John R. Obst

Lignification in Trees: Indication of Exclusive Peroxidase Participation

Syringaldazine did not turn purple on cross sections of tree branches or saplings or on cambial tissue cultures unless hydrogen peroxide was added; this indicated the absence of laccase but presence of peroxidase in lignifying cells. Peroxidase, therefore, apparently is the only en enzyme that polymerizes p-coumaryl alcohols to lignin in trees.

Syringaldazine, an effective reagent for detecting laccase and peroxidase in fungi

Das Syringaaldehyd-azin ist eine hellgelbe, kristalline und leicht zu synthetisierende Verbindung, die in verdünnter alkoholischer Lösung von Laccase + Luftsauerstoff bzw. von Peroxydase + H2O2 unter Bildung eines intensiv purpurnen Pigments rasch oxydiert wird. Diese hochempfindliche Reaktion ist als Tüpfelprobe leicht anzuwenden und ergibt einen sehr spezifischen Nachweis dieser Phenoloxydasen in Pilzkulturen: Trägt man einige Tropfen verdünnter Azinlösung auf eine Pilzkultur auf, bildet sich an der Luft bei Anwesenheit von Laccase ein purpurner Fleck. Ist nur wenig Laccase vorhanden, wird die Probe nur rosa bis rot; bleibt sie farblos, ist keine Laccase vorhanden. Im letzteren Fall kann man verdünnte H2O2-Lösung an derselben Stelle auftropfen, um Peroxydase nachzuweisen. Färbt sich der Fleck dann lila rot, ist eine Peroxydase vorhanden. Der Nachweis von Peroxydase ist bei Anwesenheit von Laccase nicht möglich. Ist keine Laccase vorhanden, kann man eine Furoguajacin-Lösung (bzw. frisch hergestellte Guajaktinktur) an einer anderen Stelle der Kultur auftragen, um Tyrosinase nachzuweisen. Die Tyrosinase oxydiert das Azin überhaupt nicht. Mit Hilfe dieser eindeutigen und schnellen Nachweismethoden wird man den Zusammenhang zwischen der Bildung der verschiedenen Phenoloxydasen durch holz-zerstörende Pilze und deren Abbau von Holz und von Lignin besser untersuchen können.

Use of syringaldazine for detection of laccase in sporophores of wood rotting fungi

Fungal laccase can be detected with the nonautooxidizable laccasespecific compound, syringaldazine, in the presence of tyrosinase and peroxidase. Data are presented from tests with the azine derivative of numerous fungous sporophores and associated decayed woody tissues from various localities in Wisconsin and Oregon. Syringaldazine is shown to be a reliable replacement and more specific reagent for the detection of extracellular fungal laccase, and in the absence of laccase and in the presence of hydrogen peroxide, for peroxidase. Laccase is further implicated in fruit-body formation.

Analytical Pyrolysis of Hardwood and Softwood Lignins and Its use in Lignin-Type Determination of Hardwood Vessel Elements

Abstract Pyrolysis-gas chromatography-mass spectrometry was performed on milled wood lignins (MWLs) and wood samples. Among the major pyrolysis products identified from loblolly pine lignin were guaiacol, 4-methylguaiacol, 4-vinylguaiacol, vanillin, coniferal-dehyde, and coniferyl alcohol. White oak lignin pyrolysis products also included guaiacol, 4-methylguaiacol, and vanillin and additionally 2,6-dimethoxyphenol, 4-methyl-2,6-dimethoxyphenol, syringaldehyde, and sinapaldehyde. By identification of these pyrolysis products from either wood or MWL it is possible to classify lignins as either guaiacyl-type or syringyl/guaiacyl-type. Pyrolysis of isolated vessel elements from white oak, white birch, and American elm indicated that vessel lignin is of the syringyl/ guaiacyl-type.

Guaiacyl and Syringyl Lignin Composition in Hardwood Cell Components

White oak cell types, fibers, rays, and vessels, were isolated from high-yield chemimechanical, neutral sulphite semichemicai (NSSC), and kraft pulps. Milled wood lignins (MWL) from NSSC fiber and ray cell fractions analyzed by ultraviolet and infrared spectroscopy were found to have similar syringyl/ guaiacyl ratios. Methoxyl analyses of MWLs were also similar and» upon comparison to the methoxyl contents of their Klason lignins and of Klason lignins from pulp fractions, it was found that the methoxyl analyses of the Klason lignins from wood and pulp fractions were representative of actual lignin methoxyl content. A middle lamella enriched lignin fraction was obtained by refining of the fibers; cell wall lignin-enriched fractions were obtained by selective removal of middle lamella lignin by oxygen and acid chlorite äs shown by ultraviolet microscopy. These lignin fractions had similar methoxyl contents indicating the relatively uniform syringyl/guaiacyl ratio of lignin in different morphological regions. Methoxyl analysis of intact vessels isolated from an oak NSSC pulp gave unambiguous proof of the syringyl/guaiacyl nature of hardwood vessel lignin. The occurrence of typical syringyl/guaiacyl ratios throughout the wood was also indicated for birch and ash.

Biodegradation of Compression Wood and Tension Wood by White and Brown Rot Fungi

Ultrastructual investigations and chemical determinations of compression wood from Abies balsamea, Picea mariana and Pinus strobus after decay by white or brown rot fungi demonstrated that this type of wood is more resistant to decay than normal wood. Hyphae colonizing compression wood were found in cell lumina and intercellular spaces whereas normal wood cell had hyphae only in cell lumina. Compression wood did not alter the type of cell wall degradation produced by the various fungi tested, but the rate and extent of decay were limited. The white rot fungus, Trametes versicolor, caused a nonselective attack of all cell wall components as indicated by erosion of secondary wall layers and middle lamellae. A selective removal of lignin occurred throughout the cell walls of wood decayed by the other white rot fungi (Phellinus pini, Phlebia tremellosa and Scytinostroma galatinum) that were evaluated. The brown rot fungi, Fomitopsis pinicola and Oligoporus placentus caused a diffuse removal of polysaccharides from both compression wood and normal wood. Tension wood from Populus temuloides and Acer rubrum decayed by white or brown rot fungi had similar amounts of decay to those observed in normal wood. Ultrastructual observations, however, showed striking differences in the progressive stages of decay. Hyphae of Tremetes versicolor located in cell lumina did not cause erosion or severe degradation of the adjacent, underlying gelatinous layer associated with tension wood. A typical nonselective degradation of the secondary wall layers and middle lamellae, however, occurred beneath this gelatinous layer. In areas of advanced degradation, the secondary walls and middle lamellae between cell walls were completely degraded leaving only the gelatinous layer and cell corner regions of the middle lamellae. Brown rot fungi were able to degrade the gelatinous layer and other cell wall layers resulting in extensive degradation of polysaccharides.

Kinetics of Alkaline Cleavage of β-Aryl Ether Bonds in Lignin Models: Significance to Delignification

The kinetics of the alkaline cleavage of 0-0-4 aryl ethers of nonphenolic units were described for the lignin models veratrylglycol-0-guaiacyI ether (I) and veratryglycerol-0-guaiacyl ether (II). The use of p-dioxane and methyl cellosolve äs cosolvents to achieve homogeneity in the alkaline treatments of the models was found to be unnecessary äs I and II were water soluble at cooking temperature (170 °C). Both methyl cellosolve and dioxane suppressed the rate of alkaline cleavage of I but only dioxane did so for II. /J-Aryl ether cleavage of an erythroand threo-mixturc of II did not follow first-order kinetics. The similarity of the apparent pseudo first-order rate of bulk delignification in alkaline pulping and the rate of first-order alkaline cleavage of certain ß-ethers in model Systems is coincidental and misleading in understanding the mechanism of delignification.

The Syringyl Content of Softwood Lignin

Abstract Representations of softwood lignins typically include small amounts, 2 to 5%, of syringylpropane units. Carbon-13 nuclear magnetic resonance and pyrolysisgas chromatography of loblolly pine milled wood lignin indicated a much lower value. Pyrolysis-gas chromatography/mass spectrometry of whole softwoods also gave lower than expected yields of syringyl products. One chemical method, alkaline nitrobenzene oxidation, was also used to determine syringyl content. Results obtained using these methods indicate that the syringyl content of lignin in typical softwoods appears to be less than 0.1% of the total phenylpropane units. Ginkgo was also examined, and its lignin was found to contain only a very small amount of syringylpropane units. Cambial extracts of three softwoods were analyzed by pyrolysis-gas chromatography/mass spectrometry, and the data indicated the possibility of the presence of a slight amount of syringin. These results suggest that lignin in typical softwoods is formed almost exclusive...

Characterization of hardwood lignin: investigation of syringyl/guaiacyl composition by 13C nuclear magnetic resonance spectroscopy

Carbon-13 nuclear magnetic resonance Spectroscopy was investigated äs a method to determine the relative syringyl/guaiacyl composition of hardwood lignins. By this technique, red oak fiber and middle lamella milled wood lignins (MWLs) were similar while white birch MWLs from different morphological regions gave apparent differences in their syringyl/guaiacyl ratios. However, methoxyl contents of MWLs were similar, indicating the unsuitability of carbon magnetic resonance Spectroscopy, äs performed, äs a quantitative method. A correlation was noted between the measured carbon magnetic resonance syringyl/ guaiacyl ratio and the molecular weight distribution of the MWLs. Molecular weight distributions of the MWLs suggested that birch middle lamella lignin was higher in molecular weight than fiber cell wall lignin.