Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase

In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O₂ during laccase and tyrosinase treatment and of H₂O₂ during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20–120 min after the addition of enzyme).     

Production of laccase, manganese peroxidase and lignin peroxidase by Brazilian marine-derived fungi

Marine-derived fungi are a potential for the search of new compounds with relevant features. Among these, the ligninolytic enzymes have potential applications in a large number of fields, including the environmental and industrial sectors. This is the work aimed to evaluate the enzymatic activities of three marine-derived fungi (Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857 and Mucor racemosus CBMAI 847) under different carbon sources and salinity conditions by using statistical experimental design. MnP, LiP and laccase were detected when these fungi were cultured in malt extract, however when grown on basal medium containing glucose and wheat bran LiP was not detected and yet an increase in MnP and laccase was observed. Statistical analysis through surface responses was performed and results showed high values of MnP and laccase activities under 12.5% and 23% (w/v) salinity, highlighting the potential use of these fungi for industrial applications and in bioremediation of contaminated sites having high salt concentrations. The highest values for LiP (75376.34 UI L⁻¹), MnP (4484.30 IU L⁻¹) and laccase (898.15 UI L⁻¹) were obtained with the fungus M. racemosus CBMAI 847 and it is the first report concerning ligninolytic enzymes production by a zygomycete from this genus.     

NMR study of the active site of resting state and cyanide-inhibited lignin peroxidase from Phanerochaete chrysosporium. Comparison with horseradish peroxidase

One- and two-dimensional 1H NMR spectroscopy has been used to probe the active site of the high spin ferric resting state and the low spin, cyanide-inhibited derivative of isozyme H2 of the lignin peroxidase, LiP, from Phanerochaete chrysosporium strain BKM 1767. One-dimensional NMR revealed a resting state LiP that is five coordinate at 25 degrees C with an electronic structure similar to that of horseradish peroxidase, HRP. Differential paramagnetic relaxivity was used to identify the C beta H signals of the axial His177. A combination of bond correlation spectroscopy and nuclear Overhauser effect spectroscopy of cyanide-inhibited LiP (LiP-CN) has allowed the assignment of all resolved heme resonances without recourse to isotope labeling, as well as those of the proximal His177 and the distal His48. The surprising effectiveness of the two dimensional NMR methods on such a large and paramagnetic protein indicates that such two dimensional experiments can be expected to have major impact on solution structure determination of diverse classes of heme peroxidases. The two dimensional NMR data of LiP-CN reveal a heme contact shift pattern that reflects a close similarity to that of HRP-CN, including the unusual in-plane trans and cis orientation of the 2- and 4-vinyls. The axial His177 also exhibits the same orientation relative to the heme as in HRP-CN. The proximal His177 contact shifted resonances of both the low spin LiP-CN and high spin LiP are shown to reflect significantly reduced hydrogen bond donation by, or imidazolate character for, the axial histidine in LiP relative to HRP, which may explain the higher redox potential of LiP. The signals are identified for a distal residue that originates from the protonated His48 with disposition relative to the heme similar to that found for the distal His42 in HRP-CN. In contrast, the absence of any resolved signals attributable to an Arg44 in LiP-CN suggest that this distal residue has an altered orientation relative to the heme compared with that of the conserved Arg38 in HRP-CN (Thanabal, V., de Ropp, J. S., and La Mar, G. N. (1987) J. Am. Chem. Soc. 109, 7516-7525).     

Oxidation of 1-hydroxybenzotriazole by laccase and lignin peroxidase

A method to measure laccase and lignin peroxidase (LiP) activity at 408 nm (402–410 nm) using 1-hydroxybenzotriazole (HBT) was developed. The assay can be performed either as a kinetic measurement or as a stopped reaction using 5 mM Na-azide which improves the spectrum. Only white-rot fungal laccases and LiP were found to oxidize HBT to give shoulders or peaks at 402-410 nm. Phanerochaete and Phlebia manganese peroxidases did not give absorbance increase at 402–410 nm. © Rapid Science Ltd. 1998     

Production of lignin peroxidase and laccase by Phlebia radiata

Summary A cultivation method using carrierbound mycelium was developed for the production of lignin-modifying enzymes by Phlebia radiata. Laccase and lignin peroxidase were produced in batch and semi-continuous cultivations. Laccase activity was clearly enhanced by veratryl alcohol. The presence of both veratryl alcohol and Tween 80 was required for lignin peroxidase production in submerged cultivations. During the course of the semi-continuous cultivations production of lignin peroxidase activity increased fourfold compared with static cultivations.     

Titration study of guaiacol oxidation by horseradish peroxidase.

Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 0.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound II. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound II. The direct two-electron transfer from guaiacol to compound I without a dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer.     

Oxidative metabolism of the anti-cancer agent mitoxantrone by horseradish, lacto-and lignin peroxidase

Mitoxantrone (MH₂X), an anthraquinone-type anti-cancer agent used clinically in the treatment of human malignancies, is oxidatively activated by the peroxidase/H₂O₂ enzyme system. In contrast to the enzymatic mechanisms of drug oxidation, the chemical transformations of MH₂X are not well described. In this study, MH₂X metabolites, produced by the horseradish, lacto- or lignin peroxidase (respectively HRP, LPO and LIP)/H₂O₂ system, were investigated by steady-state spectrokinetic and HPLC-MS methods. At an equimolar mitoxantrone/H₂O₂ ratio, the efficacy of the enzyme-catalyzed oxidation of mitoxantrone decreased in the following order: LPO > HRP > LIP, which accorded with the decreasing size of the substrate access channel in the enzyme panel examined. In all cases, the central drug oxidation product was the redox-active cyclic metabolite, hexahydronaphtho-[2,3-f]-quinoxaline-7,12-dione (MH₂), previously identified in the urine of mitoxantrone-treated patients. As the reaction progressed, data gathered in this study suggests that further oxidation of the MH₂ side-chains occurred, yielding the mono- and dicarboxylic acid derivatives respectively. Based on the available data a further MH₂ derivative is proposed, in which the amino-alkyl side-chain(s) are cyclised. With increasing H₂O₂ concentrations, these novel MH₂ derivatives were oxidised to additional metabolites, whose spectral properties and MS data indicated a stepwise destruction of the MH₂ chromophore due to an oxidative cleavage of the 9,10-anthracenedione moiety. The novel metabolites extend the known sequence of peroxidase-induced mitoxantrone metabolism, and may contribute to the cytotoxic effects of the drug in vivo. Based on the structural features of the proposed MH₂ oxidation products we elaborate on various biochemical mechanisms, which extend the understanding of mitoxantrone’s pharmaceutical action and its clinical effectiveness with a particular focus on peroxidase-expressing solid tumors, such as breast carcinoma.     

The oxidation and decarboxylation of retinoic acid by horseradish peroxidase.

The decarboxylation of retinoic acid by horseradish peroxidase was investigated. A marked increase in the yield of products was obtained. However, the data indicated the reaction was a nonenzymatic, heme catalyzed peroxidation. Previously reported requirements for phosphate, oxygen and ferrous ion were eliminated when hydrogen peroxide was provided. Peroxide also eliminated the EDTA and cyanide induced inhibition of the phosphate dependent system. In the presence of hydrogen peroxide, horseradish peroxidase was not essential to the reaction; heme equivalent amounts of hemoglobin decarboxylated retinoic acid with equal facility. However, hemoglobin was ineffective in the absence of hydrogen peroxide. Attainment of 50--60% decarboxylation represented complete utilization of the available retinoic acid. Thus the products of the reaction can be divided into two groups, products of retinoic acid oxidation and products of an oxidative decarboxylation of retinoic acid.     

Kinetic evidence of horseradish peroxidase oxidation by compound I.

The kinetics of compound II formation, obtained upon mixing a highly purified horseradish peroxidase and hydrogen peroxide, was spectrophotometrically studied at three wavelengths in the absence of an added reducing agent. Our experiments confirm George's finding that more than one mole of compound II is formed per mole of hydrogen peroxide added. The new mechanism that we propose, contrary to the mechanism of George, is only valid when compound II is obtained in the absence of an added donor. Moreover, it is not inconsistent with the classical Chance mechanism of oxidation of an added donor by the system peroxidase -- hydrogen peroxide. According to this new mechanism, in the absence of an added donor, compound II formation involved two pathways. The first pathway is the monomolecular reduction of compound I by the endogenous donor, and the second pathway is the formation of two moles of compound II through the oxidoreduction reaction between one mole of peroxidase and one mole of compound I.     

Veratryl alcohol-mediated oxidation of isoeugenyl acetate by lignin peroxidase.

The mechanism of the veratryl alcohol (VA)-mediated oxidation of isoeugenyl acetate (IEA) by lignin peroxidase, and the subsequent spontaneous Calpha-Cbeta cleavage of IEA to vanillyl acetate were studied. IEA oxidation only occurred in the presence of VA. It probably did not bind to lignin peroxidase as evidenced by an unaffected Km for VA in the presence of IEA, and by the fact that a 10-fold molar excess of the unreactive IEA counterpart, eugenyl acetate, did not affect the IEA oxidation rate. IEA was very efficient in recycling VA. Up to 34 mol of IEA were oxidized per mol VA. Formation of the predominant VA oxidation product, veratraldehyde, was postponed until IEA was almost completely oxidized. Together these findings suggest that IEA was oxidized by VA.+ rather than directly by lignin peroxidase. Thus, VA functioned as a redox mediator during IEA oxidation which is remarkable considering the high calculated ionization potential of 8.81 eV. Regardless of the presence of O2, approximately 2 mol of IEA were consumed per mol H2O2, which indicated that IEA was enzymatically oxidized by one electron to the putative radical cation (IEA.+). After formation of IEA.+, a series of O2-dependent chemical reactions were responsible for Calpha-Cbeta cleavage to the major oxidation product vanillyl acetate, as evidenced by the observation that an N2 atmosphere did not inhibit IEA oxidation, but almost completely inhibited vanillyl acetate formation. GC-MS analyses revealed that under an air atmosphere 1-(4'-acetoxy-3'-methoxyphenyl)-2-propanone, 1-(4'-acetoxy-3'-methoxyphenyl)-1-hydroxy-2-propanone, and 1-(4'-acetoxy-3'-methoxyphenyl)-2-hydroxy-1-propanone were also formed. Formation of the latter two was diminished under an N2 atmosphere.     

The selective visualization of lignin peroxidase,manganese peroxidase and laccase,produced by white rot fungi on solid media

A visual method for the selective screening of lignin degrading enzymes, produced by white rot fungi (WRF), was investigated by the addition of coloring additives to solid media. Of the additives used in the enzyme production media, guaiacol and RBBR could be used for the detection of lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. Syringaldazine and Acid Red 264 were able for the detection of both the MnP and laccase, and the LiP and laccase, respectively, and a combination of these two additives was able to detect each of the ligninases produced by the WRF on solid media.     

Mechanism of action of lignins and lignin model compounds with horseradish peroxidase

Horseradish peroxidase displayed a ping-pong kinetic reaction mechanism with lignin model compounds and lignins. Oxidation of the α carbon on acetosyringone or acetovanillone failed above pH 6.5, while conversion of α-methylsyringyl (or guaiacyl) alcohol to acetosyringone (or vanillone) occurred optimally at pH 7.8. Small MW fragments were not formed from lignins at pH 6.4 and 7.8. These observations provide evidence for the growing concept that freely soluble peroxidase is not a lignolytic enzyme.     

Anomalous oxidation of MDL 73,404 by horseradish peroxidase

3,4-Dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopyran-2-ethanaminium-4-methylbenzene sulfonate (MDL 73,404) is a cardioselective water-soluble quaternary ammonium analogue of Vitamin E which is synthesized to augment the antioxidant defence in situations of free radical injury such as myocardial infarction/reperfusion. Its oxidation by any peroxidative enzyme has not been studied kinetically. This paper describes its enzymatic oxidation by horseradish peroxidase (HRP). The activity was followed spectrophotometrically at 255nm, and the experimental results were simulated using the program "KINETIC 3.1" for Windows 3.x. The MDL 73,404 was oxidized by horseradish peroxidase in the presence of H2O2 to its corresponding MDL 73,404 quinone. During this oxidation, the horseradish peroxidase showed an unexpectedly slow kinetic response with time, which contrast with the linear product accumulation curve measured with 2,2'-azino-bis-(3-estilbenzotiazol-6-sulfonic acid) (ABTS). This response was dependent on the respective concentrations of enzyme, MDL 73,404 and H2O2. However, when the enzyme was incubated with H2O2, the slow kinetic response disappeared and a lag period was observed. Furthermore, when p-coumaric acid (PCA) was added, the activity increased and the slow kinetic response became a straight line. In order to explain this anomalous behaviour, a kinetic model has been proposed and its differential equations simulated. From the correlation between experimental and simulated results it is concluded that MDL 73,404 can act as a slow response substrate for peroxidase, probably due to the presence of a quaternary ammonium side chain that confers on it a slow capacity to convert compound III into ferriperoxidase.     

Electroenzymatic oxidation of veratryl alcohol by lignin peroxidase

This paper reports the formation of veratraldehyde by electroenzymatic oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) hybridizing both electrochemical and enzymatic reactions and using lignin peroxidase. The novel electroenzymatic method was found to be effective for replacement of hydrogen peroxide by an electrochemical reactor, which is essential for enzyme activity of lignin peroxidase. The effects of operating parameters such as enzyme dosage, pH, and electric potential were investigated. Further, the kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to oxidation when hydrogen peroxide was supplied externally.     

Phlebia radiata laccase forms induced by veratric acid and xylidine in relation to lignin peroxidase and manganese-dependent peroxidase

White-rot basidiomycete Phlebia radiata grown in the nitrogen-poor liquid medium was tested for the inducing effect of 2,5-xylidine and veratric acid on laccase production and properties of its preparations. Also lignin peroxidase and manganese-dependent peroxidase were assayed under the same conditions. The maximum of laccase activity for veratric acid as a stimulator was done much earlier than that of xylidine. It was very sharp and disappeared quickly. At that time only weak lignin peroxidase and manganese-dependent peroxidase activities were noticed. The maximum of laccase induced by xylidine was observed much later and kept longer. Both laccase preparations showed the same properties. For biotechnological reasons, the production of laccase induced by the nontoxic veratric acid is much more economic and better acceptable than that induced by xylidine.     

Decolorization of Kraft effluent by free and immobilized lignin peroxidases and horseradish peroxidase

Color removal from Kraft effluent by lignin peroxidase and horseradish peroxidase was compared. Free lignin peroxidase and horseradish peroxidase removed color from kraft effluent. Immobilization of lignin peroxidase type III, lyophilized fungal culture and horseradish peroxidase on CNBr-Sepharose 4B improved the decolorization by factor of 2.9, 4.5 and 2.6, respectively in 48 h. Lignin peroxidase type I was effective only in the immobilized form in decolorization. In general, the immobilized form all the studied systems exhibited an average value around of 30% polymer consumption and very little of depolymerization. Lignin peroxidases and lyophilized fungal culture were shown to have considerable potential for treating Kraft effluents.     

The oxidation of veratryl alcohol, dimeric lignin models and lignin by lignin peroxidase: The redox cycle revisited

Abstract: The mechanism of oxidation of veratryl alcohol and β-0–4 dimeric lignin models is reviewed. Veratryl alcohol radicals are intermediates in both oxidation pathways. The possible role of the veratryl alcohol radical cation as a mediator is discussed. The lignin peroxidase (LIP) redox cycle is analyzed in terms of the Marcus theory of electron transfer. Reduction of both LiP-Compound I (LiP-I) and LiP-Compound II (LiP-II) by veratryl alcohol occurs in the endergonic region of the driving force. The reduction of LiP-II has a higher reorganization energy due to the change in spin state and the accompanying conformational change in the protein. It is suggested that a reversible nucleophilic addition of a carbohydrate residue located at the entrance of the active site channel plays a key role in the LiP redox cycle. Moreover. (polymeric) hydroxysubstituted benzyl radicals may reduce LiP-II via long-range electron transfer.