Several model lipids representative for main paper pulp lipophilic extractives - including alkanes, fatty alcohols, fatty acids, resin acids, free and esterified sterols, and triglycerides – were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator. The reaction products were analyzed by GC and GC-MS. The laccase alone decreased the amount of some unsaturated lipids, however, the most rapid and extensive lipid modification was obtained with the laccase-mediator system. Most unsaturated lipids were largely oxidized and the dominant oxidation products detected were epoxy and hydroxy-fatty acids from fatty acids, and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. In contrast, saturated lipids were not modified, although some of them were oxidized when the enzymatic reactions were carried out in the presence of unsaturated lipids. The results obtained are discussed in the context of enzymatic control of pitch deposits, to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.This study was funded by the BIORENEW EU-project (NMP2-CT-2006-026456) and the Spanish MEC (BIO2007-28719-E). Beldem (Andenne, Belgium) is acknowledged for laccase supply. S.M. thanks the Spanish CSIC and CELESA for an I3P contract and J.R thanks the CSIC for an I3P fellowship.Peer reviewe

Gutiérrez Suárez, Ana

Lomascolo, Anne

Martínez, Ángel T.

Molina, Setefilla

Record, Eric

Rencoret, Jorge

Río Andrade, José Carlos del

Digital.CSIC

OXIDATION OF MODEL LIPIDS REPRESENTATIVE FOR MAIN PAPER PULP LIPOPHILIC EXTRACTIVES BY THE LACCASE-MEDIATOR SYSTEM  Setefilla Molina1, Jorge Rencoret1, José C. del Río1, Anne Lomascolo2, Eric Record2, Ángel T. Martínez3, Ana Gutiérrez3 1Instituto de Recursos Naturales y Agrobiología, CSIC, PO Box 1052, E-41080 Seville, Spain 2Institut National de la Recherche Agronomique, CP 925, F-13288 Marseille, France 3Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain   ABSTRACT  Several model lipids representative for main paper pulp lipophilic extractives - including alkanes, fatty alcohols, fatty acids, resin acids, free and esterified sterols, and triglycerides – were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator. The reaction products were analyzed by GC and GC-MS. The laccase alone decreased the amount of some unsaturated lipids, however, the most rapid and extensive lipid modification was obtained with the laccase-mediator system. Most unsaturated lipids were largely oxidized and the dominant oxidation products detected were epoxy and hydroxy-fatty acids from fatty acids, and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. In contrast, saturated lipids were not modified, although some of them were oxidized when the enzymatic reactions were carried out in the presence of unsaturated lipids. The results obtained are discussed in the context of enzymatic control of pitch deposits, to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.     I. INTRODUCTION Lipophilic extractives, such as fatty and resin acids, fatty alcohols, alkanes, steroids and triglycerides cause pitch deposits along the pulp and paper manufacturing processes (Back and Allen, 2000). Besides physicochemical methods, enzymes and microorganisms have been investigated to solve pitch problems (Gutiérrez et al., 2001). Lipases, which hydrolyze triglycerides, are successfully applied in softwood (mainly pine) mechanical pulping at mill scale (Fujita et al., 1992). However, pitch problems in most of the chemical and mechanical processes using other raw materials have not been solved yet. Other compounds, such as free and esterified sterols, resin acids, fatty alcohols and alkanes, are responsible for pitch problems in these processes (Gutiérrez et al., 2001; Gutiérrez and del Río, 2005). Modification of lignans and other colloidal substances in process waters and pulps from softwood pulping by laccase treatment has been reported (Buchert et al., 2002; Zhang et al., 2005). In contrast to lipases, laccases are oxidative enzymes whose action is directed toward phenols, anilines and related compounds. The interest on laccases as industrial biocatalysts has, however, increased after discovering the effect of some synthetic compounds (Bourbonnais and Paice, 1990; Call, 1994) expanding the action of laccases to non-phenolic aromatic substrates and, therefore, increasing their potential in degradation of lignin and other recalcitrant compounds. Moreover, the use of laccases in the presence of redox mediators has very recently been described for the removal of lipophilic extractives responsible for pitch deposits from wood and nonwood paper pulps (Gutiérrez et al., 2006a; 2006b). Further investigations on the chemistry of the reactions of the laccase-mediator system with several model compounds representative for the main lipophilic extractives from different pulp types, have been carried out to better understand the degradation patterns observed in pulps.  II. EXPERIMENTAL Model lipophilic compounds. Alkanes (octadecane), fatty alcohols (1-hexadecanol), fatty acids (palmitic, oleic and linoleic acids), resin acids (abietic acid), free sterols (sitosterol), sterol esters (cholesteryl palmitate, cholesteryl oleate and cholesteryl linoleate) and triglycerides (triheptadecanoin and trilinolein), were used.  Laccase and mediator. The laccase used was from Pycnoporus cinnabarinus. Laccase activity was measured during oxidation of 5 mM 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to its cation radical (ε436 29300 M-1 cm-1) in 0.1 M sodium acetate (pH 5) at 24ºC. One activity unit was defined as the amount of enzyme transforming 1 µmol of ABTS per min. 1-Hydroxybenzotriazole (HBT) was used as mediator. Enzymatic treatment of model compounds. The enzymatic treatments (five replicates) of the different model lipids (1 mg) were performed using laccase (0.5 U/mg lipid), HBT (1 mg/mg lipid), and Tween 20 as dispersant (1% v/v) at pH 4, 50ºC, and different reactions times (5, 15, and 30 min, 1, 2, and 8 h). Oxygen was bubbled through the reaction flasks. In control experiments, lipids were treated under the same conditions but without laccase and mediator. Additional controls including laccase alone and boiled laccase were also performed. Mixtures of the saturated lipids (octadecane, hexadecanol, palmitic acid or triheptadecanoin) with linoleic acid or cholesteryl linoleate were treated (2 h) with laccase-HBT under the same conditions described above. After the enzymatic treatments, the lipid dispersions were immediately evaporated, and the reaction products recovered  with chloroform:methanol (1:1), dried and redissolved in chloroform for gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. Bis(trimethylsilyl)trifluoroacetamide in the presence of pyridine was used to prepare trimethylsilyl derivatives, before and after sodium borohydride reduction.  GC and GC-MS analyses of lipids. The GC and GC-MS analyses were performed as previously described (Gutiérrez et al., 1998). Peaks were quantified by area, and data from replicates were averaged. In all cases the standard deviations were below 5% of the mean values. Longer columns (30 m) were also used when necessary.   III. RESULTS AND DISCUSSION  Reactivity of different model lipids with laccase-HBT and laccase alone. Twelve model lipids representative for the main lipophilic extractives from different hardwood, softwood and nonwoody pulps were treated with the high redox-potential laccase from P. cinnabarinus in the presence and absence of HBT as mediator, to get further insight into the chemistry of these reactions. Two-hour laccase reactions were carried out with all the model lipids. The chromatographic analyses evidenced that octadecane, hexadecanol, palmitic acid and triheptadecanoin were not modified after 2-h treatment neither with laccase alone nor with laccase-HBT. No decrease in the amount of these saturated compounds was observed even when longer (8 h) reactions were carried out (data not shown). In contrast, 60-100% decrease of the initial amount of unsaturated compounds (Fig. 1) such as abietic acid (A), trilinolein (B), linoleic (C) and oleic (D) acids, sitosterol (E), cholesteryl palmitate (F), oleate (G) and linoleate (H), was found at the end of 2-h laccase-HBT treatment. Likewise, a decrease of 20-40% of these unsaturated lipids was observed after treatment with laccase alone except in the cases of abietic acid that decreased 95%, and cholesteryl palmitate and sitosterol that were not affected (data not shown).  When shorter laccase-HBT treatments (60, 30, 15 and 5 min) of the latter lipids were analyzed, different reactivities were observed (Fig. 1). Thirty minutes of laccase-HBT treatment were enough to attain 40-100% reduction of all these unsaturated lipids. Abietic acid and trilinolein were already completely oxidized after only 5-min enzymatic treatment. High reduction (≥95%) of cholesteryl linoleate also required only 5-min treatment, whereas 30 min were required for cholesteryl oleate and linoleic acid. On the other hand, up to 75% reduction of sitosterol was attained after 1-h treatment, and complete transformation of this sterol was attained after 2 h. Therefore, sitosterol, together with cholesteryl palmitate and oleic acid, was the most refractory towards the laccase-HBT treatment among the different unsaturated lipids assayed.  Reaction products from the enzymatic treatment of unsaturated model lipids. The formation of oxidized products during 2-h treatment of eight model lipids by laccase-HBT is shown in Fig. 1, together with the decrease of the initial compounds. Although abietic acid (A), trilinolein (B), and linoleic acid (C) were completely or strongly transformed after 5 and 15 min of laccase-HBT treatment, respectively, new products were not observed. In contrast, several oxidized compounds were identified after laccase-HBT treatment of oleic acid (D) including one epoxy-fatty acid (cis and trans forms of 9,10-epoxyoctadecanoic acid), and four hydroxy-fatty acids (8-hydroxy-9-octadecenoic acid, 9-hydroxy-10-octadecenoic acid, 10-hydroxy-8-octadecenoic acid and 11-hydroxy-9-octadecenoic acid) quantified together, whose presence suggests the formation of four allylic hydroperoxides (with -OOH groups on carbons 8, 9, 10 or 11). The epoxy-fatty acid formed could be produced by hydroperoxide reaction with oleic acid (Frankel, 2005).  During free sitosterol (E) treatment with laccase-HBT the main new compound identified was 7-ketositosterol together with minor amounts of stigmasta-3,5-dien-7-one. Traces of 7α- and 7β-hydroxysitosterol were also detected. By observing the oxysterol pattern formed it could be inferred that the oxidation would be a free radical reaction with formation of 7-hydroperoxide, whose degradation forms the 7α- and 7β-hydroxysitosterol and 7-ketositosterol. On the other hand, the laccase-HBT system exhibited different degradation behaviors on the different sterol esters assayed. Cholesteryl palmitate (F) was mainly oxidized during the first hour of treatment, and 40% of the initial ester remained at the end of the reaction. The most prominent compound formed was tentatively identified as 7-ketocholesteryl palmitate. In addition, minor amounts of cholesta-3,5-dien-7-one and traces of 7α- and 7β-hydroxycholesteryl palmitate were also identified. On the other hand, cholesteryl oleate (G) and linoleate (H) exhibited relatively similar degradation patterns after the enzymatic treatment since both compounds were completely transformed by laccase-HBT after 15-30 min reaction. The above cholesta-3,5-dien-7-one was produced from both sterol esters, being the main product at the end of the reactions. 7-Ketocholesteryl oleate was also detected from cholesteryl oleate. Additional products were identified during the first 15 min of reaction, including free oleic acid from cholesteryl oleate, and several new compounds from cholesteryl linoleate. These included cholesterol or 7-ketosterol esters containing fatty-acid chains of different lengths, including those tentatively identified as cholesteryl and 7-ketocholesteryl ester core aldehydes. Traces of free cholesterol and 7-ketocholesterol were also found during the initial stages of cholesteryl linoleate transformation by laccase-HBT. By observing the oxidation products from the above cholesterol esters it could be deduced that the oxysterols formed depend on the fatty-acid moiety.    Finally, it should be mentioned that none of the oxidized compounds mentioned above was detected when the reactions were performed with laccase alone.  012345670 20 40 60 80 100 120Time (min)molsx 10-6(C)molsx 10-6120 20 40 60 80 100 120Time (min)(E)0molsx 10-6012345670 20 40 60 80 100 120Time (min)(D)01230 20 40 60 80 100 120Time (min)molsx 10-6(H)0120 20 40 60 80 100 120Time (min)molsx 10-6(G)01230 20 40 60 80 100 120Time (min)molsx 10-6(F)012345670 20 40 60 80 100 120Time (min)molsx 10-6(A)molsx 10-6120 20 40 60 80 100 120Time (min)0(B)linoleic acid oleic acidepoxy-fatty acidshydroxy-fatty acidsabietic acid trilinoleinsitosterolstigmasta-3,5-dien-7-one7-ketositosterolcholesta-3,5-dien-7-one7-ketocholesteryl oleatecholesteryl oleatecholesta-3,5-dien-7-onecholesteryl linoleate7-ketocholesteryl core aldehydescholesteryl core aldehydespalmitic acidcholesta-3,5-dien-7-onecholesteryl palmitate7-ketocholesteryl palmitatemolsx 10-6molsx 10-6molsx 10-6molsx 10-6molsx 10-6molsx 10-6molsx 10-6molsx 10-6molsx 10-6Figure 1. Oxidation of eight model lipids (Ο) and formation of oxidation products during laccase-HBT reactions: (A) abietic acid; (B) trilinolein; (C) linoleic acid; (D) oleic acid; (E) sitosterol; (F) cholesteryl palmitate; (G) cholesteryl oleate; and (H) cholesteryl linoleate.  Laccase-HBT action on model lipid mixtures. During treatment of individual lipids, it was found that octadecane, 1-hexadecanol, palmitic acid and triheptadecanoin, were not modified by laccase-HBT. However, the corresponding lipid classes were extensively removed during the laccase-HBT treatment of pulps (Gutiérrez et al., 2006b). With the aim of studying whether the transformation of saturated lipids in pulps could be influenced by the presence of other lipid compounds, 2-h reactions of saturated lipids with laccase-HBT were carried out in the presence of unsaturated ones such as linoleic acid and cholesteryl linoleate. It could be observed that 1-hexadecanol decreased near 25% with laccase-HBT in the presence of the unsaturated lipids. Moreover, octadecane decreased 52% and 26% with laccase-HBT in the presence of linoleic acid or cholesteryl linoleate, respectively. This confirmed that the presence of unsaturated lipids in paper pulp facilitates the modification of the saturated ones, suggesting that lipid radicals generated from peroxidation of unsaturated lipids by laccase-HBT participate in the oxidation of the less reactive saturated lipids. More modest decreases were observed in reactions with laccase alone. In contrast, triheptadecanoin was not modified, and only a slight decrease of palmitic acid was attained. Hexadecanoic acid was formed after the reaction of laccase-HBT with 1-hexadecanol, in the presence of either linoleic acid or cholesteryl linoleate. In contrast, no reaction product was found after laccase-HBT reaction with octadecane in the presence of the latter unsaturated lipids.  IV. CONCLUSIONS We report the reaction of several model lipids with laccase in the presence and absence of HBT. Different reactivities were observed being correlated with the number of double bonds in their structures. The search for reaction products yielded variable results. No evidence for reaction products could be obtained for the most reactive compounds (such as abietic acid, linoleic acid and trilinolein). In contrast, oxygenated compounds appeared, and subsequently decreased, in the reactions of lipids with an intermediate reactivity degree (such as cholesteryl linoleate and oleate). With the less reactive lipids (such as oleic acid, sitosterol and cholesteryl palmitate) accumulation of reaction products was observed. The nature and abundance of the above oxidation products provided some clues on the mechanisms of lipid oxidation by laccase-HBT. Finally, some compounds (namely octadecane, 1-hexadecanol, palmitic acid and triheptadecanoin) did not show any reactivity with the laccase-HBT, although they could be modified in mixtures with unsaturated lipids. These results help to understand those obtained during laccase-HBT treatment of pulps, and can contribute to the development of enzymatic methods for pitch control in pulp and paper manufacturing and other biotechnological applications of the laccase-mediator system.  V. ACKNOWLEDGEMENT This study was funded by the BIORENEW EU-project (NMP2-CT-2006-026456) and the Spanish MEC (BIO2007-28719-E). Beldem (Andenne, Belgium) is acknowledged for laccase supply. S.M. thanks the Spanish CSIC and CELESA for an I3P contract and J.R thanks the CSIC for an I3P fellowship.   VI. REFERENCES E. L. Back, L. H. Allen, Pitch control, wood resin and deresination, TAPPI Press, Atlanta, 2000. R. Bourbonnais, M. G. Paice, Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation, FEBS Lett. 1990, 267, 99-102. J. Buchert, A. Mustranta, T. Tamminen, P. Spetz, B. Holmbom, Modification of spruce lignans with Trametes hirsuta laccase, Holzforschung 2002, 56, 579-584. H.-P. Call, Verfahren zur Veränderung, Abbau oder Bleichen von Lignin, ligninhaltigen Materialien oder ähnlichen Stoffen, Patent (International) 1994, WO 94/29510. E. N. Frankel, Lipid oxidation, The Oil Press, Bridgwater, 2005. Y. Fujita, H. Awaji, H. Taneda, M. Matsukura, K. Hata, H. Shimoto, M. Sharyo, H. Sakaguchi, K. Gibson, Recent advances in enzymic pitch control, Tappi J. 1992, 75 (4), 117-122. A. Gutiérrez, J. C. del Río, Chemical characterization of pitch deposits produced in the manufacturing of high-quality paper pulps from hemp fibers, Bioresour. Technol. 2005, 96, 1445-1450. A. Gutiérrez, J. C. del Río, F. J. González-Vila, F. Martín, Analysis of lipophilic extractives from wood and pitch deposits by solid-phase extraction and gas chromatography, J. Chromatogr. 1998, 823, 449-455. A. Gutiérrez, J. C. del Río, D. Ibarra, J. Rencoret, J. Romero, M. Speranza, S. Camarero, M. J. Martínez, A. T. Martínez, Enzymatic removal of free and conjugated sterols forming pitch deposits in environmentally sound bleaching of eucalypt paper pulp, Environ. Sci. Technol. 2006a, 40, 3416-3422. A. Gutiérrez, J. C. del Río, M. J. Martínez, A. T. Martínez, The biotechnological control of pitch in paper pulp manufacturing, Trends Biotechnol. 2001, 19, 340-348. A. Gutiérrez, J. C. del Río, J. Rencoret, D. Ibarra, A. T. Martínez, Main lipophilic extractives in different paper pulp types can be removed using the laccase-mediator system, Appl. Microbiol. Biotechnol. 2006b, 72, 845-851. X. Zhang, S. Renaud, M. Paice, The potential of laccase to remove extractives present in pulp and white water from TMP newsprint mills, J. Pulp Paper Sci. 2005, 31, 175-180. 

Oxidation of model lipids representative for main paper pulp lipophilic extractives by the laccase-mediator system.

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