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Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase in relation to lignification.

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content.

Amino Acid Sequence↗

Cell-specific and conditional expression of caffeoyl-coenzyme A-3-O-methyltransferase in poplar.

Caffeoyl coenzyme A-3-O-methyltransferase (CCoAOMT) plays an important role in lignin biosynthesis and is encoded by two genes in poplar (Populus trichocarpa). Here, we describe the expression pattern conferred by the two CCoAOMT promoters when fused to the gus-coding sequence in transgenic poplar (Populus tremula x Populus alba). Both genes were expressed similarly in xylem and differentially in phloem. In xylem, expression was preferentially observed in vessels and contact rays, whereas expression was barely detectable in storage rays and fibers, suggesting different routes to monolignol biosynthesis in the different xylem types. Furthermore, after wounding, fungal infection, and bending, the expression of both genes was induced concomitantly with de novo lignin deposition. Importantly, upon bending and leaning of the stem, the cell-specific expression pattern was lost, and both genes were expressed in all cell types of the xylem. CCoAOMT promoter activity correlated well with the presence of the CCoAOMT protein, as shown by immunolocalization. These expression data may explain, at least in part, the heterogeneity in lignin composition that is observed between cell types and upon different environmental conditions.

Blotting, Western↗

Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis.

The hydroxyl group in the 3-position of the phenylpropanoid compounds is introduced at the level of coumarate shikimate/quinate esters, whose synthesis implicates an acyltransferase activity. Specific antibodies raised against the recombinant tobacco (Nicotiana tabacum) acyltransferase revealed the accumulation of the enzyme in stem vascular tissues of tobacco, in accordance with a putative role in lignification. For functional analysis, the acyltransferase gene was silenced in Arabidopsis thaliana and N. benthamiana by RNA-mediated posttranscriptional gene silencing. In Arabidopsis, gene silencing resulted in a dwarf phenotype and changes in lignin composition as indicated by histochemical staining. An in-depth study of silenced N. benthamiana plants by immunological, histochemical, and chemical methods revealed the impact of acyltransferase silencing on soluble phenylpropanoids and lignin content and composition. In particular, a decrease in syringyl units and an increase in p-hydroxyphenyl units were recorded. Enzyme immunolocalization by confocal microscopy showed a correlation between enzyme accumulation levels and lignin composition in vascular cells. These results demonstrate the function of the acyltransferase in phenylpropanoid biosynthesis.

Acyltransferases↗

Aspergilli and lignocellulosics: enzymology and biotechnological applications.

Aspergilli are versatile ascomycetes that are able to transform at a rapid rate a wide spectrum of lignin-related aromatic compounds. While it is clear that these fungi can degrade phenolic and polysaccharide components from lignocellulosic material, the status regarding degradation of high-molecular mass lignins is controversial. This review compiles data from the literature as well as that from the authors' laboratory with the aim of clarifying this point. The main body of evidence points towards the inability of aspergilli alone to degrade lignin free of low-molecular mass contaminants. Nevertheless, the ability of this genus to efficiently degrade hemicelluloses makes it an essential participant in the complex microbial system necessary for wood decay under natural conditions. Aspergilli are known to overproduce high levels of hemicellulolytic enzymes. Out of the large array of these enzymes that act in concert to degrade lignocellulosic material, only endoxylanases of aspergilli are described in so far as these are the main activities required for enzyme-aided bleaching. The biochemical features of the endoxylanases from Aspergillus niger are briefly described as these serve to illustrate how a complex family of isozymes is necessary to deal with the structural and chemical heterogeneity of xylans. Emphasis is placed on the biotechnological applications of lignocellulosic materials transformed by aspergilli. The key application areas are biopulping and biobleaching where a reduction in the use of environmentally harmful chemicals traditionally used in the pulp and paper industry is envisaged. Waste water treatment represents another vast application area where aspergilli have been shown to be effective not only in colour removal but also in the bioconversion of potentially noxious substances into useful bioproducts.

Aspergillus↗

Lignocellulose decomposition by selected streptomyces strains.

From 30 actinomycete cultures isolated by enrichment technique on agar media containing newsprint as a primary carbon and energy source, three Streptomyces strains were selected for characterization of their lignocellulose-decomposing abilities. All three streptomycetes were capable of oxidizing specifically 14C-labeled lignocelluloses to 14CO2. These Streptomyces were shown to attack primarily the cellulosic (glucan) components, of which between 25 to 40% evolved as 14CO2 during 1,025 h of incubation depending upon the culture used. Lignin labeled lignocelluloses were also attacked, but to a lesser degree, with up to about 3.5% being oxidized to 14CO2 depending upon the culture used. Additionally, it was shown that purified 14C-labeled milled-wood lignin was attacked, with recoveries of up to 17.7% of the label was 14CO2. This is the first conclusive evidence to show that streptomycetes can decompose lignin.

Biodegradation, Environmental↗

Biodegradation of crystal violet by the white rot fungus Phanerochaete chrysosporium.

Biodegradation of crystal violet (N,N,N',N',N'',N''-hexamethylpararosaniline) in ligninolytic (nitrogen-limited) cultures of the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance of crystal violet and by the identification of three metabolites (N,N,N',N',N''-pentamethylpararosaniline, N,N,N',N''-tetramethylpararosaniline, and N,N',N''-trimethylpararosaniline) formed by sequential N-demethylation of the parent compound. Metabolite formation also occurred when crystal violet was incubated with the extracellular fluid obtained from ligninolytic cultures of this fungus, provided that an H2O2-generating system was supplied. This, as well as the fact that a purified ligninase catalyzed N-demethylation of crystal violet, demonstrated that biodegradation of crystal violet by this fungus is dependent, at least in part, upon its lignin-degrading system. In addition to crystal violet, six other triphenylmethane dyes (pararosaniline, cresol red, bromphenol blue, ethyl violet, malachite green, and brilliant green) were shown to be degraded by the lignin-degrading system of this fungus. An unexpected result was the finding that substantial degradation of crystal violet also occurred in nonligninolytic (nitrogen-sufficient) cultures of P. chrysosporium, suggesting that in addition to the lignin-degrading system, another mechanism exists in this fungus which is also able to degrade crystal violet.

Basidiomycota↗

Identification of a specific manganese peroxidase among ligninolytic enzymes secreted by Phanerochaete chrysosporium during wood decay.

The specific enzymes associated with lignin degradation in solid lignocellulosic substrates have not been identified. Therefore, we examined extracts of cultures of Phanerochaete chrysosporium that were degrading a mechanical pulp of aspen wood. Western blot (immunoblot) analyses of the partially purified protein revealed lignin peroxidase, manganese-dependent peroxidase (MnP), and glyoxal oxidase. The dominant peroxidase, an isoenzyme of MnP (pI 4.9), was isolated, and its N-terminal amino acid sequence and amino acid composition were determined. The results reveal both similarities to and differences from the deduced amino acid sequences from cDNA clones of dominant MnP isoenzymes from liquid cultures. Our results suggest, therefore, that the ligninolytic-enzyme-encoding genes that are expressed during solid substrate degradation differ from those expressed in liquid culture or are allelic variants of their liquid culture counterparts. In addition to lignin peroxidase, MnP, and glyoxal oxidase, xylanase and protease activities were present in the extracts of the degrading pulp.

Alcohol Oxidoreductases↗

The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase.

The white rot fungus Pycnoporus cinnabarinus was characterized with respect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with low amounts of an unusual peroxidase. Neither lignin peroxidase nor manganese peroxidase was detected. Laccase was produced constitutively during primary metabolism. Addition of the most effective inducer, 2,5-xylidine, enhanced laccase production ninefold without altering the isoenzyme pattern of the enzyme. Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,000 Da, as determined by calibrated gel filtration chromatography, and a carbohydrate content of 9%. The enzyme displayed an unusual behavior on isoelectric focusing gels; the activity was split into one major band (pI, 3.7) and several minor bands of decreasing intensity which appeared at regular, closely spaced intervals toward the alkaline end of the gel. Repeated electrophoresis of the major band under identical conditions produced the same pattern, suggesting that the laccase was secreted as a single acidic isoform with a pI of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This appeared to be confirmed by N-terminal amino acid sequencing of the purified enzyme, which yielded a single sequence for the first 21 residues. Spectroscopic analysis indicated a typical laccase active site in the P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indicated the enzyme to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus laccase showed close homology to the N-terminal sequences determined for laccases from Trametes versicolor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The principal features of the P. cinnabarinus enzyme system, a single predominant laccase and a lack of lignin- or manganese-type peroxidase, make this organism an interesting model for further studies of possible alternative pathways of lignin degradation by white rot fungi.

Amino Acid Sequence↗

Ligninolytic characteristics of Pleurotus ostreatus strain F6 and its monokaryotic protoplast derivative P19.

A stable isolate of Pleurotus ostreatus P19 differing in some morphological and physiological characteristics from its parental wild-type strain F6 was obtained via protoplast isolation during the preparation of strains with altered ligninolytic abilities. The isolate is monokaryotic, does not form clamp-connections, and produces much higher activities of enzymes involved in lignin modification (laccase, manganese peroxidase). Cellulase activity was comparable to that of wild-type strain F6, but the xylanase activity was slightly higher in isolate P19. However, this monokaryotic derivative degrades lignin at a slightly lower rate than its parental strain F6. Electron microscopy observations of wood degradation as a function of mycelium growth were performed on three zones of birch wafers delimited according to the distance from the point of inoculation. The different stages of fungal mycelium growth showed differences in the ultrastructural patterns of the decay not only between the strains P19 and F6, but also depending on the distance from the point of inoculation. This suggests a spatio-temporally controlled secretion of enzymes along the hyphae. The enhanced ability of P19 to degrade the condensed forms of lignin in middle lamellae is correlated to its higher laccase activity.

Biodegradation, Environmental↗

Fourier transform infrared studies of heterogeneity, photodegradation, and lignin/hemicellulose ratios within hardwoods and softwoods.

There is little information available on the variation in lignin content of growth rings in hardwoods. This study examines whether infrared microscopy can detect intra-incremental differences in the chemical composition of three hardwoods (R. pseudoacacia, P. Americana, and G. triacanthos) and the effect of such differences on the delignification of the hardwoods during weathering. Earlywood has higher lignin content than latewood in R. pseudoacacia and P. americana, but the opposite was found for G. triacanthos. The delignification of the earlywood and latewood during weathering varied for the three species. It was greater in the earlywood of R. pseuoacacia, whereas in P. americana and G. triacanthus it was more pronounced in latewood. Differences in density and lignin content of earlywood and latewood help explain these differences. In addition, a deconvoluting software package was used to determine whether it is possible to estimate the lignin/hemicellulose ratio in softwoods and hardwoods. Results from the 1760-1580 cm(-1) region provided data that can be used to estimate the lignin/hemicellulose ratio of softwoods and hardwoods. This information can be obtained far more easily using infrared microscopy than with conventional wet chemical techniques, potentially allowing characterization of greater numbers of species than has hitherto been possible.

Gleditsia↗

Enhanced enzymatic hydrolysis of steam-exploded Douglas fir wood by alkali-oxygen post-treatment.

Good enzymatic hydrolysis of steam-exploded Douglas fir wood (SEDW) cannot be achieved owing to the very high lignin content ( >40%) that remains associated with this substrate. Thus, in this study, we investigated the use of alkali-oxygen treatment as a posttreatment to delignify SEDW and also considered the enzymatic hydrolyzability of the delignified SEDW. The results showed that under optimized conditions of 15% NaOH, 5% consistency, 110 degrees C, and 3 h, approx 84% of the lignin in SEDW could be removed. The resulting delignified SEDW had good hydrolyzability, and cellulose-to-glucose conversion yields of over 90 and 100% could be achieved within 48 h with 20 and 40 filter paper units/g of cellulose enzyme loadings, respectively. It was also indicated that severe conditions, such as high NaOH concentration and high temperature, should not be utilized in oxygen delignification of SEDW in order to avoid extensive condensation of lignin and significant degradation of cellulose.

Biotechnology↗

Characterization of low molecular weight organic acids from beech wood treated in supercritical water.

Japanese beech (Fagus crenata Blume), its cell wall components, and model compounds were treated by supercritical water (380 degrees C, 100 MPa) for 5 s using a batch-type reactor to investigate the production behavior of low molecular weight organic acids. It was found that cellulose and hemicellulose were decomposed to formic acid, pyruvic acid, glycolic acid, acetic acid, and lactic acid, whereas lignin was barely decomposed to such organic acids under the given conditions. However, after prolonged treatment (380 degrees C, 100 MPa, 4 min) of lignin, some organic acids were recovered owing perhaps to the decomposition of the propyl side chain of lignin. It was additionally revealed that the predominant organic acid recovered was acetic acid, which might be derived from the acetyl group of hemicellulose in Japanese beech.

Acids↗

The lignol approach to biorefining of woody biomass to produce ethanol and chemicals.

Processes that produce only ethanol from lignocellulosics display poor economics. This is generally overcome by constructing large facilities having satisfactory economies of scale, thus making financing onerous and hindering the development of suitable technologies. Lignol Innovations has developed a biorefining technology that employs an ethanol-based organosolv step to separate lignin, hemicellulose components, and extractives from the cellulosic fraction of woody biomass. The resultant cellulosic fraction is highly susceptible to enzymatic hydrolysis, generating very high yields of glucose (>90% in 12-24 h) with typical enzyme loadings of 10-20 FPU (filter paper units)/g. This glucose is readily converted to ethanol, or possibly other sugar platform chemicals, either by sequential or simultaneous saccharification and fermentation. The liquor from the organosolv step is processed by well-established unit operations to recover lignin, furfural, xylose, acetic acid, and a lipophylic extractives fraction. The process ethanol is recovered and recycled back to the process. The resulting recycled process water is of a very high quality, low BOD5, and suitable for overall system process closure. Significant benefits can be attained in greenhouse gas (GHG) emission reductions, as per the Kyoto Protocol. Revenues from the multiple products, particularly the lignin, ethanol and xylose fractions, ensure excellent economics for the process even in plants as small as 100 mtpd (metric tonnes per day) dry woody biomass input a scale suitable for processing wood residues produced by a single large sawmill.

Alcohol Dehydrogenase↗

Fenton's reagent-mediated degradation of residual Kraft black liquor.

In this work, the effect of Fenton's reagent on the degradation of residual Kraft black liquor was investigated. The effect of Fenton's reagent on the black liquor degradation was dependent on the concentration of H2O2. At low concentrations (5 and 15 mM) of H2O2, Fenton's reagent caused the degradation of phenolic groups (6.8 and 44.8%, respectively), the reduction of reaction medium pH (18.2%), and the polymerization of black liquor lignin. At a high concentration (60 mM) of H2O2, Fenton's reagent induced an extensive degradation of lignin (95-100%) and discoloration of the black liquor. In the presence of traces of iron, the addition of H2O2 alone induced mainly lignin fragmentation. In conclusion, Fenton's reagent and H2O2 alone can degrade residual Kraft black liquor under acidic conditions at room temperature.

Biotechnology↗

Ligninolysis. A very peculiar microbial process.

Lignin, the most abundant renewable source of aromatic carbon on earth, consists in a highly irregular three dimensional biopolymer of oxygenated phenylpropanoid units. In natural environments, lignin is only degraded efficiently by some basidioamycetes that secrete an array of enzymes for this purpose. Recent advances in our understanding of the mechanism of lignin breakdown have revealed several features that make this process highly unique from a biochemical standpoint. This article summarizes some of them.

Biopolymers↗

Sorption and desorption of naphthalene by soil organic matter: importance of aromatic and aliphatic components.

Nonlinear isotherm behavior has been reported for the sorption of hydrophobic organic compounds (HOCs) in soil organic matter (SOM), but the exact mechanisms are unknown. Our objective was to provide insight into the sorption mechanism of HOCs in SOM by studying the sorption-desorption processes of naphthalene in a mineral soil, its humic fractions, and lignin. Additionally, humin and lignin were used for studying the effects of temperature and cosolvent on HOC sorption. All isotherms were nonlinear. The humin and lignin isotherms became more linear at elevated temperatures and with the addition of methanol indicating a condensed to expanded structural phase transition. Isotherm nonlinearity and hysteresis increased in the following order: soil humic acid (HA) < soil < soil humin. Of the samples, aliphatic-rich humin exhibited the largest degree of nonlinearity and had the highest sorption capacity for naphthalene. High nonlinearity and hysteresis in humin were most likely caused by its condensed structure. A novel aliphatic, amorphous condensed conformation is proposed. This conformation can account for both high sorption capacities and increased nonlinearity observed for aliphatic-rich samples and can explain many sorption disparities discussed in the literature. This study clearly illustrates the importance of both aliphatic and aromatic moieties for HOC sorption in SOM.

Adsorption↗

Effects of intake and forage level on site and extent of digestion of plant cell wall monomeric components by sheep.

The objective of this experiment was to study the effects of level of intake and proportion of forage in the diet on site and extent of digestion of plant cell wall monomeric components (phenolics, neutral sugars) by sheep. Four Suffolk wethers (65 kg) with ruminal and duodenal cannulas were fed diets containing 75% (75A) or 25% (25A) alfalfa hay at two levels of intake, 1,700 (high intake, HI) or 1,100 (low intake, LI) g DM/d in a 4 X 4 latin-square design with a 2 X 2 factorial arrangement of treatments. The remainder of the diet consisted primarily of ground corn. Wethers had lower (P less than .05) total tract NDF and ADF digestibilities when fed 75A or at HI. An intake X forage level interaction (P less than .05) was noted for non-core lignin ferulic acid (FA) total tract disappearance, with wethers at LI having the highest, 25A-HI having the lowest, and 75A-HI having an intermediate FA disappearance. Wethers fed at LI had higher (P less than .05) total tract disappearance of non-core lignin p-coumaric acid (PCA). Wethers fed 75A had greater (P less than .05) total tract disappearances of core lignin vanillic acid (VA) and vanillin (VAN). Total tract digestibilities of the neutral monosaccharides glucose (GLC), xylose (XYL), arabinose (ARA) and galactose (GAL) were greater (P less than .05) for LI vs HI treatments. Wethers consuming 75A had greater (P less than .05) total tract ARA and GAL digestibilities, whereas wethers fed 25A had greater (P less than .05) total tract digestibilities of GLC and mannose (MAN). Diet composition and level of intake appear to influence site and extent of digestion of cell wall monomeric components.

Animals↗

Higher intake of DK265 corn silage by dairy cattle.

In this experiment, intake of DK265 3-way corn hybrid by dairy cattle was compared specifically with intake 1) of its bm3 isogenic form, 2) of its 2 related single-way hybrids, and 3) of 2 controls that were registered hybrids of similar earliness. Both dry matter (DM) and lignin contents were similar in all hybrids except for the bm3 hybrid, which was less lignified. There was a tendency for lower starch content and, correlatively, higher neutral detergent fiber content in DK265 and in the 2 related single-way hybrids. Significant intake differences were observed between hybrids; the highest intake was recorded for the bm3 hybrid. Among normal hybrids, DK265 and one of its related single-way hybrids registered significantly higher intakes than other hybrids. Among normal hybrids, cell wall digestibility and/or lignin content did not explain all of the variations observed for intake, whereas the higher intake of DK265 bm3 could be related to its lower lignin content as compared with isogenic DK265. It was hypothesized that the higher intake observed for the DK265 hybrid was probably related to specific friability traits that are not relevantly measured through the usual tests used in corn breeding.

Animals↗