unbleached flax Soda–AQ pulp. Klason lignin (%) 1.39 Relative carbohydrate composition (%) Ara Xyl Man Gal Glu 0.5 5.0 0.6 0.6 93.3 Ash (%) 0.51 3914 E.M. Cadena et al. / Bioresource Technology 102 (2011) 3911–3917 Table 2 Kappa number, brightness, extractive content and HexA content of flax soda–AQ pulps before and after laccase–mediator treatment (TvL, Trametes villosa; PcL, Pycnoporus cinnabarinus; LG, lauryl gallate; PCA, p-coumaric acid). Sample IniPulp TvL TvL + LG PcL PcL + PCA TCF pulp TCF TvL TCF TvL + LG Descriptions Industrial unbleached flax soda AQ pulp; initial pulp Treated unbleached pulp with TvL without mediator Treated unbleached pulp with TvL with LG mediator Treated unbleached pulp with PcL without mediator Treated unbleached pulp with PcL with PCA mediator Industrial TCF bleached flax soda AQ pulp Treated TCF pulp with TvL without mediator Treated TCF pulp with TvL with LG mediator Kappa number Initial Acetone extracted 9.2 8.3 20.5 8.5 13.4 1.8 2.6 14.4 9.2 8.0 16.5 8.3 12.3 ND 2.6 9.9 Brightness % ISO HexA content (lmol/g) Extractives (%) 38.8 33.6 22.9 44.0 41.7 77.0 78.5 24.8 12.4 3.7 2.2 10.5 9.6 ND 4.5 1.6 0.05 0.34 1.39 0.11 0.62 ND 0.03 1.73 ND, not determined. outer parts of the fiber. The combination of TvL + LG or PcL + PCA, on the other hand, yielded further reduction of the HexA content, showing that the oxidized form of the mediator could penetrate deeper into the fiber wall and attack the HexA located in these parts. The remainder of HexA present in the fiber after the laccase–mediator treatment can therefore be considered as nonaccessible by the mediator, probably due to its location deep inside the fiber wall, which is supported by observations after xylanase treatment of TvL + LG pulps, where it was found that xylanase hydrolysis could remove substantial amounts of xylose while the HexA content was not affected. The large increase in kappa number observed for both the unbleached and bleached pulp after treatment with either TvL + LG or PcL + PCA is indicative of a coupling of the mediator to the pulp. Due to the aromatic/phenolic structure of the two mediators, these should be prone to oxidation by acidic permanganate, thus contributing to the kappa number (Li and Gellerstedt, 1998). It was experimentally shown that a kappa number determination of each mediator alone gave values of 1.25 and 0.81 kappa number units per mg of LG and PCA, respectively. Increased kappa number after laccase-induced coupling of natural phenols onto sisal and flax fibers has been observed before in work on fiber functionalization (Aracri et al., 2009; Fillat et al., 2010). In order to distinguish between physically absorbed and chemically linked mediators, all pulp samples were extracted with acetone after laccase or laccase/mediator treatment (Table 2). In pulps treated in the presence of mediator, increased levels of extractives were found, which were indicative of the presence of physically adsorbed mediator, as shown in Table 2. In all cases, a portion of the mediator could be removed in the acetone extraction, as revealed by GC–MS analysis of the extracts. For analysis of fiberlinked mediator, thioacidolysis was employed as an analytical technique (Rolando et al., 1992). Although normally used as a method for detection of b-O-40 linkages in lignin, the combination of a hard Lewis acid, boron trifluoride, together with a soft nucleophile, ethanethiol, should be an efficient system for cleavage of various ethers and esters. Application of this method to the unbleached pulp treated with TvL + LG and followed by extraction with acetone revealed after silylation of the reaction mixture, a new peak identified as dodecyloxy-trimethyl silane, i.e., the aliphatic portion of the original lauryl gallate (LG). This observation supports the chemical bonding of LG onto the pulps. A coupling reaction between the mediator and pulp should involve at least one of the components cellulose, hemicellulose or lignin. In order to find out the details of this reaction, almost pure cellulose (dissolving pulp) was first treated with TvL + LG. However, after extraction of the pulp with acetone, no significant change of kappa number was noticed (Table 3). Industrial flax lignin was also treated with TvL + LG and, after purification by filtration and washing, analyzed by 2D HSQC NMR. Again, no significant Table 3 Kappa number of dissolving pulp and eucalyptus TCF bleached kraft pulp before and after laccase–mediator treatment (TvL, Trametes villosa; LG, lauryl gallate). Kappa number after TvL + LG treatment followed by acetone extraction Sample Description Initial kappa number Dissolving pulp Eucalyptus TCF bleached kraft pulp HexA- and lignin-free Lignin-free, with 68 lmol/ g HexA 0.3 0.5 6.8 14.0 structural changes could be observed, thus also ruling out lignin as a reaction partner. The remaining reactive component in the flax pulps, HexA, was therefore considered the likely coupling partner to the mediator, which was further confirmed by reaction of a TCF-bleached eucalypt pulp with a high content of HexA with the TvL + LG-system. As shown in Table 3, a pronounced increase in kappa number after acetone extraction of the pulp was encountered. Thus, it can be concluded that HexA seems to be the sole structure that is able to give a coupling product with the mediator lauryl gallate and, presumably, also with p-coumaric acid. Because the degree of reaction between HexA and LG is high (Table 2), these results further imply that by making an optimal choice of phenolic structure and laccase, an efficient fiber functionalization reaction should be possible, while at the same time a substantial reduction of the detrimental HexA could be achieved. For further characterization of the coupling product between pulp-HexA and mediator, the original unbleached as well as the TvL + LG-treated unbleached and TCF-bleached flax pulp was fractionated into two different LCCs by employing the separation scheme depicted in Fig. 1 (Li et al., 2009, 2011). After thioacidolysis of all fractions, it was found that the major portion of the uncondensed lignin was present in the glucan-lignin fraction (around 20–30 lmol/g), whereas the xylan–lignin fraction only contained 2–5 lmol/g of sample. All LCC fractions were found to contain mainly guaiacyl units (Day et al., 2005). On 1H NMR analysis of the various LCCs, it was observed that only the xylan–lignin LCC showed differences before and after the TvL + LG treatment. Thus, as expected, the intensity of the signal centered at 5.8 ppm and attributed to the C4-proton in HexA (Teleman et al., 1995) showed a considerable decrease in the LCC isolated after TvL + LG treatment. In the same sample, a new signal centered about 1.3 ppm was present and assigned by comparison with authentic lauryl gallate to the long aliphatic hydrocarbon chain present. The xylan–lignin LCC isolated from the TvL + LG treated unbleached flax pulp was also subjected to analysis by SEC. Two major and partly overlapping fractions were obtained as shown in Fig. 2. From the elution profile, three different fractions (after 35.6, 39.2 and 42.6 min, respectively, as shown in the figure) were 3915 E.M. Cadena et al. / Bioresource Technology 102 (2011) 3911–3917 Fig. 2. Size exclusion chromatography (SEC) (upper) and UV spectra (middle) extracted out from three fractions from the SEC of lignin–carbohydrate complex (LCC1, xylan– lignin) from unbleached flax soda–AQ pulp after incubation with Trametes villosa in the presence of lauryl gallate (TvL + LG) in comparison with the UV spectrum (lower) of commercial lauryl gallate (LG). analyzed using a photo diode array detector. In the first of these, a spectrum similar to the one of authentic lauryl gallate was obtained, whereas only indications of similar absorbance maxima were seen in the second. The third spectrum was diffuse with only broad absorbance over the whole UV range (Fig. 2). Based on the analytical data obtained, it can thus be concluded that the mediator lauryl gallate (in its phenoxy radical form) seems to couple to the xylan–HexA double bond, giving rise to an adduct, as depicted in Fig. 3. The difference in reactivity between the two mediators was further studied using isolated xylan from a HexA-rich eucalypt pulp. Treatment of the xylan with either TvL + LG or TvL + PCA revealed noticeable differences, as shown by 1H NMR analysis. Thus, treatment with TvL + LG showed a substantial decrease in COOH H R R HO O OH Laccase R O Xylan R1 R2 COOH O R1 R2 OH R1 O R2 O HO O OH Xylan Fig. 3. Suggested reaction mechanism leading to the coupled structure between lauryl gallate (LG) (R1@R2@OH, R@COO-n-dodecyl) and hexenuronic acid (HexA). 3916 E.M. Cadena et al. / Bioresource Technology 102 (2011) 3911–3917 intensity of the HexA-signal at 5.8 ppm, indicative of HexA elimination in agreement with the treatment of flax pulp with either TvL or TvL + LG (Table 2). However, no coupling of LG to the xylan took place because no signals due to the aliphatic side chain were present. Therefore, HexA has a higher reactivity towards TvL than LG. Consequently, the observed coupling product between HexA and LG found in the flax pulps should only originate from those parts of the fiber wall where TvL itself has no direct accessibility. Treatment of the xylan with TvL + PCA, on the other hand, resulted in no or very little elimination of HexA, while a substantial amount of polymeric aromatic structures could be observed giving rise to 1H NMR signals in the region 6–8 ppm. These signals were assigned to polymerization products of the mediator itself caused by the laccase TvL, thus showing that PCA has a higher reactivity towards TvL than HexA. Although laccases are thought to have non-specific substrate requirements, the results obtained here demonstrate that the laccase or a phenoxy radical, once formed by oxidation of a phenolic mediator by laccase, will preferentially react with a conjugated double bond (PCA), if present. In the absence of this functional group, an isolated double bond like in HexA will show higher reactivity than the phenolic aromatic ring (LG). It has been reported before that the reactivity of laccase, as measured by an ABTS test, can be influenced by the applied mediator and by the presence of pulp. A ‘‘deactivation’’ of laccase by mediator has been observed depending on the structure of the mediator. Without the presence of pulp, the ‘‘deactivation’’ was more severe (Fillat et al., 2010). Although attack of laccase by the oxidized mediator has been assumed to be the reason for the ‘‘deactivation’’, it cannot be ruled out that the ‘‘deactivation’’ in fact depends on the preference of the laccase/phenoxy radical attacking the mediator and/or pulp components over the ABTS. Different laccases having, e.g., different oxidation potentials will also give different reaction patterns with the mediator and with the substrate (pulp or hexenuronic acid). It could therefore be concluded from this study that the choice of laccase/mediator system will determine the outcome of the reaction with pulp fibers, and depending on laccase type and mediator structure, HexA removal or fiber modification through mediator coupling as well as delignification or extractive removal can be achieved to a maximum extent. 4. Conclusions The laccase/mediator system TvL + LG results in the elimination of a major portion of HexA, partly through a direct oxidation of accessible HexA and partly by a coupling between LG and HexA after oxidation of LG. As a result, pulps that are low in HexA but have a coupled structure are obtained. The PcL + PCA, on the other hand, did not give much HexA removal, and upon oxidation by laccase, polymerization of PCA was the preferred reaction. The fact that the mediator LG can couple to HexA demonstrates that fiber functionalization can be achieved by laccase together with the proper choice of mediator. Such effects could be of interest for the introduction of new fiber properties such as hydrophobation. Acknowledgements Financial support from the EU, Framework Program 6, through the project White biotechnology for added value products from renewable plant polymers: design of tailor-made biocatalysts and new industrial bioprocesses (Biorenew, NMP2-CT-2006-026456), is gratefully acknowledged. The Education and Science Ministry (MEC) Spanish Programme FUNCICEL CTQ2009-12904 is also acknowledged. The authors would further like to give thanks to NovozymesÒ and INRA and Celesa for supplying laccases and flax pulps, respectively. References Aracri, E., Colom, J.F., Vidal, T., 2009. Application of laccase-natural mediator systems to sisal pulp: an effective approach to biobleaching or functionalizing pulp fibres? Biores. Technol. 100, 5911. Aracri, E., Fillat, A., Colom, J.F., Gutierrez, A., del Rio, J.C., Martinez, A.T., Vidal, T., 2010. Enzymatic grafting of simple phenols on flax and sisal pulp fibres using laccases. Biores. Technol. 101, 8211–8216. Bourbonnais, R., Paice, M.G., 1992. 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