Relationship between production of hydroxyl radicals and degradation of wood, crystalline cellulose, and a lignin-related compound or accumulation of oxalic acid in cultures of brown-rot fungi

The degradation of wood, filter paper cellulose, and a lignin-substructure model, was measured in cultures of seven fungi usually regarded as brown-rot fungi. Hydroxyl radical production and the accumulation of oxalic acid in the cultures were also measured. Four of the fungi, Gloeophyllum trabeum, Tyromyces palustris, Laetiporus sulphureus, and Postia placenta, were typical brown-rot fungi, in that they preferentially degraded and eliminated the polysaccharides in wood and produced large amounts of hydroxyl radical. The rates of hydroxyl radical generation in cultures of the four fungi were directly proportional to the degradation rates of wood, cellulose, and the lignin-related compound, and inversely proportional to the amount of oxalic acid in the cultures. Two of the fungi, Daedalea dickinsii and Lentinus lepideus, did not degrade any of the substrates significantly and produced very little hydroxyl radical. Coniophora puteana had the highest rate of cellulose degradation, but did not degrade wood or the lignin model significantly and produced only negligible amounts of hydroxyl radical. These results indicate that brown-rot fungi produce large amounts of hydroxyl radical for the degradation of wood and crystalline cellulose.     

Consumption of O<Subscript>2</Subscript>, evolution of CO<Subscript>2</Subscript> and reduction of Cr(VI) during fixation of chromium based wood preservatives in wood

Norway spruce dust was impregnated with aqueous solutions of chromated copper wood preservatives. Immediately after treatment, observation of CO₂ evolution and O₂ consumption were performed. Significant quantities of CO₂ were released during reaction of chromium (K₂Cr₂O₇) containing solutions with wood or brown rotted wood. Nevertheless, during reaction of cellulose with these preservatives we did not observe evolution of CO₂. The presence of copper did not influence on concentration of CO₂. Opposite to CO₂ evolution, treatment of wood and brown rotted wood resulted in O₂ consumption. The oxygen concentration decrease in the measuring chamber was approximately 5 times greater than increase of concentration of carbon dioxide. Electron paramagnetic resonance (EPR) observations of chromium fixation showed that chromium is reduced from Cr(VI) to Cr(III) with Cr(V) as an intermediate on wood, brown rotted wood and cellulose. However, the reduction on wood and brown rotted wood was faster than the reduction on cellulose, as determined from changes of Cr signals in EPR spectra. So, evolution of CO₂ and consumption of O₂ as well as EPR signals of Cr species thus indicate that brown rotted wood, consisting of lignin and hemicelluloses in contact with Cr(VI) reacts more intensively than cellulose, and possibly, oxidation mechanisms of lignin and cellulose with Cr(VI) are different. Received: 20 July 2000     

Characterization of acetylated wood decayed by brown-rot and white-rot fungi

The objective of this study was to characterize the decay of acetylated wood due to brown-rot and white-rot fungi by analysis of chemical composition, X-ray measurements, and¹³C-NMR spectroscopy. The decay by brown-rot fungus became inhibited at a weight percent gain (WPG) due to acetylation of more than 10%, and the mass loss (LOSS) due to decay became zero at a WPG of about 20%. The LOSS due to white-rot fungus decreased slowly with the increase in WPG, reaching zero at a WPG of about 12%. The losses of lignin by brown-rot decay increased initially with the decrease in LOSS owing to the progressing acetylation and then decreased at a LOSS of less than 60%. Polysaccharides were more easily decomposed than lignin during the decay of acetylated wood due to brown-rot fungus. The losses of both components due to white-rot decay decreased as the LOSS decreased with progressing acetylation. The white-rot fungus tended to preferentially decompose the lignin during the decay of acetylated wood. The brown-rot fungus decomposed the cellulose in the crystalline region to a large degree when the LOSS was more than 40%, whereas the white-rot fungus decomposed the crystalline region and the noncrystalline region in acetylated wood to the same degree. The brown-rot fungus preferentially decomposed unsubstituted xylose units in acetylated wood and partly decomposed the mono-substituted xylose units. It was suggested that the mono- and disubstituted cellulose were partly decomposed by brown-rot fungus.This paper was presented at the 46th and 47th annual meetings of the Japan Wood Research Society at Kumamoto and Kochi in April 1996 and April 1997, respectively     

Mineralization of the methoxyl carbon of isolated lignin by brown-rot fungi under solid substrate conditions

Summary The objectives of this work were to begin developing an experimental system for studying the demethylation of lignin by brown-rot fungi and to examine the influence of selected culture parameters. As substrate for demethylation, we used partially 3-O-demethylated lignin that had been isolated earlier from brown-rotted spruce wood; we remethylated with¹⁴CH₃I, giving a lignin with both [3-¹⁴C]methoxyl and [4-¹⁴C]methoxyl groups. This lignin was added to pine wood flakes, which were incubated with selected brown-rot fungi, and the evolved¹⁴CO₂ was trapped and measured. Of eight fungi examined,Gloeophyllum trabeum andWolfiporia cocos gave the highest rates of mineralization of the¹⁴C-methoxyl carbons. With the former but not the latter fungus, methoxyl mineralization was over twice as fast in an atmosphere of O₂ than in air. Amending the cultures with ammonium tartrate suppressed mineralization to some extent. Further studies withG. trabeum showed that glutamate lowered the rate of mineralization and that glucose and glycerol sharply suppressed it. Addition of Fe²⁺ and Mn²⁺ slightly increased the rate of mineralization. Our results suggest that in unsupplemented cultures the rate-limiting step in methoxyl mineralization is the initial demethylation. Thus the two likely initial C₁ products, methanol and formaldehyde (as¹⁴C compounds), were mineralized much more rapidly than the methoxyl carbon of the lignin (as was formic acid), and no low molecular weight labeled intermediates from the [¹⁴C]-methoxyl lignin accumulated in the cultures. Our results also provide evidence that the spruce lignin was partially polymerized byG. trabeum. Mineralization of the methoxyl carbon of a synthetic [3-¹⁴C]-methoxyl lignin was slower than that of the spruce lignin, suggesting either that the synthetic lignin was more recalcitrant or that the [4-¹⁴C]methoxyl group in the [3,4-¹⁴C]-methoxyl spruce lignin was attacked more readily.We thank Karen L. Martinson and Michael D. Mozuch for excellent technical help, and Tor P. Schultz for valuable suggestions. This research was supported in part by the U.S. Department of Agriculture, Wood Utilization Research Program, Project No. 350-0612, to Mississippi State University.The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain and not subject to copyright in the United States.     

Micromorphological characteristics of decayed wood and laccase produced by the brown-rot fungus <Emphasis Type="Italic">Coniophora puteana</Emphasis>

Microscopic examination showed the cell wall decay pattern produced by the brown-rot fungus Coniophora puteana to be different from the degradation pattern known to be typical for brown-rot fungi. Erosion and thinning of cell walls in patterns considered to be characteristic of white-rot decay were observed. In particular, the fungal strain COP 20242 degraded secondary cell wall layers extensively, and also degraded lignin-rich middle lamellae. Some strains of C. puteana produced soft-rot type cavities in the S2 layer. All strains of C. puteana employed in the present work showed a positive reaction to tannic acid in the Bavendamm test, indicating the production of laccase. Microscopic and enzymatic studies provided evidence to suggest that the wood decay by C. puteana is unique both in terms of micromorphological and enzymatic patterns of cell wall degradation. This is because brown-rot fungi are not generally known to form cavities in the cell walls or to produce lignin-degrading enzymes. These observations suggest that lignin degradation capacity of brown-rot fungi may be greater than previously considered.     

Lignin biochemistry: Biosynthesis and biodegradation

Summary Lignin biosynthesis via shikimate-cinnamate pathways in plants, and the biosynthetic differences of guaiacyl-and syringyl lignins between gymnosperms and angiosperms have been elucidated by tracer experiments using ¹⁴C labeled precursors and the following enzyme reactions. The formation of guaiacyl lignin but not syringyl lignin in gymnosperms was attributed to the following factors; absence of ferulate-5-hydroxylase, poor affinity of O-methyltransferase toward 5-hydroxyferulate, and lack of activation and/or reduction of sinapatc. A mechanism of lignin-carbohydrate complexes formation in wood cell walls was elucidated based on the reaction of the quinone methide of guaiacylglycerol--guaiacyl ether with sugars, and the analysis of DHP-polysaccharide complexes.The main cleavage mechanisms of side chains and aromatic rings of lignin model compounds and synthetic lignin (DHP) by white-rot fungi and their enzymes, lignin peroxidase and laccase have been elucidated using ²H, ¹³C and ¹⁸O-labeled lignin substructure dimcrs with ¹⁸O₂ and H₂ ¹⁸O. Side chains and aromatic rings of these substrates were cleaved via aryl cation radical and phenoxy radical intermediates, in reaction mediated only by lignin peroxidase/H₂O₂ and laccase/O₂.Academy Lecture presented at the Cellucon 88 in Japan, International Symposium on New Functionalisation Developments in Cellulosics and Wood, held in Kyoto, Japan, November 28 to December 1, 1988This paper is a conclusion of our investigations on the biosynthesis and microbial degradation of lignin for 30 years in the Research Section of Lignin Chemistry, Wood Research Institute, Kyoto University. The author is greatly indebted to Drs. M. Shimada, F. Nakatsubo, T. Yamasaki, H. Ohashi, M. Tanahashi, Y. Nakamura, H. Kuroda, H. Kutsuki, T. Katayama, Y. Kamaya, T. Umezawa and Messrs. H. Fushiki, M. Ohta, A. Noguchi, H. Namba, T. Habe, S. Kawai, S. Yokota, and T. Hattori for their kind cooperation in these investigations. These invstigations were supported in part by Grant-in-Aid Nos. 548047, 57480058, 59760124, 60760130, 60440015, 61760142, 61560193, 62790250 for Scientific Research, and 1980 Grant-in-Aid for Environmental Science (R-33-8), No. 403064 from the Ministry of Education, Science and Culture of Japan, and a 1980 Weyerhaeuser grant     

Recycled ionic liquid 1-ethyl-3-methylimidazolium acetate pretreatment for enhancing enzymatic saccharification of softwood without cellulose regeneration

In this work, pretreatment of wood meals using a recycled ionic liquid (IL), 1-ethyl-3-methylimidazolium acetate ([Emim]Ac), enhanced glucose liberation by enzymatic saccharification, without dissolution of cellulose and lignin. In contrast, previous studies on IL pretreatment have mostly focused on lignocellulosic dissolution to regenerate cellulose and removing lignin. Softwood (Cryptomeria japonica) was pretreated with [Emim]Ac at 60–100 °C for 2–8 h without collecting regenerated cellulose. The pretreatment did not have a strong effect on wood component dissolution (weight of residues: 91.7–98.8%). The residues contained relatively high amounts of lignin (26.6–32.6%) with low adsorption of [Emim]Ac (0.9–2.7%). Meanwhile, the crystallinity index (C _r I) of cellulose in the wood was significantly reduced by pretreatment, from 50.9% to 28.4–37.1%. In spite of the high lignin contents in the residues, their glucose liberation values by enzymatic saccharification using a cellulase mixture were 3–16 times greater than that of untreated wood. A good correlation was found between the saccharification effectiveness of pretreated samples and the C _r I. Although lignin dissolved in [Emim]Ac continued to accumulate after repeated use of [Emim]Ac, the pretreatment was found to be effective for three consecutive cycles without the need to remove the dissolved materials.     

In vitro decay of Aextoxicon punctatum and Fagus sylvatica woods by white and brown-rot fungi

Summary The in vitro decay of Aextoxicon punctatum and Fagus sylvatica wood by the fungi Trametes versicolor, Ganoderma australe, Phlebia chrysocrea and Lentinus cyathiformis was studied by the agar-block method, and then the decayed woods were analyzed by chemical and spectroscopic techniques. The results demonstrated the strong resistance of the A. punctatum wood to the brown-rot fungus L. cyathiformis; the resistance might be related to the low S/G lignin ratio in this Austral hardwood. Wood decay by the Austral white-rot fungi G. australe and P. chrysocrea was rather limited, and preferential degradation of lignin was not produced although all the fungi studied increased wood digestibility. The most characteristic white and brown-rot decay patterns were observed during the in vitro decay with T. versicolor and L. cyathiformis, respectively. Trametes versicolor caused high weight losses and reduced the lignin content of the wood, whereas L. cyathiformis produced a preferential removal of xylan. No important changes in the solid-state ¹³C NMR spectra were observed after wood degradation by T. versicolor, but this technique evidenced an increase in aromatic carbon by L. cyathiformis. This increase was higher than that found in the Klason lignin content, suggesting the presence of altered lignin fractions in the brown-rotted wood.The authors are indebted to Prof. H. D. Lüdemann for the facilities at the Institut für Biophysik und physikalische Biochemie (Regensburg), to A. Navarrete (INIA, Madrid) for her collaboration, and to C. F. Warren (ICE, Alcalá de Henares) for her linguistic assistance. The computer program for spectra treatment was developed by G. Almendros (Centro de Ciencias Medioambientales, CSIC, Madrid). This investigation has been funded by the Spanish Biotechnology Program (Grant BIO88-0185)     

Semi-quantitative method to evaluate the α-carbonyl content in lignin

A method to estimate the content of -carbonyl structures in lignin was developed. This method consists of two successive treatments: NaBD₄ treatment of pulp to reduce an -carbonyl structure in lignin, and nitrobenzene oxidation. NaBD₄ was used to convert an -carbonyl structure to a deuterium-labeled hydroxymethine structure. The ratio of D-vanillin [(HO)(H₃CO)C₆H₃CDO] to H-vanillin [(HO)(H₃CO)C₆H₃CHO] or that of their syringyl analogues obtained by nitrobenzene oxidation was used as the measure of the content of -carbonyl structure. Model experiments demonstrated that when sodium hydroxide was used as alkali for the nitrobenzene oxidation, the retention of deuterium at the side chain -position was very low due to the displacement of deuterium with hydrogen by an unknown reaction mechanism. In order to depress this unexpected displacement, the reaction conditions of the nitrobenzene oxidation were modified. The modified nitrobenzene oxidation employs 0.5mol/l of lithium hydroxide as a reaction medium instead of 2.0mol/l sodium hydroxide. By this modification, this method could successfully trace the formation and the degradation of the -carbonyl structure in milled wood lignins.This paper was presented in part at the 11th International Symposium on Wood and Pulping Chemistry, Nice, France, June 2001 and at the 46th Lignin Symposium, Kyoto, Japan, November 2001     

木材白腐菌对木质素生物降解机制的初步研究

通过测定6种白腐菌火木层孔菌Phellinus igniarius及粗毛盖菌Funalia gallica、三色革裥菌Lenzites tricolor、冬拟多孔菌Polyporellus brumalis、偏肿拟栓菌Pseudotrametes gibbosa和血红密孔菌Pycnoporus sanguineus分解山杨材一定时间后的木质素含量，研究木材白腐菌对山杨材木质素生物降解机制。测定结果表明，按照木质素的减少百分率，这6种白腐菌对山杨材木质素的分解能力依次为血红密孔菌、偏肿拟栓菌、三色革裥菌、冬拟多孔菌、冬拟多孔菌、火木层孔菌；6种白腐菌对山杨材木质素及综纤维素的分解量X1、X2及分解时间Y这3个量之间存在多元回归关系；冬拟多孔菌是较多分解木质素、较少分解纤维素的木材白腐菌。     

Look back over the studies of lignin biochemistry

The role of the cinnamate pathway in monolignol biosynthesis based on feeding experiments with lignifying plant stems and characterization of the enzymes in the pathway, O-methyltransferase (OMT), cinnamyl alcohol dehydrogenase (CAD), etc. is discussed. Monolignol biosynthesis via metabolic grids according to newly characterized enzymes in the pathway is also reviewed and discussed. The cleavage mechanisms of side chains and aromatic rings by lignin peroxidase and laccase elucidated by using ¹⁸O, ²H, and ¹³C labeled lignin substructure dimers and DHP with ¹⁸O₂ and H₂ ¹⁸O are reviewed. Finally, the prospects of lignin biochemistry in the wood and paper industries are discussed according to the recent progress on gene technology on wood formation and microbial degradation of lignin.     

Recent progress in the chemistry and topochemistry of compression wood

Summary A review of the chemistry and topochemistry of compression wood with 200 references. Compression wood contains on the average 30% cellulose, 35–40% lignin, 10% galactan, 9% galactoglucomannan, 8% xylan, and 2% of a 1,3-glucan (laricinan). The cellulose is less crystalline, and the xylan has fewer arabinose side chains than in normal wood. The lignin is composed of guaiacylpropane and p-hydroxyphenylpropane units. It is more condensed, has a higher proportion of carbon-carbon bonds, and contains fewer arylglycerol--aryl ether structures than a normal conifer lignin. The ray cells and the primary wall of the tracheids have the same chemical composition in normal and compression woods. The galactan is largely located in the outer region of the secondary wall. Only 5–10% of the lignin in compression wood tracheids is extracellular. The middle lamella is less lignified than in normal wood, while the S₁ and inner S₂ layers have a lignin concentration of 30–40% which is twice as high as in normal wood. The lignin content of the S₂ (L) layer is equal to or higher than that of the intercellular region along the wall. The review is concluded with a brief reference to areas where present information is incomplete or lacking.A portion of an Academy Lecture of the International Academy of Wood Science, presented at the International Symposium on Wood and Pulping Chemistry (Ekmandagarna 1981), held in Stockholm, Sweden, June 9–12, 1981. Reprints of the unabridged review, published under the title Recent Progress in the Chemistry, Ultrastructure, and Formation of Compression Wood in the preprints of the symposium (SPCI Report 38, Vol. 1, p. 99–147) are available from the author. I wish to express my gratitude to my colleague Professor Robert A. Zabel for generous travel assistance     

Impact of thermal modification on color and chemical changes of spruce and oak wood

Thermal modification of wood is an environment-friendly alternative method for improving several properties of wood without the use of chemicals. This paper deals with the examination of color and chemical changes in spruce (Picea abies L.) and oak wood (Quercus robur F.) that occur due to thermal treatment. The thermal modification was performed at 160, 180, and 210 °C according to thermowood process. The color changes were measured by the spectrophotometer and described in the L*a*b* color system. Chemical changes were examined by wet chemistry methods, infrared spectroscopy and liquid chromatography. During the experiment, oak samples showed smaller color changes than spruce samples at all temperature values. During thermal modification, the content of cellulose, lignin, and extractives increases; however, the hemicellulose content drops by 58.85% (oak) and by 37.40% (spruce). In addition to deacetylation, new carbonyl and carboxyl groups are formed as a result of oxidation. Bonds in lignin (mainly β-O-4) and methoxyl groups are cleaved, and lignin is condensed at higher temperatures.     

Origin of free radicals produced from the syringyl end groups in lignins

Enzyme lignin was prepared from reed; it was acid-hydrolyzed and reduced with NaBH₄. Spin concentrations of radicals produced by oxidation of the lignins with K₃[Fe(CN)₆] or H₂O₂ were determined from electron spin resonance spectra. The radicals were observed by the oxidation of sinapyl alcohol. It was found that the two radicals observed by the oxidation of dioxane lignins were deprotonated 2,6-dimethoxyp-benzosemiquinone and 6-hydroxy-2-methoxy-p-benzosemiquinone, and that they were produced from syringyl end groups. The production rate of radicals from syringyl end groups possessing an-carbonyl group was greater than that possessing an-hydroxyl group. It was suggested that stilbene-type syringyl end groups were produced from phenylcoumaran moieties in lignins by acidolysis. The radicals produced from guaiacyl end groups were not observed by the oxidation process.     

Study on low mass thermal degradation products of milled wood lignins by thermogravimetry-mass-spectrometry

Summary Thermogravimetry-mass-spectrometry (TGMS) as a sophisticated analytical instrument is described for the thermal analysis of milled wood lignins from spruce (Picea abies L.), beech (Fagus sylvatica L.) and bamboo (Bambus sp.). The samples were heated on the thermobalance in an inert gas atmosphere (Ar) with 20°C/min heating rate. The weight loss curves (TG) and their 1st derivatives (DTG) were recorded. The evolution of 10 low mass degradation products with m/z below 44 was monitored as a function of the temperature by means of a quadrupol mass spectrometer. Thus the intensity profiles of H₂, CH₃ , H₂O, CO, CHO, HCHO, CH₃O, CH₃OH, CH₃CO and CO₂ were recorded and interpreted in terms of lignin structure and the course of carbonization. The results are in agreement with the results of differencial scanning calorimetry (DSC) and pyrolysis-gas-chromatography mass-spectrometry (PyGCMS) of the phenolics.     

Study on biodegradated ability of thirteen edible fungi to straw

The biodegradated abilities of 13 edible fungi to straw were studied. The results showed that all the experimental fungi except Tricholama mongolicum had definite biodegradated abilities to the lignin and cellulose of straw. The Ideal fungus for straw degradation was screened out as Pleurotus ostreatus, which showed a higher degradation ability for lignin (17.86%) and lower degradation rate for cellulose (2.24%), with a Selection Factor (SF) of 7.97. The degradation rates of lignin and cellulose for other fungi ranged from 2.30% to 16.54% and 5.60% to 17.32%, respectively, and the SF was very low in range of 0.14 to 2.24.The ratio of colony‘s diameters to the color-zone (d1/d2) and SF are negative correlation, with a correlation coefficient of -0.1476.     

Delignification of cell walls of <Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis> during alkaline nitrobenzene oxidation

To clarify the behavior of whole lignins in wood cell walls during alkaline nitrobenzene oxidation, the delignification process from cell walls in normal and compression woods of Chamaecyparis obtusa Endl. (Cupressaceae) was observed using ultraviolet and transmission electron microscopies. The lignin content conspicuously decreased to around 10% after 35min in normal wood. The lignin content in compression wood finally leveled off at aroumd 10% after 50min. In gel filtration of oxidation products in ethyl acetate, a high molecular weight fraction was prominent in extracts from the early stage of the reaction. As the oxidation progressed, the high molecular weight fraction became less prominent in both normal and compression wood. Changes in the weights of cell wall residues during reaction indicated that approximately half of the components other than lignin were also removed from the cell walls. This shows that the majority of lignin with relatively high molecular weight is removed from the cell walls together with polysaccharides in the early stage of the reaction and that further oxidative degradation occurs in solution in later stages. Only a small amount of the lignin with low molecular weight could be analyzed by gas chromatography.Parts of this report were presented at the 47th (Kochi, April 1997) and 48th (Shizuoka, April 1998) Annual Meetings of the Japan Wood Research Society, and at the Lignin Symposium, Sapporo, October 1997     

Characterization of the lignin-derived products from wood as treated in supercritical water

Sugi (Cryptomeria japonica D. Don) and buna (Fugus crenata Blume) woods were treated with supercritical water (>374°C, >22.1 MPa) and fractionated into a water-soluble portion and a water-insoluble residue. The latter was washed with methanol to be fractionated further into a methanol-soluble portion and a methanol-insoluble residue. Whereas the carbohydrate-derived products were in the water-soluble portion, most of the lignin-derived products were found in the methanol-soluble portion and methanol-insoluble residue. The lignin-derived products in the methanol-soluble portion were shown to have more phenolic hydroxyl groups than lignin in original wood. The alkaline nitrobenzene oxidation analyses, however, exhibited much less oxidation product in the methanol-soluble portion and methanol-insoluble residue. These lines of evidence suggest that the ether linkages of lignin are preferentially cleaved during supercritical water treatment. To simulate the reaction of lignin, a study with lignin model compounds was performed;-O-4-type lignin model compounds were found to be cleaved, whereas biphenyl-type compounds were highly stable during supercritical water treatment. These results clearly indicated that the lignin-derived products, mainly consisting of condensed-type linkages of lignin due to the preferential degradation of the ether linkages of lignin, occurred during supercritical water treatment.This study was presented in part at the 45th lignin symposium, Ehime, Japan, October, 2000; and the 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1988     

Immunogold labeling of an extracellular substance producing hydroxyl radicals in wood degraded by brown-rot fungusTyromyces palustris

A fraction containing low-molecular-weight peptides that catalyzes redox reactions between electron donors and O₂ to produce ·OH, was partially purified from wood-decaying cultures of the brown-rot fungusTyromyces palustris. Polyclonal antibodies raised to the fraction were used for immunogold labeling of transverse sections of sapwood of spruce in various stages of degradation byT. palustris to demonstrate the cellular localization of the ·OH-producing substance. Initially, the wood cell wall was attacked primarily by fungal hyphae growing in the cell lumen. During the early stages of degradation, the gold label was localized in the fungal cytoplasm and cell wall and in the extracellular slime sheath surrounding the fungal cell wall. The gold label also was found throughout the wood cell wall, although the cell wall remained almost intact so long as the fungal hyphae remained in the lumen. Thus, the ·OH-producing substance is secreted by the hyphae into the lumen, and it diffuses through the S3 layer into the S2 layer and the middle lamella. The role of this ·OH-producing system in wood degradation byT. palustris is discussed.     

5α-Reductase inhibitory compounds produced by polymerization of resveratrol with horseradish peroxidase

To produce 5-reductase inhibitory compounds, resveratrol was enzymatically oxidized in a horseradish peroxidase (HRP)/H₂O₂ system. Ethyl acetate extract of the oxidation products showed strong 5-reductase inhibitory activity with 10%–15% organic solvents in the system, whereas without organic solvent little inhibitory activity was exhibited. The optimum pH of enzymatic oxidation for acquisition of the inhibitory activity was 4.5. The inhibitory compounds were isolated and identified as resveratroltrans-dehydrodimer and resveratrolcis-dehydrodimer by comparing with published nuclear magnetic resonance data. The two resveratrol dehydrodimers have stronger inhibitory activity than natural resveratrol dimers and trimers found inShorea species.