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New insights into the structural organization of the plant polymer lignin.

The organizational features of lignin structure and the mechanism of its synthesis have significant implications for the response of the plant to stress. It was unknown whether the enzymic formation of lignin in the cell wall is an uncontrolled process or finely regulated in time and space. In vitro scanning tunneling microscopy (STM), atomic force microscopies (AFM), near-field scanning optical microscopy (NSOM). and the novel environmental scanning electron microscopy (ESEM) imaging studies of the lignin model compounds have directly shown its highly ordered structure and elucidated its modular and fractal organization. Direct evidence was presented for the existence of strong intermolecular forces responsible for holding lignin globules together in highly ordered structures. Fractal analysis was applied as a theoretical approach, to show regularity and modular organization of lignin. Surface chemistry studies of the lignin monolayer reveal intrinsic properties that may be a key to osmotic pressure and cell size control mechanism in the higher plant cells. The obtained data contribute to the explanation of the mechanisms of cell wall synthesis in vivo.

Cell Size↗

Genetic and biochemical investigations on bacterial catabolic pathways for lignin-derived aromatic compounds.

Lignins are the most abundant aromatic compounds in nature, and their decomposition is essential to the terrestrial carbon cycle. White rot fungi secreting phenol oxidases are assumed to be involved in the initial degradation of native lignin, whereas bacteria play a main role in the mineralization of lignin-derived low-molecular-weight compounds in soil. There are a number of reports on the degradation pathways for lignin-derived aromatic compounds, but their catabolism has not been enzymatically or genetically characterized. Sphingomonas paucimobilis SYK-6 is one of the best-characterized lignin-degrading bacteria. It can grow on a wide variety of lignin-related biaryls and monoaryls, including beta-aryl ether, biphenyl, diarylpropane, and phenylpropane. These compounds are degraded via the protocatechuate (PCA) 4,5-cleavage pathway or multiple 3-O-methylgallate (3MGA) catabolic pathways. In this review, the enzyme systems for beta-aryl ether and biphenyl degradation, O demethylation linked with one carbon metabolism, the PCA 4,5-cleavage pathway, and the multiple 3MGA catabolic pathways in SYK-6 are outlined.

Bacteria↗

Strategies to enhance the enzymatic hydrolysis of pretreated softwood with high residual lignin content.

Pretreatment of Douglas-fir by steam explosion produces a substrate containing approx 43% lignin. Two strategies were investigated for reducing the effect of this residual lignin on enzymatic hydrolysis of cellulose: mild alkali extraction and protein addition. Extraction with cold 1% NaOH reduced the lignin content by only approx 7%, but cellulose to glucose conversion was enhanced by about 30%. Before alkali extraction, addition of exogenous protein resulted in a significant improvement in cellulose hydrolysis, but this protein effect was substantially diminished after alkali treatment. Lignin appears to reduce cellulose hydrolysis by two distinct mechanisms: by forming a physical barrier that prevents enzyme access and by non-productively binding cellulolytic enzymes. Cold alkali appears to selectively remove a fraction of lignin from steam-exploded Douglas-fir with high affinity for protein. Corresponding data for mixed softwood pretreated by organosolv extraction indicates that the relative importance of the two mechanisms by which residual lignin affects hydrolysis is different according to the pre- and post-treatment method used.

Cell Culture Techniques↗

Treatment with lignin residue: a novel method for detoxification of lignocellulose hydrolysates.

Acid hydrolysis of lignocellulose to hydrolysates intended for production of fuel ethanol results in the formation of byproducts in addition to fermentable sugars. Some of the byproducts, such as phenolic compounds and furan aldehydes, are inhibitory to the fermenting microorganism. Detoxification of the hydrolysates may be necessary for production of ethanol at a satisfactory rate and yield. The lignin residue obtained after hydrolysis is a material with hydrophobic properties that is produced in large amounts as a byproduct within an ethanol production process based on lignocellulosic raw materials. We have explored the possibility of using this lignin residue for detoxification of spruce dilute-acid hydrolysates prior to fermentation with Saccharomyces cerevisiae. Three dilute-acid hydrolysates of spruce were treated with lignin residue, which in all cases resulted in improved fermentability in terms of productivity and yield of ethanol. The effect was improved by washing the lignin before treatment, by using larger amounts of lignin in the treatment, and by performing the treatment at low temperature. Treatment with the lignin residue removed up to 53% of the phenolic compounds and up to 68% of the furan aldehydes in a spruce dilute-acid hydrolysate. A larger fraction of furfural was removed compared to the less hydrophobic 5-hydroxymethylfurfural.

Biotechnology↗

Lignin radicals in the plant cell wall probed by Kerr-gated resonance Raman spectroscopy.

Lignin radicals are crucial intermediates for lignin biosynthesis in the cell wall of vascular plants. In this work they were for the first time, to our knowledge, selectively observed in wood cell walls by laser-based Kerr-gated resonance Raman spectroscopy, and the observations were supported by density functional theory prediction of their vibrational properties. For dry wood cells a lignin radical Raman band is observed at 1,570 cm(-1) irrespective of species. For wet beech cells they were generated in situ and observed at 1,606 cm(-1). DFT/B3LYP/6-31+G(d) modeling results support that in beech they are formed from syringyl (S) phenolic moieties and in spruce from guaiacyl (G) phenolic moieties. The observed lignin radical band is predicted as G is approximately 1,597 cm(-1) and S is approximately 1,599 cm(-1), respectively, and is assigned the (Wilson notation) nu(8a) phenyl ring mode. The RR band probes lignin radical properties, e.g., spin density distribution, and these respond to charge polarization or hydrogen bonding to proximate water molecules. These observations can be crucial for an understanding of the factors that control cell wall structure during biosynthesis of vascular plants and demonstrate the unique potential of RR spectroscopy of lignin radicals.

Cell Wall↗

In vitro simulation studies of silica deposition induced by lignin from rice.

To reveal the possible mechanism of silica deposition in higher plants, lignin was isolated from rice straw following a modified method to conduct a simulation experiment in vitro. UV and infrared absorption spectra showed that the substance had the unique characteristics of pure lignin. The presence of silicon in the precipitation was revealed by TEM (transmission electron microscopy) with EDXA (energy dispersive X-ray analysis) device. It was found that in the borax solution where lignin precipitation occurred silica-lignin co-precipitation was produced but not in the DMSO solution where lignin was broken into its composition compounds and did not precipitate. This means that it is macromolecular lignin itself but not its compounds that could induce silica deposition in higher plants.

Lignin↗

Hydrogenolysis of lignins: influence of the pretreatment using microwave and ultrasound irradiations.

Hydrolytic eucalyptus lignin was converted to oils by hydrogenolysis. The lignin was obtained by acid hydrolysis of eucalyptus chips from two semi-industrial sources in Brazil: FTI and Coalbra. Hydrogenolysis was performed in an 1-L reactor using lignin/water ratio 1/4 (w/v), pH 9 (adjusted with NaOH), 0.1 g sodium formate/g lignin, 20 MPa (argon pressure) at 280 degrees C for 15 min. After reaction, the products were filtered and the solids extracted with chloroform/ethanol 3/1 (v/v). The solvent was evaporated from the organic phase and a dark oil was obtained. The solid remaining after extraction was weighed to calculate the conversion. Without pretreatment conversions were 41.3 and 47.9% for Coalbra and FTI, respectively. The oil yields were 22.7-27.6% for Coalbra and FTI, respectively. Using microwave pretreatment (30 min of irradiation at 490 W) the conversions were 41.6-50.5% and the oil yields increased slightly to 25.2-31.4%. The polymeric chains in the lignin breakdown due to the action of water near the boiling point under microwave irraditation. On the other hand, by using ultrasound (30 min at 50 degrees C in a ultrasound bath of 25 kHz and 0.8 W/cm2) the conversions were 35.2-46.9% and oil yields were 22.0-27.1%. Ultrasound favors the formation of radicals that probably caused the reticulation of lignins, decreasing the conversion and yield. Oils analyzed by infrared spectroscopy showed an increase in C=O bond intensities, compared with the original lignins.

Carbohydrates↗

[Preparation of spherical lignin cation adsorption resin with black pulping liquor].

Spherical lignin cation adsorption resin was obtained by two-step method with black liquor of paper mill and characterized by the Scanning electron microscopy (SEM) and Fourier transform infrared spectrometry (FTIR). Firstly, the spherical lignin beads were prepared with black liquor of paper mill by applying reverse-phase suspension polymerization technique, and the preparation conditions were also optimized. The suitable conditions include 200 r/min of stirring speed, 90 degrees C of reaction temperature, 1.0 h of reaction time and use epoxy chloropropane (1.5% by weight of lignin) as cross-linking agent, the ideal volume ratio between oil phase and water phase was 3:1 by using kerosene oil as disperse phase, and the perfect dispersant agent was Tween 80 (3% by weight of lignin). Secondly, the spherical lignin cation adsorption resin was developed by grafting the acrylamide onto the back-bone of the spherical lignin beads. The best concentration of acrylamide was 0.72 mol/L at ambient temperature for 2.0 h, and the ideal initiator system was the Fenton reagent of H2O2/Fe2+. Under the above conditions, the ion exchange capacity of the prepared resin could reach 1.640 5 mmol/g.

Adsorption↗

Ligninolysis by a purified lignin peroxidase.

The lignin peroxidases (LiPs) of white-rot basidiomycetes are generally thought to catalyze the oxidative cleavage of polymeric lignin in vivo. However, direct evidence for such a role has been lacking. In this investigation, 14C- and 13C-labeled synthetic lignins were oxidized with a purified isozyme of Phanerochaete chrysosporium LiP. Gel permeation chromatography of the radiolabeled polymers showed that LiP catalyzed their cleavage to give soluble lower-M(r) products. To a lesser extent, the enzyme also polymerized the lignins to give soluble higher-M(r) products. This result is attributable to the fact that purified LiP, unlike the intact fungus, provides no mechanism for the removal of lignin fragments that are susceptible to repolymerization. LiP catalysis also gave small quantities of insoluble, perhaps polymerized, lignin, but in lower yield than intact P. chrysosporium does. 13C NMR experiments with 13C-labeled polymer showed that LiP cleaved it between C alpha and C beta of the propyl side chain to give benzylic aldehydes at C alpha, in agreement with the cleavage mechanism hypothesized earlier. The data show that LiP catalysis accounts adequately for the initial steps of ligninolysis by P. chrysosporium in vivo.

Benzyl Alcohols↗

Purification of major lignin peroxidase isoenzymes from Phanerochaete chrysosporium by chromatofocusing.

The basidiomycete Phanerochaete chrysosporium produces several isoforms of lignin peroxidase, which catalyzes the oxidative depolymerization of lignin To date, ion-exchange chromatography and preparative isoelectric focusing (IEF) have been commonly used for isolation of lignin peroxidase isoenzymes. In this work we have purified major lignin peroxidases to high purity by a one-step chromatographic method, chromatofocusing. The purified isoenzymes were identified by analytical IEF using isoenzymes purified by preparative IEF as standards. The specific activities and spectral properties of the isoenzymes were comparable with the previously published data. The predominant isoenzyme under the growth conditions used was LiP 4.65. Almost 50% of the lignin peroxidase activity applied into the column was recovered in the LiP 4.65 fraction. The total recovery of the lignin peroxidase activity was over 80%.

Chromatography, Ion Exchange↗

Synthesis and properties of lignin peroxidase from Streptomyces viridosporus T7A.

The production of lignin peroxidase by Streptomyces viridosporus T7A was studied in shake flasks and under aerobic conditions in a 7.5-L batch fermentor. Lignin peroxidase synthesis was found to be strongly affected by catabolite repression. Lignin peroxidase was a non-growth-associated, secondary metabolite. The maximum lignin peroxidase activity was 0.064 U/mL at 36 h. In order to maximize lignin peroxidase activity, optimal conditions were determined. The optimal incubation temperature, pH, and substrate (2,4-dichlorophenol) concentration for the enzyme assays were 45 degrees C, 6, and 3 mM, respectively. Stability of lignin peroxidase was determined at 37, 45, and 60 degrees C, and over the pH range 4-9.

Enzyme Repression↗

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.

Cloning, Molecular↗

Lignin-degrading enzyme production by Bjerkandera adusta immobilized on polyurethane foam.

Production of the lignin-degrading enzymes lignin peroxidase (Lip), manganese peroxidase (MnP), and laccase (Lac) by the white-rot fungus Bjerkandera adusta was investigated experimentally using polyurethane foam (PUF) as a carrier of immobilized fungal mycelia. An immobilized cell culture with a low-nitrogen medium yielded significantly greater LiP, MnP, and Lac activities in comparison with those obtained in a liquid culture. The maximum activities of the three enzymes were 450, 370, and 100 U/ml, respectively, under the following incubation condition: glucose concentration, 20 g/l; temperature, 30 degrees C; pH 4.5. The activities of MnP and Lac were significantly higher than those reported using other incubation methods. Lignin was degraded to the extent of 40% and its decolorization ratio was about 70% at an incubation time of 40 h using lignin-degrading enzymes from B. adusta. Six different isozymes of MnP were synthesized by B. adusta, two of which exhibited high MnP activity. Our preliminary finding that extracellular enzymes from B. adusta are capable of degrading and decoloring lignin makes these enzymes attractive for further research aimed at their large-scale application in lignin depolymerization, pulp biobleaching, and the degradation of toxic pollutants.

Journal Article↗

Lignin peroxidases can also oxidize manganese.

The peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium include lignin peroxidases and manganese-dependent peroxidases. The major isozymes, called lignin peroxidases, are thought to oxidize chemicals directly. The manganese-dependent peroxidases (H3, H4, H5, and H9) are relatively minor, making up only a fraction of the total peroxidase protein. However, we have found that lignin peroxidases will also catalyze the H2O2-dependent oxidation of Mn2+ to Mn3+. We have used lignin peroxidase isozyme H2 (LiPH2) to characterize the manganese peroxidase activity of lignin peroxidases. Transient state kinetic studies were used to obtain a second-order rate constant of 4.2 x 10(4) M-1 S-1 for the reaction of LiPH2-compound I with free or chelated Mn2+ at pH 6.0. This reaction was too fast to monitor at pH 4.5. Only chelated Mn2+ could reduce LiPH2-compound II to ferric enzyme. The Mn(2+)-chelate (oxalate) first bound LiPH2-compound II with a Kd of (1.5 +/- 0.3) x 10(-5) M and then reduced LiPH2-compound II to ferric enzyme with a first order rate constant of 215 +/- 6 S-1. Steady-state kinetic studies on LiPH2 were performed by directly monitoring the formation of Mn(3+)-oxalate. These results show that oxidation of Mn2+ by a lignin peroxidase does not occur through free radical mediation as proposed previously [Popp et al. (1990) Biochemistry 29, 10475-10480). Electron spin resonance and oxygen evolution studies also indicate that Mn2+ is directly oxidized by LiPH2.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗

Haloperoxidase activity of Phanerochaete chrysosporium lignin peroxidases H2 and H8.

Monochlorodimedone (MCD), commonly used as a halogen acceptor for haloperoxidase assays, was oxidized by hydrogen peroxide in the presence of lignin peroxidase isoenzymes H2 and H8. When oxidized, it produced a weak absorption band with an intensity that varied with pH. This absorbance was used as a simple method for the product analysis because it disappeared when MCD was brominated or chlorinated. We assessed the activity of the lignin peroxidases for oxidation of bromide by measuring the bromination of MCD, the formation of tribromide, the bromide-mediated oxidation of glutathione, and the bromide-mediated catalase-like activity. We analyzed the reaction products of MCD and the halide-mediated oxidation of glutathione when bromide was replaced by chloride. These enzymes demonstrated no significant activity for oxidation of chloride. Unlike other peroxidases, the lignin peroxidases exhibited similar pH-activity curves for the iodide and bromide oxidations. The optimum pH for activity was about 2.5. Surprisingly, this pH dependence of lignin peroxidase activity for the halides was nearly the same in the reactions with hydrogen donors, such as hydroquinone and guaiacol. The results suggested that protonation of the enzymes with pKa approximately 3.2 is necessary for the catalytic function of lignin peroxidases, irrespective of whether the substrates are electron or hydrogen donors. These unique reaction profiles of lignin peroxidases are compared to those of other peroxidases, such as lactoperoxidase, bromoperoxidase, chloroperoxidase, and horseradish peroxidase. Isozyme H2 was more active than isozyme H8, but isozyme H8 was more stable at very acidic pH.

Basidiomycota↗

Lignin peroxidase: resonance Raman spectral evidence for compound II and for a temperature-dependent coordination-state equilibrium in the ferric enzyme.

Resonance Raman (RR) spectroscopy of lignin peroxidase (ligninase, dairylpropane oxygenase) from the basidiomycete Phanerochaete chrysosporium suggests two different coordination states for the native ferric enzyme. Evidence for a high-spin, hexacoordinate ferric protoporphyrin IX was presented by Andersson et al. [Andersson, L. A., Renganathan, V., Chiu, A.A., Loehr, T. M., & Gold, M. H. (1985) J. Biol. Chem. 260, 6080-6087], whereas Kuila et al. [Kuila, D., Tien, M., Fee, J. A., & Ondrias, M. R. (1985) Biochemistry 24, 3394-3397] proposed a high-spin, pentacoordinate ferric system. Because the two RR spectral studies were performed at different temperatures, we explored the possibility that lignin peroxidase might exhibit temperature-dependent coordination-state equilibria. Resonance Raman results presented herein indicate that this hypothesis is indeed correct. At or near 25 degrees C, the ferric iron of lignin peroxidase is predominantly high spin, pentacoordinate; however, at less than or equal to 2 degrees C, the high-spin, hexacoordinate state dominates, as indicated by the frequencies of well-documented spin- and coordination-state marker bands for iron protoporphyrin IX. The temperature-dependent behavior of lignin peroxidase is thus similar to that of cytochrome c peroxidase (CCP). Furthermore, lignin peroxidase, like horseradish peroxidase (HRP) and CCP, clearly has a vacant coordination site trans to the native fifth ligand at ambient temperature. High-frequency RR spectra of compound II of lignin peroxidase are also presented. The observed shifts to higher frequency for both the oxidation-state marker band v4 and the spin- and coordination-state marker band v10 are similar to those reported for the compound II forms of HRP and lactoperoxidase and for ferryl myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗

Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants.

Different transgenic tobacco lines down-regulated for either one or two enzymes of the monolignol pathway were compared for their lignin content and composition, and developmental patterns. The comparison concerned CCR and CAD down-regulated lines (homozygous or heterozygous for the transgene) and the hybrids resulting from the crossing of transgenic lines individually altered for CCR or CAD activities. Surprisingly, the crosses containing only one allele of each antisense transgene, exhibit a dramatic reduction of lignin content similar to the CCR down-regulated parent but, in contrast to this transgenic line, display a normal phenotype and only slight alterations of the shape of the vessels. Qualitatively the lignin of the double transformant displays characteristics more like the wild type control than either of the other transgenics. In the transgenics with a low lignin content, the transformations induced other biochemical changes involving polysaccharides, phenolic components of the cell wall and also soluble phenolics. These results show that the ectopic expression of a specific transgene may have a different impact depending on the genetic background and suggest that the two transgenes present in the crosses may operate synergistically to reduce the lignin content. In addition, these data confirm that plants with a severe reduction in lignin content may undergo normal development at least in controlled conditions.

Alcohol Oxidoreductases↗

Mechanistic features of lignin peroxidase-catalyzed oxidation of substituted phenols and 1,2-dimethoxyarenes.

The steady state kinetic parameters Km and kcat for the oxidation of phenolic substrates by lignin peroxidase correlated with the presteady state kinetic parameters Kd and k for the reaction of the enzyme intermediate compound II with the substrates, indicating that the latter is the rate-limiting step in the catalytic cycle. ln Km and ln Kd values for phenolic substrates correlated with redox properties, unlike ln kcat and ln k. This finding suggests that in contrast to horseradish peroxidase, electron transfer is not the rate-limiting step during oxidation by lignin peroxidase compound II. A mechanism is proposed for lignin peroxidase compound II reactions consisting of an equilibrium electron transfer step followed by a subsequent rate-limiting step. Analysis of the correlation coefficients for linear relationships between ln Kd and ln Km and different calculated redox parameters supports a mechanism in which the acidic forms of phenols are oxidized by lignin peroxidase and electron transfer is coupled with proton transfer. 1,2-Dimethoxyarenes did not comply with the trend for phenolic substrates, which may be a result of more than one substrate binding site on lignin peroxidase and/or alternative binding modes. This behavior was supported by analogue studies with the 1,2-dimethoxyarenes veratric acid and veratryl aldehyde, both of which are not oxidized by lignin peroxidase. Inclusion of either had little effect on the rate of oxidation of phenolic substrates yet resulted in a decrease in the oxidation rate of 1,2-dimethoxyarene substrates, which was considerable for veratryl alcohol and less pronounced for 3,4-dimethoxyphenethylalcohol and 3,4-dimethoxycinnamic acid, in particular in the presence of veratric acid.

Benzyl Alcohols↗