Lignin and Mn peroxidase-catalyzed oxidation of phenolic lignin oligomers

The oxidation of phenolic oligomers by lignin and manganese peroxidases was studied by transient-state kinetic methods. The reactivity of peroxidase intermediates compound I and compound II was studied with the phenol guaiacol along with a beta-O-4 phenolic dimer, trimer, and tetramer. Compound I of both peroxidases is much more reactive than compound II. The rate constants for these substrates with Mn peroxidase compound I range from 1.0 x 10(5) M-1 s-1 for guaiacol to 1.1 x 10(3) M-1 s-1 for the tetramer. Reactivity is much higher with lignin peroxidase compound I with rate constants ranging from 1.2 x 10(6) M-1s-1 for guaiacol to 3.6 x 10(5) M-1 s-1 for the tetramer. Rate constants with compound II are much lower with Mn peroxidase exhibiting very little reactivity. The rate constants dramatically decreased with both peroxidases as the size of the substrate increased. The extent of the decrease was much more dramatic with Mn peroxidase, leading us to conclude that, despite its ability to oxidize phenols, Mn2+ is the only physiologically significant substrate. The rate decrease associated with increasing substrate size was more gradual with lignin peroxidase. These data indicate that whereas Mn peroxidase cannot efficiently directly oxidize the lignin polymer, lignin peroxidase is well suited for direct oxidation of polymeric lignin.     

Biomimetic oxidation of nonphenolic lignin models by Mn(III): new observations on the oxidizability of guaiacyl and syringyl substructures

Mn(III) is a one-electron oxidant, produced in vivo by the Mn peroxidases of white-rot fungi, and thought to be involved in lignin degradation by these organisms. However, Mn(III) has not been shown to oxidize the major nonphenolic substructures of lignin under mild conditions. We have used Mn(III) acetate as a biomimetic model for enzymatically generated Mn(III), and report that low concentrations of this oxidant suffice to oxidize nonphenolic lignin models at physiological temperatures and pH values. Under these conditions, the monomeric lignin model veratryl alcohol was oxidized to veratraldehyde, and the diarylpropane model 1-(3,4-dimethoxyphenyl)-2-phenylpropanol was oxidatively cleaved to veratraldehyde, 1-phenylethanol, and acetophenone. In an attempt to identify other lignin models that might be oxidized by Mn(III) more rapidly, we compared the rates at which Mn(III) was reduced by two guaiacyl models, veratryl alcohol and 1-(3-methoxy-4-isopropoxyphenyl)ethanol, vs two syringyl models, 3,4,5-trimethoxybenzyl alcohol and 1-(3,5-dimethoxy-4-isopropoxyphenyl)ethanol. The results were the opposite of those predicted: the syringyl models were oxidized slower than the guaiacyl models by Mn(III). To investigate the basis for this unexpected result, we recorded the visible absorption spectra of charge-transfer complexes prepared between each of the lignin models and an electron acceptor, tetracyanoethylene or p-chloranil. The results, in general agreement with the kinetic findings, showed that the nonphenolic syringyl lignin models had higher ionization potentials than the guaiacyl models.     

Preferential degradation of phenolic lignin units by two white rot fungi.

The differential biodegradation of phenolic and nonphenolic (C-4-etherified) lignin units in wheat straw treated with the white rot fungi Pleurotus eryngii and Phanerochaete chrysosporium was investigated under solid-state fermentation conditions. Two analytical techniques applied to permethylated straw were used for this purpose, i.e., alkaline CuO degradation and analytical pyrolysis (both followed by gas chromatography-mass spectrometry for product identification). Despite differences in the enzymatic machinery produced, both ligninolytic fungi caused a significant decrease in the relative amount of phenolic lignin units during the degradation process. Nevertheless, no differences in the biodegradation rates of phenolic and etherified cinnamic acids were observed. Changes in lignin composition and cinnamic acid content were also analyzed in the phenolic and nonphenolic lignin moieties. The results obtained are discussed in the context of the enzymatic mechanisms of lignin biodegradation.     

Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell homogenate from monkey.

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3.5-(125)I]-diiodo-L-thyronine or 3-[3',5'-(125)I]triiodo-L-thyronine (phenolic ring-labeled 'reverse' triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, O.9 X approximately 1 cm) to measure 125I-. Both deiodinases were destroyed by boiling for 1 min. Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1-5mM), and slightly by 5 mM beta-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 MM) and inhibited by Mg(2+) (5mM). Methylmercaptoimidazol and Mg(2+), Ca(2+) and Mn(2+) (0.1-1.0 mM) had little or no effect on either reaction, but Zn(2+) (0.1 mM) strongly inhibited both. Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10 micron M, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3',5'-triiodo-L-thyronine(rT3) greater than L-thyroxine(T4) greater than 3,5,3'-triiodo-L-thyronine(T3), whereas for the nonphenolic ring deiodinase the order is T3 greater than T4 greater than rT3. Diiodotyrosine did not affect their deiodination.     

Initial Steps in the Pathway for Bacterial Degradation of Two Tetrameric Lignin Model Compounds

We investigated the metabolic route by which a lignin tetramer-degrading mixed bacterial culture degraded two tetrameric lignin model compounds containing β—O—4 and 5—5 biphenyl structures. The α-hydroxyl groups in the propane chain of both phenolic and nonphenolic tetramers were first oxidized symmetrically in two successive steps to give monoketones and diketones. These ketone metabolites were decomposed through C_α(=O)—C_β cleavage, forming trimeric carboxyl acids which were further metabolized through another C_α(=O)—C_β cleavage. Dehydrodiveratric acid, which resulted from the cleavage of the carbon bonds of the nonphenol tetramer, was demethylated twice. Four metabolites of the phenolic tetramer were purified and identified. All of these were stable compounds in sterile mineral medium, but were readily degraded by lignin tetramer-degrading bacteria along the same pathway as the phenol tetramer. No monoaromatic metabolites accumulated. All metabolites were identified by mass and proton magnetic resonance spectrometry. The metabolic route by which the mixed bacterial culture degraded tetrameric lignin model compounds was different from the route of the main ligninase-catalyzed C_α—C_β cleavage by Phanerochaete chrysosporium.     

Evidence That Ceriporiopsis subvermispora Degrades Nonphenolic Lignin Structures by a One-Electron-Oxidation Mechanism

The white-rot fungus Ceriporiopsis subvermispora is able to degrade nonphenolic lignin structures but appears to lack lignin peroxidase (LiP), which is generally thought to be responsible for these reactions. It is well established that LiP-producing fungi such as Phanerochaete chrysosporium degrade nonphenolic lignin via one-electron oxidation of its aromatic moieties, but little is known about ligninolytic mechanisms in apparent nonproducers of LiP such as C. subvermispora. To address this question, C. subvermispora and P. chrysosporium were grown on cellulose blocks and given two high-molecular-weight, polyethylene glycol-linked model compounds that represent the major nonphenolic arylglycerol-(beta)-aryl ether structure of lignin. The model compounds were designed so that their cleavage via one-electron oxidation would leave diagnostic fragments attached to the polyethylene glycol. One model compound was labeled with (sup13)C at C(inf(alpha)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(alpha))-C(inf(beta)) cleavage after one-electron oxidation. The other model compound was labeled with (sup13)C at C(inf(beta)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(beta))-O-aryl cleavage after one-electron oxidation. To assess fungal degradation of the models, the high-molecular-weight metabolites derived from them were recovered from the cultures and analyzed by (sup13)C nuclear magnetic resonance spectrometry. The results showed that both C. subvermispora and P. chrysosporium degraded the models by routes indicative of one-electron oxidation. Therefore, the ligninolytic mechanisms of these two fungi are similar. C. subvermispora might use a cryptic LiP to catalyze these C(inf(alpha))-C(inf(beta)) and C(inf(beta))-O-aryl cleavage reactions, but the data are also consistent with the involvement of some other one-electron oxidant.     

Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound.

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3, 4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both kcat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect kcat and the oxidation rate of dimer I.     

Tyrosinase reaction/chitosan adsorption for selectively removing phenols from aqueous mixtures

The goal of this work was to explore the technical feasibility of an enzymatic approach as an alternative to traditional approaches for phenol separations. Specifically, we examined a two-step approach to selectively remove phenols from mixtures containing nonphenolic isomers. Our model solutes, of molecular formula C(7)H(8)O, were the phenol, cresol; the alkyl aryl ether, anisol; and the alcohol, benzyl alcohol. The first step is this two-step approach employed the enzyme mushroom tyrosinase to selectively convert the phenolic, presumably to an o-quinone product. The tyrosinase was specific for the phenol and was not observed to react with either the ether or the alcohol. The second step of this two-step approach employed a sorbent of an appropriate surface chemistry to bind the products of the tyrosinase-catalysed reaction of phenols. The sorbent used for this study was chitosan. Chitosan was observed to be unable to adsorb either nonphenol and was unable to adsorb unreacted cresol. However, Chittosan effectively adsorbs UV-absorbing reaction products of the tyrosinase-catalysed reaction of phenols. When mixtures of cresol and either anasol or benzyl alcohol were studied, the two-step approach was effective for completely removing the phenolic without loss of either the ether or alcohol or the ether (i.e., phenols were removed with high separation factors).     

Effects of cycloheximide on inverse regulation of phenolic and nonphenolic ring deiodinases in monkey hepatocarcinoma cells

Both phenolic and nonphenolic ring deiodinase activities in monkey hepatocarcinoma cells (NCLP-6E) were increased by addition of serum in a concentration-dependent manner: the stimulatory effect of serum was evident at a concentration as low as 1.5%, and was maximal at 5%. Lineweaver-Burk analysis showed that the increases in the deiodinase activities are due to the increase in Vmax, but not in Km. The addition of cycloheximide at concentrations ranging from 0.1 to 50 micrograms/ml inhibited the stimulatory effect of serum on phenolic ring deiodinase activity progressively. On the other hand, nonphenolic ring deiodinase activity was increased as much as 4-fold by the addition of 0.5-5 micrograms/ml cycloheximide together with 0.5% serum; a high concentration of the drug, 50 micrograms/ml, however, did not elicit such an increase. Actinomycin D at 5 micrograms/ml completely abolished the increase in nonphenolic ring deiodinase activity by serum or cycloheximide. In addition, actinomycin D inhibited the increase in phenolic ring deiodinase activity by serum in a dose-dependent manner at concentrations ranging from 0.05 to 5 micrograms/ml. It is concluded that phenolic and nonphenolic ring deiodinases are regulated by different mechanisms in monkey hepatocarcinoma cells (NCLP-6E).     

Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system

Phenolic and nonphenolic (permethylated) synthetic [14C]lignins were depolymerized by Trametes villosa laccase in the presence of a radical mediator, 1-hydroxybenzotriazole (HOBT). Gel permeation chromatography of the treated lignins showed that approximately 10% of their substructures were cleaved. The system also cleaved a beta-O-4-linked model compound, 1-(4-ethoxy-3-methoxy-ring-[14C]phenyl)-2-(2-methoxyphenoxy)-propane- 1,3-diol, and a beta-1-linked model, 1, 2-bis-(3-methoxy-4-[14C]methoxyphenyl)-propane-1,3-diol, that represent nonphenolic substructures in lignin. High performance liquid chromatography of products from the oxidized models showed that they were produced in sufficient yields to account for the ability of laccase/HOBT to depolymerize nonphenolic lignin.     

DFT study on gas-phase hydrodeoxygenation of guaiacol by various reaction schemes

Guaiacol is an important phenolic component present in pyrolytic bio-oils; and in this work its hydrodeoxygenation (HDO) by various reaction schemes has been considered within the framework of density functional theory. In this computational study, primarily three reaction schemes for the HDO of guaiacol are considered. In the first reaction scheme (RS 1), guaiacol undergoes hydrogenolysis at O–CH₃ bond site of methoxy group to produce catechol and methane followed by HDO of catechol forming phenol and water, followed by HDO of phenol producing benzene and water and finally benzene leading to cyclohexane formation. In the second reaction scheme (RS 2), guaiacol undergoes hydrogenolysis at C_aromatic–O bond of methoxy group producing phenol and methanol followed by hydrotreatment of phenol to form cyclohexane along with same intermediates as in the first reaction scheme. In the third reaction scheme (RS 3), HDO of guaiacol compound at C_aromatic–OH sigma bond produces anisole and water; and then anisole follows two secondary pathways to produce cyclohexane. In this computational study, the transition state optimisations, vibrational frequency and IRC calculations are carried out by B3LYP functional with 6-311+g(d,p) basis set using Gaussian 09 and Gauss View 5 software package.     

Carbonate recrystallization in root-free soil and rhizosphere of Triticum aestivum and Lolium perenne estimated by 14C labeling

The photo- and bio-degradation of dissolved organic matter (DOM) in water from the Broad River were investigated in laboratory experiments using a solar simulator to control the intensity and exposure of samples to irradiation. The water samples included a natural assemblage of microorganisms, and the daily exposure of samples to irradiation was varied to distinguish the relative contributions of photochemical and biological degradation. Concentrations of dissolved organic carbon (DOC) and specific components of DOM, including chromophoric DOM (CDOM), lignin phenols and amino acids, were monitored to investigate the reactivity and predominant pathway of degradation of these DOM components. Biodegradation was primarily responsible for the overall remineralization of DOC and losses of the amino acid component of DOM, whereas photodegradation was primarily responsible for losses of the chromophoric and lignin phenol components of DOM. The rates of photodegradation of lignin phenols were strongly influenced by the presence of methoxy groups on the aryl ring. Syringyl (S) phenols have two methoxy substitutions, vanillyl (V) phenols have one methoxy substitution, and p-hydroxy (P) phenols are not substituted with methoxy groups. Photochemical decay constants were highest for S phenols, lowest for P phenols and followed a consistent pattern (S > V > P) in the experiments. The carbon-normalized yields of amino acids and lignin phenols were found to be useful molecular indicators of the highly reactive (i.e. labile) components of biodegradable and photodegradable DOM, respectively.     

Manganese-Dependent Cleavage of Nonphenolic Lignin Structures by Ceriporiopsis subvermispora in the Absence of Lignin Peroxidase

Many ligninolytic fungi appear to lack lignin peroxidase (LiP), the enzyme generally thought to cleave the major, recalcitrant, nonphenolic structures in lignin. At least one such fungus, Ceriporiopsis subvermispora, is nevertheless able to degrade these nonphenolic structures. Experiments showed that wood block cultures and defined liquid medium cultures of C. subvermispora rapidly depolymerized and mineralized a (sup14)C-labeled, polyethylene glycol-linked, high-molecular-weight (beta)-O-4 lignin model compound (model I) that represents the major nonphenolic structure of lignin. The fungus cleaved model I between C(inf(alpha)) and C(inf(beta)) to release benzylic fragments, which were shown in isotope trapping experiments to be major products of model I metabolism. The C(inf(alpha))-C(inf(beta)) cleavage of (beta)-O-4 lignin structures to release benzylic fragments is characteristic of LiP catalysis, but assays of C. subvermispora liquid cultures that were metabolizing model I confirmed that the fungus produced no detectable LiP activity. Three results pointed, instead, to the participation of a different enzyme, manganese peroxidase (MnP), in the degradation of nonphenolic lignin structures by C. subvermispora. (i) The degradation of model I and of exhaustively methylated (nonphenolic), (sup14)C-labeled, synthetic lignin by the fungus in liquid cultures was almost completely inhibited when the Mn concentration of the medium was decreased from 35 (mu)M to approximately 5 (mu)M. (ii) The fungus degraded model I and methylated lignin significantly faster in the presence of Tween 80, a source of unsaturated fatty acids, than it did in the presence of Tween 20, which contains only saturated fatty acids. Previous work has shown that nonphenolic lignin structures are degraded during the MnP-mediated peroxidation of unsaturated lipids. (iii) In experiments with MnP, Mn(II), and unsaturated lipid in vitro, this system mimicked intact C. subvermispora cultures in that it cleaved nonphenolic (beta)-O-4 lignin model compounds between C(inf(alpha)) and C(inf(beta)) to release a benzylic fragment.     

Mn-dependent peroxidase from the lignin-degrading white rot fungus Phlebia radiata

A homogeneous Mn-dependent peroxidase (MnP) was purified from the extracellular culture fluid of the lignin-degrading white rot fungus Phlebia radiata by anion exchange chromatography. The enzyme had a molecular weight of 49,000 and pI 3.8. It was a glycoprotein, containing carbohydrate moieties accounting for 10% of the molecular weight. Mn-peroxidase was capable of oxidizing phenolic compounds in the presence of H2O2, whereas the effect on nonphenolic lignin model compounds was insignificant. MnP contained protoporphyrin IX as a prosthetic group. During enzymatic reactions H2O2 converted the native MnP to compound II. Mn2+ was essential in completing the catalytic cycle by returning the enzyme to its native state. The oxidation of ultimate substrates was dependent on superoxide radicals, O2- and probably on Mn3+ generated during the catalytic cycle. MnP exhibited high activity of NADH oxidation without exogenously added H2O2. It was shown to produce H2O2 at the expense of NADH.     

Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both k_cat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect k_cat and the oxidation rate of dimer I.     

一株新分离的云芝栓孔菌Z-1应用于木质素降解及染料脱色

木质素是一种非结晶性的复杂三维网状酚类高分子聚合物,被认为是木质纤维素生物质抗降解的天然屏障。探索并开发高效降解木质素的微生物资源已成为近些年来的研究焦点。本研究对新分离的一株具有潜在木质素降解能力的菌株Z-1开展了系列研究。首先通过形态学和系统发育分析将菌株Z-1鉴定为云芝栓孔菌Trametes versicolor。平板定性检测初步表明云芝栓孔菌T. versicolor Z-1具有较强的产过氧化物酶和漆酶能力。以木质素为唯一碳源时,T. versicolor Z-1对木质素的降解率和脱色率分别可达13.38%和26.43%。酶活检测分析表明该菌主要是通过分泌漆酶和锰过氧化物酶（manganese peroxidase,MnP）降解木质素。利用傅里叶变换红外光谱（fourier transform-infrared spectroscopy,FT-IR）、扫描电镜（scanning electron microscope,SEM）及气相色谱-质谱联用（gas chromatography-mass spectroscopy,GC-MS）对云芝栓孔菌T. versicolor Z-1降解后木质素残渣结构以及代谢物的鉴定分析结果证实了该菌对木质素的强降解能力,并表明该菌对木质素的降解途径包括酚醚键的断裂、芳香环侧链氧化裂解以及芳香环开环反应等。此外,该菌还对多种芳香类染料展现出了强的脱色能力,其中对刚果红、孔雀石绿和考马斯亮蓝R-250 3种染料的脱色率达到100%。本研究表明云芝栓孔菌T. versicolor Z-1具有应用于工业化木质素降解与芳香化合物类染料脱色的开发前景。     

Aromatic ring cleavage of 4,6-di(tert-butyl)guaiacol, a phenolic lignin model compound, by laccase of Coriolus versicolor

It was found that 2,4-di(tert-butyl)-4-(methoxycarbonylmethyl)-2-buten-4-ol ide (II) was formed as an aromatic ring cleavage product of a phenolic lignin model compound, 4,6-di(tert-butyl)guaiacol (I), by laccase of Coriolus versicolor. Based on isotopic experiments with 18O2 and H2 18O, the mechanism of formation of II from I is discussed.     

Increases in phenolic and nonphenolic ring deiodinase activity due to serum in monolayer liver cell cultures

When monkey hepatocarcinoma cells (NCLP-6E) were treated with 10% of various serum preparations for 24 h at 37 degrees C, nonphenolic ring deiodinase activity increased 2.0- to 2.3-fold. Phenolic ring deiodinase activity also increased 0.9- to 2.1-fold. Dialysis of the sera enhanced the effect on deiodinase activities in some preparations, but reduced activity in other serum preparations. Similarly, a 1.3- to 3.1-fold increase in phenolic ring deiodinase activity was observed in rat hepatoma cells (R-Y121B). In this case, dialysis usually reduced the effect of the sera. It is concluded that both large molecule(s) (undialyzable) and small molecule(s) (dialyzable) in serum contribute to the regulation of phenolic and nonphenolic ring deiodinase activity in NCLP-6E and R-Y121B cells.     

Polymerization of lignin fragments contained in a model effluent by polyphenoloxidases and horseradish peroxidase/hydrogen peroxide system*

An effluent containing soluble lignin fragments was treated with potato-polyphenoloxidases (PPO) or horseradish peroxidase/hydrogen peroxide system (HRP/H(2)O(2)). In both cases the reaction was evidenced by the formation of a brown precipitate that was a consequence of the polymerization of lignin fragments. The effect of reaction time, pH, and amount of soluble lignin per unit of enzyme activity on the insolubilization yield was evaluated for PPO-initiated reactions. For HRP-initiated reactions, the amount of H(2)O(2) per unit of enzyme activity was also evaluated. Mathematical models were calculated to predict the insolubilization yield as a function of the significant variables. Based on these models, the insolubilization reaction was optimized and reached maximal values of ca. 50% in both reaction systems. Higher insolubilization yields were not achieved. Chemical characterization of the soluble lignin fragments indicated that the insolubilization yield could not be improved, because the lignin fragments had limited amounts of free phenolic substructures available for the initial steps of the polymerization reaction.     

Enzymatic modification of kraft lignin through oxidative coupling with water-soluble phenols

The aromatic polymer lignin can be modified through promotion of oxidative coupling between phenolic groups on lignin and various phenols. The reaction is initiated by an oxidation of both components, e.g., by using the oxidoreductases laccase or peroxidase. Coupling between phenolic monomers and lignin has previously been studied by the use of radio-labeled phenols. In this study, incorporation of water-soluble phenols into kraft lignin, using laccase as catalyst, was investigated. Several phenols with carboxylic or sulfonic acid groups were used as markers for the incorporation. The modified lignin was isolated and the amount of phenol incorporated was characterized by means of titration, quantitative 1H-NMR, and quantitative 31P-NMR after modification with 2-chloro-4,4,5,5-tetramethyl-1,2,3-dioxaphospholane. Only a few of the phenols studied were found to be incorporated into lignin. When the phenol guaiacol sulfonate was incorporated into kraft lignin, the lignin became water-soluble at pH 2.4 and a low ionic strength due to the introduction of sulfonic acid groups. The content of sulfonic acid groups in the product was 0.5-0.6 mmol/g lignin. A lower amount of 4-hydroxyphenylacetic acid was incorporated under similar conditions.