Phenolic compounds as likely natural mediators of laccase: A mechanistic assessment

The oxidation mechanism of non-phenolic substrates induced by laccase under catalysis by two phenolic mediators is shown to be radical.     

Whole-cell fungal transformation of precursors into dyes

Background  

Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes.     

Do the extracellular enzymes cellobiose dehydrogenase and manganese peroxidase form a pathway in lignin biodegradation?

The extracellular enzyme manganese peroxidase is believed to degrade lignin by a hydrogen peroxide-dependent oxidation of Mn(II) to the reactive species Mn(III) that attacks the lignin. However, Mn(III) is not able to directly oxidise the non-phenolic lignin structures that predominate in native lignin. We show here that pretreatment of a non-phenolic lignin model compound with another extracellular fungal enzyme, cellobiose dehydrogenase, allows the manganese peroxidase system to oxidise this molecule. The mechanism behind this effect is demethoxylation and/or hydroxylation, i.e. conversion of a non-phenolic structure to a phenolic one, mediated by hydroxyl radicals generated by cellobiose dehydrogenase. This suggests that cellobiose dehydrogenase and manganese peroxidase may act in an extracellular pathway in fungal lignin biodegradation. Analytical techniques used in this paper are reverse-phase high-pressure liquid chromatography, gas chromatography connected to mass spectroscopy and UV-visible spectroscopy.     

Characterization of two new multiforms of Trametes pubescens laccase

Electrochemical properties of two multiforms of laccase from Trametes pubescens basidiomycete (LAC1 and LAC2) have been studied. The standard redox potentials of the T1 sites of the enzymes were found to be 746 and 738 mV vs. NHE for LAC1 and LAC2, respectively. Bioelectroreduction of oxygen based on direct electron transfer between each of the two forms of Trametes pubescens laccase and spectrographic graphite electrodes has been demonstrated and studied. It is concluded that the T1 site of laccase is the first electron acceptor, both in solution (homogeneous case) and when the enzymes are adsorbed on the surface of the graphite electrode (heterogeneous case). Thus, the previously proposed mechanism of oxygen bioelectroreduction by adsorbed fungal laccase was additionally confirmed using two forms of the enzyme. Moreover, the assumed need for extracellular laccase to communicate directly and electronically with a solid matrix (lignin) in the course of lignin degradation is discussed. In summary, the possible roles of multiforms of the enzyme based on their electrochemical, biochemical, spectral, and kinetic properties have been suggested to consist in broadening of the substrate specificity of the enzyme, in turn yielding the possibility to dynamically regulate the process of lignin degradation according to the real-time survival needs of the organism.     

Peroxyl radicals are potential agents of lignin biodegradation

Past work has shown that the extracellular manganese-dependent peroxidases (MnPs) of ligninolytic fungi degrade the principal non-phenolic structures of lignin when they peroxidize unsaturated fatty acids. This reaction is likely to be relevant to ligninolysis in sound wood, where enzymes cannot penetrate, only if it employs a small, diffusible lipid radical as the proximal oxidant of lignin. Here we show that a non-phenolic beta-O-4-linked lignin model dimer was oxidized to products indicative of hydrogen abstraction and electron transfer by three different peroxyl radical-generating systems: (a) MnP/Mn(II)/linoleic acid, (b) arachidonic acid in which peroxidation was initiated by a small amount of H(2)O(2)/Fe(II), and (c) the thermolysis in air of either 4,4'-azobis(4-cyanovaleric acid) or 2,2'-azobis(2-methylpropionamidine) dihydrochloride. Some quantitative differences in the product distributions were found, but these were attributable to the presence of electron-withdrawing substituents on the peroxyl radicals derived from azo precursors. Our results introduce a new hypothesis: that biogenic peroxyl radicals may be agents of lignin biodegradation.     

Degradation of non-phenolic lignin by the white-rot fungus Pycnoporus cinnabarinus

High-molecular-weight lignin was methylated with diazomethane. The lignin (i.e., phenolic lignin) and methylated lignin (i.e., non-phenolic lignin) were mixed with fully bleached softwood pulp. Degradation of the lignin preparations by the white rot fungus Pycnoporus cinnabarinus was studied. After a 3-month incubation with the fungus, over 40% of the non-phenolic lignin and about 70% the phenolic lignin were degraded. The presence of phenolic hydroxyl groups in lignin greatly enhanced the degradation rate of lignin. This study reveals that P. cinnabarinus, an exclusively laccase-producing fungus, is capable of oxidatively degrading both phenolic and non-phenolic lignins. The ability of the fungus to degrade non-phenolic lignin suggests that a laccase/mediator system is involved in the complete degradation of lignin. After the fungal degradation of lignins, the content of carboxylic acids substantially increased for both phenolic and non-phenolic lignins.     

Significant enhancement in radical-scavenging activity of curcuminoids conferred by acetoxy substituent at the central methylene carbon

For a compound to be a radical-trapping antioxidant, the antioxidant-derived radical must be sufficiently inert to molecular oxygen as this would generate harmful chain-propagating peroxyl radicals. Curcumin has a unique structure with phenolic hydroxyl group as well as β-diketone moiety in the same molecule, both of which are able to donate electrons to free radicals. However, due to the reactivity toward molecular oxygen, the carbon-centered radical derived from β-diketone moiety do not serve as radical-trapping antioxidants. In this study, we reasoned that stabilization of the carbon-centered radical through substitution with an electron-withdrawing group would enhance the radical-scavenging antioxidative activity of the resulting curcuminoids. Thus, various substituents (methyl, allyl, methoxy, xanthate, and acetoxy) covering broad spectrum of the polar substituent effect were introduced to the central methylene position of both phenolic and non-phenolic curcuminoids. With the free phenolic hydroxyl groups present, the methylene-substituent did not exert significant effect on the antioxidant activity of the curcuminoids (EC(50)=23.2-30.3 μM) with the exception of the acetoxy-substituted derivative (EC(50)=8.7 μM) which showed more potent activity than curcumin (EC(50)=22.6 μM). When substituted to the non-phenolic curcumin scaffold, however, the methylene-substituent enhanced antioxidant activity of the otherwise inactive curcuminoids in the increasing order of methyl相似文献   

Catalytic Efficiency of some Mediators in Laccase-Catalyzed Alcohol Oxidation

The enzyme laccase oxidises phenolic groups of lignin but not the non-phenolic ones. Redox mediators activate laccase towards the non-phenolic groups, particularly the benzyl alcohols. The oxidation step is performed by the oxidised form of the mediator, generated on its interaction with laccase. The oxidised mediator can follow an electron transfer, a radical hydrogen atom transfer or an ionic mechanism in the oxidation of the non-phenolic subunits. Support for these conclusions is provided by (i) investigating the product pattern with suitable probe substrates, (ii) measuring the intramolecular kinetic isotope effect. Determination of electrochemical properties and bond dissociation energies via semiempirical calculations enabled us to rationalise the origin of the different mechanistic behaviour of the mediators. Finally, a comparison of different laccase-mediator-systems (LMS), when applied to the delignification of wood pulp, indicates violuric acid as the most efficient mediator, in an oxidation that is selectively directed towards lignin only.     

Catalytic Efficiency of some Mediators in Laccase-Catalyzed Alcohol Oxidation

The enzyme laccase oxidises phenolic groups of lignin but not the non-phenolic ones. Redox mediators activate laccase towards the non-phenolic groups, particularly the benzyl alcohols. The oxidation step is performed by the oxidised form of the mediator, generated on its interaction with laccase. The oxidised mediator can follow an electron transfer, a radical hydrogen atom transfer or an ionic mechanism in the oxidation of the non-phenolic subunits. Support for these conclusions is provided by (i) investigating the product pattern with suitable probe substrates, (ii) measuring the intramolecular kinetic isotope effect. Determination of electrochemical properties and bond dissociation energies via semiempirical calculations enabled us to rationalise the origin of the different mechanistic behaviour of the mediators. Finally, a comparison of different laccase-mediator-systems (LMS), when applied to the delignification of wood pulp, indicates violuric acid as the most efficient mediator, in an oxidation that is selectively directed towards lignin only.     

Effect of phenolic acids on manganese peroxidase-dependent peroxidation of linoleic acid and degradation of a non-phenolic lignin model compound

The influence of aromatic phenolic and non-phenolic acids on manganese peroxidase (MnP)-dependent peroxidation of linoleic acid, and oxidation of a non-phenolic lignin model compound (LMC) was studied. Phenolic compounds inhibited both the MnP-dependent lipid peroxidation (LPO) and non-phenolic LMC degradation in the system. The antioxidant activity of the aromatic compounds in the enzymatic system with MnP-dependent LPO depends on the presence of the phenolic hydroxyl groups attached to the aromatic ring structure, the methoxylation of the hydroxyl group in the ortho position in diphenolics, and number of carbon atoms in the side chain. Natural phenolic compounds inhibit the oxidation of non-phenolic lignin in the system based on MnP-mediated LPO, but do not prevent it. This result indicates that MnP-mediated LPO may play an important role in lignin degradation even in the presence of the phenolic antioxidant compounds, and supports the possibility of the involvement of LPO in the degradation of lignin in wood.     

Characterization of a low redox potential laccase from the basidiomycete C30.

A new exocellular laccase was purified from the basidiomycete C30. LAC2 is an acidic protein (pI = 3.2) preferentially produced upon a combined induction by copper and p-hydroxybenzoate. The spectroscopic signature (UV/visible and EPR) of this isoform is typical of multicopper oxidases, but its enzymatic and physico-chemical properties proved to be markedly different from those of LAC1, the constitutive laccase previously purified from the same organism. In particular, the LAC2 kcat values observed for the oxidation of the substrates syringaldazine (kcat = 65 600 min-1), ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (kcat = 41 000 min-1) and guaiacol (kcat = 75 680 min-1) are 10-40 times those obtained with LAC1 and the redox potential of its T1 copper is 0.17 V lower than that of LAC1 (E degrees = 0.73 V). This is the first report on a single organism producing simultaneously both a high and a low redox potential laccase. The cDNA, clac2, was cloned and sequenced. It encodes a protein of 528 amino acids that shares 69% identity (79% similarity) with LAC1 and 81% identity (95% similarity) with Lcc3-2 from Polyporus ciliatus (AF176321-1), its nearest neighbor in database. Possible reasons for why this basidiomycete produces, in vivo, enzyme forms with such different behaviors are discussed.     

Reactions of blue and yellow fungal laccases with lignin model compounds

Laccases of white-rot fungi Panus tigrinus, Phlebia radiata, and Phlebia tremellosa were isolated from cultures grown in liquid media which did not contain lignin and from the cultures grown on wheat straw. The physical and chemical properties of the laccases grown in submerged cultures were typical for blue fungal laccases. The laccases of the same fungi isolated from the solid-state cultures differed from the blue forms by lack of an absorption maximum at 610 nm. The typical blue laccases of P. tigrinus, Ph. radiata, and Ph. tremellosa acquired an ability to oxidize veratryl alcohol and a non-phenolic dimeric lignin model compound of beta-1-type only in the presence of a redox mediator, 2, 2'-azinobis(3-ethylbenzthiazolinesulfonic acid). The P. tigrinus and Ph. radiata yellow laccases catalyzed the oxidation of the same substrates without any mediator. The rate of the reaction of the blue laccases with a phenolic dimeric lignin model compound of beta-O-4-type was higher than that of the yellow laccases. The yellow laccases are apparently formed by the reaction of the blue laccases with low-molecular-weight lignin decomposition products.     

The reactivity of phenolic and non-phenolic residual kraft lignin model compounds with Mn(II)-peroxidase from Lentinula edodes

Three phenolic model compounds representing bonding patterns of residual kraft lignin were incubated with manganese peroxidase from Lentinula edodes. Extensive degradation of all the phenolic models, mainly occurring via side-chain benzylic oxidation, was observed. Among the tested model compounds the diphenylmethane alpha-5 phenolic model was found to be the most reactive, yielding several products showing oxidation and fragmentation at the bridging position. The non-phenolic 5-5' biphenyl and 5-5' diphenylmethane models were found unreactive.     

Laccases from Basidiomycetes: physicochemical characteristics and substrate specificity towards methoxyphenolic compounds

Laccases from the Basidiomycetes Coriolus hirsutus, Coriolus zonatus, Cerrena maxima, and Coriolisimus fulvocinerea have been isolated and purified to homogeneity and partially characterized. The kinetics of oxidation of different methoxyphenolic compounds by the fungal laccases has been studied. As laccase substrates, such methoxyphenolic compounds as 4-hydroxy-3,5-dimethoxycinnamic acid (sinapinic acid), 4-hydroxy-3-methoxycinnamic acid (ferulic acid), and 2-methoxyphenol (guaiacol) were used. The stoichiometries of the enzymatic reactions were determined: guaiacol and sinapinic acid are one-electron donors and their oxidation apparently results in the formation of dimers. It was established that k _cat/K _m, which indicates the effectiveness of catalysis, increases in the series guaiacol, ferulic acid, and sinapinic acid. This fact might be connected with the influence of substituents of the phenolic ring of the substrates. This phenomenon was established for fungal laccases with different physicochemical properties, amino acid composition, and carbohydrate content. This suggests that all fungal laccases possess the same mechanism of interaction between organic substrate electron donors and the copper-containing active site of the enzyme and that this interaction determines the observed values of the kinetic parameters.     

Oxidation of phenolic and non-phenolic substrates by the lignin peroxidase of Streptomyces viridosporus T7A

Summary Numerous single-ring, aromatic, phenolic and non-phenolic compounds were tested as substrates of Streptomyces viridosporus T7A extracellular lignin peroxidase. Oxidations were monitored by spectroscopy, with and without 4-aminoantipyrine (4-AAP) as a color-forming reagent. The oxidation of phenols containing one or no carbon groups in the para position resulted in coupling with 4-AAP to form a red color. Thin layer chromatography and mass spectroscopy showed that the oxidation of vanillic acid (4-hydroxy-3-methoxybenzoic acid) and syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) resulted in a direct coupling between 4-AAP and the phenol ring to form a quinone structure. In the reaction with vanillyl acetone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) and 4-AAP, 4-AAP coupled to Á-carbon of vanillyl acetone. As shown by UV-visible spectroscopy, S. viridosporus T7A peroxidase oxidized phenolic compounds, but was unable to oxidize non-phenolic ones.Paper no. 91 517 of the Idaho Agricultural Experiment Station Correspondence to: D. L. Crawford     

Oxidation of Benzidine and Its Derivatives by Thyroid Peroxidase

Human thyroid peroxidase (hTPO) catalyzes a one-electron oxidation of benzidine derivatives by hydrogen peroxide through classical Chance mechanism. The complete reduction of peroxidase oxidation products by ascorbic acid with the regeneration of primary aminobiphenyls was observed only in the case of 3,3',5,5'-tetramethylbenzidine (TMB). The kinetic characteristics (k(cat) and K(m)) of benzidine (BD), 3,3'-dimethylbenzidine (o-tolidine), 3,3'-dimethoxybenzidine (o-dianisidine), and TMB oxidation at 25 degrees C in 0.05 M phosphate-citrate buffer, pH 5.5, catalyzed by hTPO and horseradish peroxidase (HPR) were determined. The effective K(m) values for aminobiphenyls oxidation by both peroxidases raise with the increase of number of methyl and methoxy substituents in the benzidine molecule. Efficiency of aminobiphenyls oxidation catalyzed by either hTPO or HRP increases with the number of substituents in 3, 3', 5, and 5' positions of the benzidine molecule, which is in accordance with redox potential values for the substrates studied. The efficiency of HRP in the oxidation of benzidine derivatives expressed as k(cat)/K(m) was about two orders of magnitude higher as compared with hTPO. Straight correlation between the carcinogenicity of aminobiphenyls and genotoxicity of their peroxidation products was shown by the electrophoresis detecting the formation of covalent DNA cross-linking.     

Engineering and Applications of fungal laccases for organic synthesis

Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis.     

MAO inhibition by arylisopropylamines: the effect of oxygen substituents at the beta-position

Twenty-nine arylisopropylamines, substituted at the beta-position of their side chain by an oxo, hydroxy, or methoxy group, were evaluated in vitro as MAO-A and MAO-B inhibitors. The oxo derivatives ('cathinones') were in general less active as MAO-A inhibitors than the corresponding arylisopropylamines, but exhibited an interesting MAO-B inhibiting activity, which was absent in the hydroxy, methoxy, and beta-unsubstituted analogues. These results suggest that selective affinity for the two MAO isoforms in this family of compounds is modulated not only by the aryl substitution pattern but also by the side-chain substituents on the arylalkylamine scaffold.     

Antioxidative and antiradical properties of plant phenolics

The plant phenolic compounds such as flavonoids, tannins and phenolic acids appeared to be strong antiradical and antioxidant compounds. The number of hydroxy groups and the presence of a 2,3-double bond and orthodiphenolic structure enhance antiradical and antioxidative activity of flavonoids. The glycosylation, blocking the 3-OH group in C-ring, lack of a hydroxy group or the presence of only a methoxy group in B-ring have a decreasing effect on antiradical or antioxidative activity of these compounds. Tannins show strong antioxidative properties. Some tannins in red wine or gallate esters were proved to have antioxidative effect in vivo. The number of hydroxy groups connected with the aromatic ring, in ortho or para position relative to each other, enhance antioxidative and antiradical activity of phenolic acids. The substitution of a methoxy group in ortho position to the OH in monophenols seems to favour the antioxidative activity of the former.     

Salmonella typhimurium histidinol dehydrogenase: complete reaction stereochemistry and active site mapping

The stereochemistry of the L-histidinol dehydrogenase reaction was determined to be R at NAD for both steps, confirming previous results with a fungal extract [Davies, D., Teixeira, A., & Kenworthy, P. (1972) Biochem. J. 127, 335-343]. NMR analysis of monodeuteriohistidinols produced by histidinol/NADH exchange reactions arising via reversal of the alcohol oxidation reaction indicated a single stereochemistry at histidinol for that step. Comparison of vicinal coupling values of the exchange products with those of L-alaninol and a series of (S)-2-amino-1-alcohols allowed identification of the absolute stereochemistry of monodeuteriohistidinols and showed that histidinol dehydrogenase removes first the pro-S then the pro-R hydrogens of substrate histidinol. The enzyme stereochemistry was confirmed by isotope effects for monodeuteriohistidinols as substrates for the pro-R-specific dehydrogenation catalyzed by liver alcohol dehydrogenase. Active site mapping was undertaken to investigate substrate-protein interactions elsewhere in the histidinol binding site. Critical binding regions are the side-chain amino group and the imidazole ring, whose methylation at the 1- or 2-position caused severe decreases in binding affinity. Use of alternative substrates further clarified active site interactions with the substrate. Compounds in which the alpha-amino group was replaced by chloro, bromo, or hydrogen substituents were not substrates of the overall reaction at 1/10,000 the normal rate.(ABSTRACT TRUNCATED AT 250 WORDS)