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PubMed1987

Selection and improvement of lignin-degrading microorganisms: potential strategy based on lignin model-amino Acid adducts.

The purpose of this investigation was to test a potential strategy for the ligninase-dependent selection of lignin-degrading microorganisms. The strategy involves covalently bonding amino acids to lignin model compounds in such a way that ligninase-catalyzed cleavage of the models releases the amino acids for growth nitrogen. Here we describe the synthesis of glycine-N-2-(3,4-dimethoxyphenyl)ethane-2-ol (I) and demonstrate that growth (as measured by mycelial nitrogen content) of the known lignin-degrading basidiomycete Phanerochaete chrysosporium Burds. with compound I as the nitrogen source depends on its production of ligninase. Ligninase is shown to catalyze the oxidative C-C cleavage of compound I, releasing glycine, formaldehyde, and veratraldehyde at a 1:1:1 stoichiometry. P. chrysosporium utilizes compound I as a nitrogen source, but only after the cultures enter secondary metabolism (day 3 of growth), at which time the ligninase and the other components of the ligninolytic system (lignin --> CO(2)) are expressed. Compound I and related adducts have potential not only in the isolation of lignin-degrading microbes but, perhaps of equal importance, in strain improvement.

Journal Article↗
PubMed1990

Mn(II) Regulation of Lignin Peroxidases and Manganese-Dependent Peroxidases from Lignin-Degrading White Rot Fungi.

Two families of peroxidases-lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)-are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of CO(2) from C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of CO(2) release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium, two other species of Phanerochaete, three species of Phlebia, Lentinula edodes, and Phellinus pini.

Journal Article↗
PubMed1999

Coupling of manganese peroxidase-mediated lipid peroxidation with destruction of nonphenolic lignin model compounds and 14C-labeled lignins.

Linoleic acid, the predominant unsaturated fatty acid (UFA) in the lipids of wood-rotting fungi, was oxidized by manganese peroxidase (MnP) from the white-rot fungus Phlebia radiata through a peroxidation mechanism. The peroxidation was markedly stimulated by hydrogen peroxide. UFAs that are substrates for lipid peroxidation and surfactants that emulsify water-insoluble components were essential for the MnP-catalyzed destruction of a nonphenolic beta-O-4-linked lignin model compound (LMC). Moreover, both components stimulated the MnP-catalyzed mineralization of 14C-labeled synthetic lignin and 14C-labeled wheat straw. A high level of destruction was obtained in reaction systems with Tween 80 acting both as surfactant and source of UFAs. The presence of the linoleic acid in reaction systems with MnP and Tween 80 additionally enhanced rate and level of LMC destruction and lignin mineralization. The results indicate that lipid peroxidation may play an important role in lignin biodegradation by wood-rotting basidiomycetes and support the hypothesis of coupling between the processes.

Basidiomycota↗
PubMed1986

Transformations of arylpropane lignin model compounds by a lignin peroxidase of the white-rot fungus Phanerochaete chrysosporium.

Various lignin model compounds of the O-arylpropane type were oxidized with purified lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium, and oxidation products were identified by gas-chromatography/mass-spectroscopy procedures. Our results are in accord with the theory that lignin peroxidase catalyzes one-electron oxidations of its substrates with formation of cation radicals, and that these radicals undergo degradative reactions that are predictable from a knowledge of cation radical and oxygen chemistry. Cation radicals formed from O-arylpropane model compounds appeared to undergo the following types of degradative transformations: addition of water to ring-centered radicals, followed by proton loss yielding quinones and alcohols; nucleophilic attack by hydroxy functions on propanoid moieties giving cyclic ketals as intermediates which decompose to yield side chain migration products; transfer of the charge of a radical from a ring to the associated alkyl moiety through an ether bond, with loss of a proton from the latter, forming a new carbon-centered radical. The new alkyl-centered radicals apparently were able to abduct dioxygen to form peroxyl radicals which decomposed giving a variety of oxidation products and probably superoxide anion. Specific examples of the above transformations are presented, and their relevance to lignin degradation is discussed.

Basidiomycota↗
PubMed2000

Altered lignin composition in phenylalanine ammonia-lyase-inhibited radish seedlings: implications for seed-derived sinapoyl esters as lignin precursors.

We earlier reported that when phenylalanine ammonia-lyase (PAL) activity in radish seedlings was inhibited by the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP), soluble sinapoyl esters carried over from the seed were converted to wall-bound esters in young cotyledons. We now report that these soluble sinapoyl esters may also be converted into lignin in the cotyledons. When radish seedlings were grown in the presence of 100 microM AIP, lignin formation (determined as lignothioglycolic acid) was inhibited ca. 74% in the cotyledons and ca. 80% in hypocotyls plus roots. The syringyl to guaiacyl (S/G) ratio in the lignin of AIP-grown plants, as determined by alkaline cupric oxidation and from Fourier-transform infrared (FT-IR) spectra, was higher in cotyledons, but lower in hypocotyls plus roots, as compared to plants grown on distilled water. These results support the view that soluble sinapoyl esters preformed in seeds may contribute to the syringyl moiety of lignin in cotyledons during early seedling development and that there is no appreciable transport of soluble sinapoyl esters from cotyledons to the hypocotyls and roots.

Brassica↗
PubMed1994

H2O2 recycling during oxidation of the arylglycerol beta-aryl ether lignin structure by lignin peroxidase and glyoxal oxidase.

Oxidative C alpha-C beta cleavage of the arylglycerol beta-aryl ether lignin model 1-(3,4-dimethoxy-phenyl)-2-phenoxypropane-1,3-diol (I) by Phanerochaete chrysosporium lignin peroxidase in the presence of limiting H2O2 was enhanced 4-5-fold by glyoxal oxidase from the same fungus. Further investigation showed that each C alpha-C beta cleavage reaction released 0.8-0.9 equiv of glycolaldehyde, a glyoxal oxidase substrate. The identification of glycolaldehyde was based on 13C NMR spectrometry of reaction product obtained from beta-, gamma-, and beta,gamma-13C-substituted I, and quantitation was based on an enzymatic NADH-linked assay. The oxidation of glycolaldehyde by glyoxal oxidase yielded 0.9 oxalate and 2.8 H2O2 per reaction, as shown by quantitation of oxalate as 2,3-dihydroxyquinoxaline after derivatization with 1,2-diaminobenzene and by quantitation of H2O2 in coupled spectrophotometric assays with veratryl alcohol and lignin peroxidase. These results suggest that the C alpha-C beta cleavage of I by lignin peroxidase in the presence of glyoxal oxidase should regenerate as many as 3 H2O2. Calculations based on the observed enhancement of LiP-catalyzed C alpha-C beta cleavage by glyoxal oxidase showed that approximately 2 H2O2 were actually regenerated per cleavage of I when both enzymes were present. The cleavage of arylglycerol beta-aryl ether structures by ligninolytic enzymes thus recycles H2O2 to support subsequent cleavage reactions.

Alcohol Oxidoreductases↗
PubMed1990

Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: role of veratryl alcohol in lignin biodegradation.

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H2O2-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H2O2 concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H2O2 to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H2O2 and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H2O2. AA competitively inhibited the oxidation of VA by LiP; the Ki for AA inhibition was 32 microM. Addition of VA to LiPIII* resulted in its conversion to the native enzyme. In contrast, AA did not convert LiPIII* to the native enzyme; instead, LiPIII* was bleached in the presence of AA. Thus, AA does not protect LiP from inactivation by H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Anisoles↗
PubMed2004

Solution-state nuclear magnetic resonance study of the similarities between milled wood lignin and cellulolytic enzyme lignin.

The structures of milled wood lignin (MWL) and cellulolytic enzyme lignin (CEL) have been analyzed using traditional chemical methods and solution-state NMR techniques. Comparisons of the results obtained reveal that subtle differences exist between the two lignin preparations. Thioacidolysis produced higher monomer yields from CEL than MWL, suggesting MWL has a more condensed structure. Quantitative (13)C NMR determined the degree of condensation in MWL to be 0.43 unit per aromatic moiety as compared to 0.36 in CEL. The MWL also contained a lower amount of beta-O-4' substructures per aromatic ring than CEL, 0.41 versus 0.47, respectively. Carbohydrate analysis revealed that the MWL may contain a higher proportion of middle lamella material as compared to the CEL. Because the middle lamella is considered to have a more condensed lignin structure, on the basis of the bulk polymerization theory, these results could explain the differences in beta-O-4' and degree of condensation.

Carbon↗
PubMed2002

Singlet oxygen mediated degradation of lignin--isolation of oxidation products from steam-exploded lignin from pine.

Lignin obtained from steam explosion of pine was fully characterized. Elemental analysis, GPC, and ultraviolet and 1H and 13C NMR spectra revealed that the obtained lignin contains both guaiacyl and syringyl units. Lignin was dissolved in acetonitrile-ethanol and treated with visible light in the presence of both oxygen and Rose Bengal for different irradiation times. Column chromatography of the residue showed the presence of six compounds: trans-sinapyl alcohol, 4-hydroxy-3,5-dimethoxybenzaldehyde, 4-hydroxy-3,5-dimethoxyphenylacetone, 4-hydroxy-3-methoxybenzaldehyde, cis-sinapyl alcohol, and sinapyl aldehyde. The total amount of fine chemicals increases with the irradiation time. However, it increases rapidly during the first eight hours, but increases slowly after this period. The most important compounds obtained were sinapyl alcohol and 4-hydroxy-3,5-dimethoxybenzaldehyde, but sinapyl alcohol was obtained in the highest amounts after eight hours' irradiation, while the highest amounts of 4-hydroxy-3,5-dimethoxybenzaldehyde were obtained after irradiation for 4 h. After 48 h irradiation comparable amounts of sinapyl aldehyde were obtained. We obtained only compounds derived from the syringyl units in lignin in agreement with the hypothesis that the guaiacyl units are more easily oxidised.

Lignin↗
PubMed1998

The DFRC Method for Lignin Analysis. 4. Lignin Dimers Isolated from DFRC-Degraded Loblolly Pine Wood.

Sixteen lignin dimers were directly isolated by gel permeation and reversed-phase TLC and HPLC from loblolly pine (Pinus taeda L.) sapwood following large-scale application of the new derivatization followed by reductive cleavage (DFRC) procedure. Their structures were elucidated by GC/MS and NMR. These dimers included representatives from all of the common interunit linkages in softwood lignins (beta-1, beta-beta, 5-5, beta-5, and 5-O-4). The DFRC procedure efficiently cleaves beta-aryl ethers, the major structural unit, so beta-ethers were not found among the dimeric products. All but two of the isolated dimers were guaiacyl-guaiacyl (from coniferyl alcohol-derived units in lignin); small amounts of p-hydroxyphenyl/guaiacyl beta-5 and beta-1 dimers were characterized. Several dimers have benzaldehydes as one unit-since aldehydes are not created by the procedure, they are either present in the native lignin or are formed during the minimal preparation of the wood. The dimers isolated and identified here will eventually allow their quantitation to aid in the structural analyses of wide-ranging plant materials.

Journal Article↗
PubMed1997

Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase.

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.

Journal Article↗
PubMed1993

Lignin peroxidase L3 from Phlebia radiata. Pre-steady-state and steady-state studies with veratryl alcohol and a non-phenolic lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol.

The catalytic cycle of lignin peroxidase (LiP, ligninase) isozyme L3 from the white-rot fungus Phlebia radiata was investigated using stopped-flow techniques. Veratryl (3,4-dimethoxybenzyl) alcohol and a lignin model compound, non-phenolic beta-O-4 dimer 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol, were used as electron donors. This is the first report on the detailed kinetic analysis of a LiP-catalysed C alpha-C beta bond cleavage of the dimer, representing the major depolymerisation reaction in the lignin polymer. The native enzyme showed a typical heme peroxidase absorbance spectrum with a Soret maximum at 407 nm. Following the reaction with H2O2, the Soret band decreased in absorbance, shifted to 403 nm and then to 421 nm, demonstrating the formation of compound I followed by the formation of compound II, respectively. Similar results have been reported for the LiP from Phanerochaete chrysosporium upon reaction with H2O2. However, compound I of L3 was more stable in the absence of additional electron donors. The second-order rate constant of compound I formation by H2O2 was determined to be 6 x 10(5) M-1 s-1 and was the same at pH 3.0 and 6.0. Compound I was rapidly reduced to compound II and further to native enzyme when either veratryl alcohol or the beta-O-4 dimer was supplied as electron donor and in both cases veratraldehyde appeared as the major product. At pH 6.0, the second-order rate constant for compound II formation was similar with either veratryl alcohol or the beta-O-4 dimer (6.7 x 10(3) and 6.5 x 10(3) M-1 s-1, respectively). At pH 3.0 formation of compound II with either reductant proceeded so rapidly that determination of the respective rate constants was not possible. The results point to identical catalytic cycles of L3 with veratryl alcohol or the beta-O-4 dimer involving both compounds I and II as intermediates and participation of the same veratryl alcohol radical as the most appropriate reductant for compound II. Chemical evidence of such a radical, formed after the initial LiP-catalysed one-electron oxidation of beta-O-4 dimeric lignin models, is presented in a separate article [Lundell, T., Schoemaker, H., Hatakka, A. & Brunow, G. (1993) Holzforschung, in the press]. The catalytic redox-cycle and the oxidation mechanism presented here reconcile seemingly contradictory results obtained in previous studies on LiP kinetics during the last decade.

Benzyl Alcohols↗
PubMed1985

Simplified procedure for recovery of lignin acidolysis products for determining the lignin-degrading abilities of microorganisms.

A simplified procedure for the identification and measurement of single-ring aromatic products of lignin acidolysis is described. The procedure employed a 6-h hydrolysis of spruce milled wood lignin in acidic dioxane at 87 degrees C, followed by a series of organic extractions to recover acidolysis products which were quantified by gas chromatography of trimethylsilyl derivatives. Complex gel permeation chromatography procedures utilized by other workers were avoided in the modified procedure, but equivalent results were obtained. The simplified procedure was utilized to hydrolyze sound and actinomycete-decayed spruce milled wood lignins and was shown to be useful as a technique for the rapid screening of microorganisms for their ability to alter lignin.

Journal Article↗
PubMed1990

Mineralization of C-Ring-Labeled Synthetic Lignin Correlates with the Production of Lignin Peroxidase, not of Manganese Peroxidase or Laccase.

Recently, Mn(II) has been shown to induce manganese peroxidases (MnPs) and repress lignin peroxidases (LiPs) in defined liquid cultures of several white rot organisms. The present work shows that laccase is also regulated by Mn(II). We therefore used Mn(II) to regulate production of LiP, MnP, and laccase activities while determining the effects of Mn(II) on mineralization of ring-labeled synthetic lignin. At a low Mn(II) level, Phanerochaete chrysosporium and Phlebia brevispora produced relatively high titers of LiPs but only low titers of MnPs. At a high Mn(II) level, MnP titers increased 12- to 20-fold, but LiPs were not detected in crude broths. P. brevispora formed much less LiP than P. chrysosporium, but it also produced laccase activity that increased more than sevenfold at the high Mn(II) level. The rates of synthetic lignin mineralization by these organisms were similar and were almost seven times higher at low than at high Mn(II). Increased synthetic lignin mineralization therefore correlated with increased LiP, not with increased MnP or laccase activities.

Journal Article↗
PubMed2004

Lignin dehydrogenative polymerization mechanism: a poplar cell wall peroxidase directly oxidizes polymer lignin and produces in vitro dehydrogenative polymer rich in beta-O-4 linkage.

An investigation was performed to determine whether lignin dehydrogenative polymerization proceeds via radical mediation or direct oxidation by peroxidases. It was found that coniferyl alcohol radical transferred quickly to sinapyl alcohol. The transfer to syringaresinol was slower, however, the transfer to polymeric lignols occurred very slightly. This result suggests that the radical mediator theory does not sufficiently explain the mechanism for dehydrogenative polymerization of lignin. A cationic cell wall peroxidase (CWPO-C) from poplar (Populus alba L.) callus showed a strong substrate preference for sinapyl alcohol and the sinapyl alcohol dimer, syringaresinol. Moreover, CWPO-C was capable of oxidizing high-molecular-weight sinapyl alcohol polymers and ferrocytochrome c. Therefore, the CWPO-C characteristics are important to produce polymer lignin. The results suggest that CWPO-C may be a peroxidase isoenzyme responsible for the lignification of plant cell walls.

Alcohols↗
PubMed2001

Bonding of hydroxycinnamic acids to lignin: ferulic and p-coumaric acids are predominantly linked at the benzyl position of lignin, not the beta-position, in grass cell walls.

A suspension in dichloromethane-water (18:1, v/v) of various fractions containing hydroxycinnamic acid ester-ether bridges between lignin and polysaccharides prepared from cell walls of matured oat (Avena sativa L.) intemodes, and a solution of their acetates in the same solvent, were treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). This reagent selectively cleaves benzyl ether and ester linkages of negatively charged aromatic nuclei. The sample treated with DDQ was directly hydrolysed either under mild (1 M NaOH, overnight at 37 degrees C) or severe (4 M NaOH, for 2 h at 170 degrees C) conditions. The hydroxycinnamic acids released in the hydrolysate were methylated with diazomethane and analysed quantitatively using gas chromatography. Significant portions of ether linkages between hydroxycinnamic acids and lignin were cleaved with DDQ, which suggests that most of the hydroxycinnamic acids were ether-linked at the benzyl position, and not the beta-position, of the lignin side chain as previously claimed.

Avena↗
PubMed2006

Differences between lignin in unprocessed wood, milled wood, mutant wood, and extracted lignin detected by 13C solid-state NMR.

Solid-state 13C nuclear magnetic resonance (NMR) spectroscopy was applied to intact and isolated loblolly pine wood samples to identify potential structural changes induced by tree age, milling, lignin extraction, or naturally occurring mutations. Special attention was paid to ketone and aldehyde as well as nonpolar alkyl groups, which could be observed at low concentrations (<2 in 1000 C) using improved spinning-sideband suppression with gated decoupling. Carbonyl structures were present in intact wood, and there are more keto groups than aldehydes. Their concentrations increased from juvenile to mature wood and with milling time, whereas extraction did not alter the C=O fraction. Significant amounts of aldehyde and dihydroconiferyl alcohol residues were present in coniferyl aldehyde dehydrogenase-deficient wood, confirming solution-state NMR spectra of the corresponding lignin. These results demonstrate the utility of solid-state NMR as an assay for changes in the lignin structure of genetically modified plants.

Alcohols↗