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Accessibility of hydroxyl groups in anhydride modified wood as measured by deuterium exchange
2017 - IRG/WP 17-40784
Acetylated wood shows improved properties largely due to the reduced amount of water in the acetylated cell wall. However, the exact mechanism by which water is excluded in acetylated wood remains unclear. Acetylation reduces hydroxyl content by substitution of hydroxyl groups in wood polymers but may also hinder access to unmodified hydroxyls by physical bulking. This work assesses hydroxyl accessibility in Radiata pine (Pinus radiata) samples that were acetylated and propionylated to different levels by means of deuterium exchange. The deuterium exchange experiments were performed in a novel way using an instrument designed for thermogravimetric analysis. Both acetylated and propionylated samples tend to reduce hydroxyl accessibility, but the relationship between accessibility and weight percentage gain for both modifications deviates from the expected theoretical relationship. The acetylated results suggest that, in addition to hydroxyl substitution, blocking of unmodified hydroxyl groups may play a role in hydroxyl accessibility reduction. The propionylated samples seemed to be damaged by the propionylation reaction, showing higher than expected accessibility. This may be a result of molecular restructuring within the cell wall which exposes new hydroxyl groups after propionylation.
G Beck, S Strohbusch, E Larnøy, H Militz, C Hill

Recent findings clarifying the mechanism of brown-rot decay protection in acetylated wood
2018 - IRG/WP 18-40827
This paper summarizes the findings obtained during my PhD research over the past three years. The results provide insights which help to understand the enhanced brown-rot decay resistance of acetylated wood with particular emphasis on the role of water. The protection acetylation provides is derived from moisture exclusion within the acetylated cell wall, but the exact mechanism by which water is excluded remained unclear. The first two studies conducted elucidate the moisture exclusion mechanism. It was demonstrated that hydroxyl (OH) accessibility is reduced in acetylated wood both by direct substitution of OH groups with acetyl groups and by hindering access of unmodified OH groups, due to steric hindrance. Characterisation of water in the various void structures of the wood anatomy by means of low-field nuclear magnetic resonance (LFNMR) relaxometry revealed that acetylation causes greater water mobility due to weaken interactions between water molecules and the acetylated pore walls. Both these initial studies also compared the moisture properties of the acetylated samples to samples modified with propionic anhydride. The idea was to demonstrate the importance of steric hindrance relative to direct OH substitution by modifying the wood with a larger anhydride molecule. However, the results indicated the propionylated cell walls were damaged in some way. The last two studies investigate acetylated wood decayed by the brown-rot fungus, Rhondonia placenta. Moisture development and chemical changes were assessed by means of LFNMR, OH accessibility and acetyl content throughout brown-rot degradation and revealed a markedly different degradation pattern for acetylated wood compared to unmodified samples, suggesting de-acetylation occurs within the cell wall prior to decomposition. Lastly, brown-rot decay in acetylated wood was investigated from the fungal perspective by determining how acetylation affects expression of fungal enzymes involved in oxidative chemistry and polysaccharide hydrolysis. Clear trends were difficult to observe for the oxidative genes selected, but genes involved in polysaccharide degradation showed a delayed response for acetylated samples and the most highly acetylated samples had the lowest overall expression levels.
G Beck

Hydroxyl accessibility - A brief review of deuterium exchange studies on modified wood
2019 - IRG/WP 19-20656
Wood modification imparts dimensional stability and decay resistance. The mechanism(s) by which this occurs are thought to be related to the bulking of the wood cell wall and subsequent moisture exclusion. Hence, quantification of accessible hydroxyl (OH) groups is of great interest when evaluating and increasing the understanding of the performance of modified wood. The aim of this study was to briefly review previous studies on the use of deuterium exchange as a method to measure OH group accessibility in modified wood with particular focus on preliminary data from a study on furfurylated wood. The experiment included furfurylated wood at three different weigh present gains (WPGs) and at different levels of mass loss caused by exposure to the brown-rot fungus Rhodonia placenta. The main findings were that: 1) OH accessibility in furfurylated samples did not change with increasing WPGs in sound samples, 2) OH accessibility in furfurylated samples increased significantly after initiation of R. placenta decay at commercial treatment level (mean WPG 32%). The increase in OH accessibility in R. placenta decaying wood was attributed to opening of cell wall microfibrils and formation of new OH groups in the brown rot modified lignin and the furfuryl alcohol polymer.
G Beck, C Hill, G Alfredsen

Non-enzymatic Gloeophyllum trabeum decay mechanisms: Further study
2001 - IRG/WP 01-10395
Information will be presented on the mechanisms involved in, and potential application of, non-enzymatic wood decay by brown rot decay fungi. Specifically, the hypothesized role of low molecular weight phenolate derivatives will be discussed in relation to non-enzymatic degradation of wood. The mechanism of binding of iron by cellulose, and binding and reduction of iron by fungal derivatives and model compounds is examined. Positive and negative aspects of potential application of these compounds in the generation of free radicals will be discussed.
B Goodell, J Jellison

The effect of low molecular weight chelators on iron chelation and free radical generation as studied by ESR measurement
2000 - IRG/WP 00-10367
The focus of this work was to improve our current knowledge of the non-enzymatic mechanisms involved in brown-rot decay. Electron Spin Resonance (ESR), also known as Electron Paramagnetic Resonance (EPR), is an attractive technique for the identification and study of chemical species containing unpaired electrons (such as radicals and certain transition metal species). ESR spin-trapping techniques are also commonly used to study very reactive and short-lived free radical species. It has been proposed that low MW chelators as well as Fenton reagents are involved in wood brown-rot decay, at least in early non-enzymatic stages. In this work, the binding between a chelator model compound and ferric iron was studied by ESR spectroscopy. The effects of the chelator model compound, Fenton reagents, as well as the reaction conditions on free radical generation were also studied using ESR spin-trapping techniques. The results indicate: 1. The relative amount of ferric iron bound to chelators is directly related to the chelator / iron ratio in the system. The relative quantity of the chelator-iron complex can be determined by measuring the intensities of the characteristic g4.3 ESR signal. 2. The effects of the chelator/iron ratio, the pH, and other reaction parameters on the hydroxyl radical generation in a Fenton type system could be determined using ESR spin-trapping techniques. 3. Data support the hypothesis that superoxide radicals are involved in the chelator mediated Fenton processes.
Yuhui Qian, B Goodell

Wood degradation mechanisms by the brown rot fungus Gloeophyllum trabeum
1997 - IRG/WP 97-10229
A mechanism for the degradation of wood by the brown rot fungus Gloeophyllum trabeum is outlined. The mechanism includes the function of redox-cycling, low molecular weight phenolic derivatives which sequester and reduce iron in acidic environments. The role of oxalate for the sequestration of iron (hydr)oxides and the pH dependent transfer of iron to the G. trabeum phenolic chelators, as well as for the maintenance of a pH gradient within the cell lumen and wood cell wall is discussed. A hypothesis for the generation of reactive oxygen species from the redox cycling of the phenolate compounds produced by G. trabeum as well as from free phenolics derived from the wood cell wall is outlined. Site specific production of hydroxyl radicals within the wood cell wall is discussed.
B Goodell, J Jellison

Characterization of glycopeptide from white-, brown- and soft rot fungi
2002 - IRG/WP 02-10424
Extracellular low-molecular-weight substances that catalyze a redox reaction between O2 and electron donors to produce hydroxyl radicals have been isolated from wood degrading cultures of white-rot, brown-rot, and soft-rot fungi. They contained protein, neutral carbohydrate, and Fe(II). These substances were glycosylated (glycated) peptides and suggested to form Amadori product (ketoamine). The glycopeptides are involved in all three types of decay.
H Tanaka, S Itakura, A Enoki

Generation of hydroxyl radical by the brown-rot fungus, Postia placenta
1988 - IRG/WP 1360
In an electron spin resonance (ESR) survey of various liquid cultures and wood slivers innoculated with the brown-rot fungus, Postia placenta, the spin trap dimethyl-l-pyrroline N-oxide (DMPO) was used to detect the presence of the hydroxyl radical. The ESR spectra for the paramagnetic DMPO- hydroxyl radical adduct was observed in (1) nitrogen-limited, liquid cultures having 1.0% glucose or 0.1% cellobiose as the carbohydrate source and (2) fungal infected wood slivers of Douglas-fir (Pseudotsuga menziesii) and white fir (Abies concolor). The 4-line ESR signal had a 1:2:2:1 intensity ratio, 15 G line splitting, and a g-factor of 2.003. The signal was stronger and more stable in wood slivers than in liquid cultures. The effect of free radical scavengers on the DMPO-hydroxyl adduct signal is currently being studied.
B Illman, D C Meinholtz, T L Highley

Preservative ability of wood to be fixed hydroxyl apatite substituted for antimicrobial metals
2001 - IRG/WP 01-30272
We succeeded in forming hydroxy apatite (HAp) in wood. HAp is non-toxicity and safe. Preservative ability of the wood, which substituted one part of Ca of constituent element of this HA p for antibacterial metals was measured. When Ca was substituted for Ag or Zn, mass loss in decay by brown-rot fungus F. palustris was restrained in about 50% (Ag) ~30% (Zn) of value of control specimen. However, when Ca was substituted for Cu, because F. palustris was copper-resistant microorganism, there was hardly the preservative ability. In case of decay by white-rot fungus T. versicolor, when Ca was substituted for Ag or Cu, the preservative ability was shown. In particular high preservative ability was provided when Ca was substituted for Cu + Zn or Ag + Zn. However, there was hardly the preservative ability when Ca was substituted for only Zn.
Y Haruhiko, I Sumaru

Destaining wood sapstains caused by Ceratocystis coerulescens
1996 - IRG/WP 96-10159
Fungal sapstain does not reduce the strength of wood, but it does discolor the wood, detracting from its appearance and decreasing the value of wood and wood products. The purpose of this investigation was to assess whether wood sapstain caused by Ceratocystis coerulescens could be destained and existing growth eradicated. The hydroxyl radicals generation under optimized conditions destained the sapstain on southern yellow pine veener disks caused by Ceratocystis coerulescens and killed the existing growth of sapstain fungi. Results indicate that the sapstain, melaninlike pigments of hyphae in pine disks, was destained and the existing growth of Ceratocystis coerulescens (Munch) Bakeshi [C-262] eliminated. As a result, sapstained wood and wood products with sapstain fungi can be salvaged, thus expanding our supply of usable wood.
S C Croan

Extracellular hydrogen peroxide producing and hydrogen peroxide reducing compounds of wood decay fungi
1991 - IRG/WP 1516
Extracellular H2O2-producing and H2O2-reducing compounds were isolated from wood-containing cultures of all the white-rot and brown-rot fungi and Ascomycetes which well degraded wood, but were not detected in the culture of the fungi which degraded little wood. The compounds are glycopeptides with a low molecular weight, require H2O2 for one-electron oxidation, catalyze the redox reaction between an electron donor such as NADH or ascorbic acid and O2 to produce H2O2 via O2·-, and produce ·OH by Fenton's reaction between the ferrous iron bound to the ligands and H2O2. The compounds show no phenol-oxidase activity and catalyze the hydroxylation of phenol to catechol and hydroquinone in the presence of H2O2.
A Enoki, G Fuse, H Tanaka

Relationship between degradation of wood, cellulose or lignin-related compounds and production of hydroxyl radical or accumulation of oxalic acid in cultures of brown-rot fungi
1994 - IRG/WP 94-10062
The degradation activities of brown rot fungi against wood, cellulose, and lignin-related compounds were measured in cultures containing glucose or wood as a carbon source. Also the activities of one-electron oxidation and hydroxyl radical production and the amount of oxalic acid present in the cultures were measured. The degradation activities of the fungi against wood, crystalline cellulose and the lignin model compounds were in direct proportion to the activities of one-electron oxidaton and hydroxyl radical production. The amount of oxalic acid win the cultures was inversely proportional to those activities and oxalic acid was almost not detectable in the cultures in which wood was actively degraded. On the basis of these results it is concluded that one-electron systems including hydroxyl radicals play important roles in the initial stage of wood decay by brown rot fungi and degrade oxalic acid produced by the fungi to trace amounts.
S Itakura, T Hirano, H Tanaka, A Enoki

Preliminary studies of the performance of iron chelators as inhibitors of brown rot (Coniophora puteana) attack
1996 - IRG/WP 96-10185
This paper describes experiments to examine the proposal that the presence of iron is essential for brown rot fungi to utilize hydroxyl radicals remote from the hyphae as a means of converting the wood into a food source. reliminary test results are presented from trials using three different iron chelators impregnated into Scots pine (Pinus sylvestris) sapwood blocks. Their relative effects on the resistance of the blocks to brown rot attack under laboratory mycology trials are detailed. In general, very dilute aqueous solutions of all the chelators studied were capable of inhibiting the attack of brown rot fungus. indications for the application of this branch of basidiomycete control and areas for further study are discussed.
E D Suttie, R J Orsler, P M Wood

Extracellular hydrogen peroxide-producing and one-electron oxidation system of brown-rot fungi
1990 - IRG/WP 1445
Wood-component-degrading compounds involved in the initial degradation of the cellulose and lignin in wood were isolated from wood-containing culture of brown-rot fungi, Gloeophyllum trabeum and Tyromyces palustris and partially purified by gel filtration on Sephadex G-25 and DEAE-Sepharose ion-exchange chromatography. The compounds were glycoproteins. The molecular weights of the glycoproteins as determined by gel filtration were very small and about 1,600-2,000. The one-electron oxidation activity of the peptides was determined by measuring ethylene production from 2-keto-4-thiomethylbutyric acid (KTBA). The peptides contained ferrous iron,required H2O2 for KTBA oxidation, were capable of catalyzing the oxidation of NADH to produce H2O2 in the presence of 02 and showed little phenol-oxidase activity under conditions giving high activity against KTBA. The ferrous iron combined with the glycopeptides was oxidized to the ferric state by H2O2.
A Enoki, S Yoshioka, H Tanaka, G Fuse

The effect of a chelator mediated fenton system on activation of TMP fibres and decolorization of synthesized dyes
2004 - IRG/WP 04-50223
The purpose of this work is to improve our current knowledge of the non-enzymatic mechanisms involved in the brown rot degradation of wood, but also to study the potential applications of a chelator-mediated Fenton system in activation of wood fibers and decolorization of synthesized dyes. In this work, Electron Spin Resonance (ESR) spin-trapping techniques were used to study the generation of hydroxyl radicals in a mediated Fenton system. The activation of Thermal Mechanical Pulp (TMP) fibers was also evaluated by ESR measurement of free phenoxy radical generation on solid fibers. The results indicate that low molecular weight chelators can improve Fenton reactions, thus in turn stimulating the free radical activation of TMP fibers. However, the data also show that excessive and prolonged free radical treatment may cause the destruction of fiber phenoxy radicals. A mediated Fenton system was evaluated for decolorization of several types of synthesized dyes as well. The results show that, compared to a neat Fenton process, the mediated Fenton process increased the production of .OH species to increase the decolorization efficiency. The color of a dilute liquid dyes (Carta Yellow RW, Carta Yellow G, or Cartasol Red 2GF) was effectively reduced to a colorless level after 90 minutes of treatment at room temperature by a mediated Fenton process. In conclusion, this study demonstrates the potential for application, but also the complexity of free radical chemistry in biological materials, especially with regard to the chelation of transition metals and the interaction between free radicals. The complexity of the dyes is similar to that of some organic wood preservatives and may provide a means for remediation of preservative contaminants in soils.
Yuhui Qian, B Goodell, J Jellison

Wood and filter paper degradation, phenol oxidase and one-electron oxidation activities by the white rot fungus Ceriporiopsis subvermispora
2003 - IRG/WP 03-10486
The activities of one-electron oxidation and phenol oxidase during incubation of cultures of the white-rot basidiomycete Ceriporiopsis subvermispora containing either glucose or wood were periodically measured. Further, the degradation activities against wood and filter paper were examined during the course of cultivation. Weight losses of Japanese beech wood and Japanese cedar wood after 12 weeks were about 20% and 15%, respectively. Weight loss of filter paper was about 23% after 9 weeks. The one-electron oxidation and phenol oxidase activities in wood-containing cultures were higher than those in glucose-containing cultures. Extracellular low-molecular-weight substance has been isolated and has been characterized to compare with the substances from other wood degrading fungi that catalyze a redox reaction between O2 and electron donors to produce hydroxyl radicals. The mechanism on wood degradation caused by the white-rot fungus C. subvermispora is discussed.
H Tanaka, S Itakura, A Enoki

The involvement of extracellular substances for the generation of hydroxyl radical during wood degradation by white-rot fungi
1997 - IRG/WP 97-10218
The activities of one-electron oxidation, hydroxyl radical generation, and phenol oxidase during the incubation of cultures of four white-rot fungi containing either glucose or wood were periodically measured. Further, their degradation activities against wood were examined during the course of cultivation. The generation of hydroxyl radical was correlated to the activity of wood degradation and it was not dependent upon the activity of oxidants such as phenol oxidase. The generation of hydroxyl radical was mostly due to the extracellular low molecular weight substances that catalyzed redox reaction between electron donors and O2 to produce H2O2 via O2 and to reduce H2O2 to HO·.The substances reduced Fe(III) to Fe(II) and strongly absorbed Fe(II).
H Tanaka, S Itakura, A Enoki

A model for attack at a distance from the hyphae based on studies with the brown rot Coniophora puteana
1995 - IRG/WP 95-10104
In timber infested by brown rot fungi, a rapid loss in strength is attributed to production of hydroxyl radicals (HO·) at a distance from the hyphae. The immediate precursor is Fenton's reagent (Fe(II)/H2O2), but the pathways leading to Fe(II) and H2O2 have remained unclear. Cellobiose dehydrogenase, purified from cultures of Coniophora puteana, will couple oxidation of cellodextrins to reduction of Fe(III). Two characteristics of brown rot are release of oxalic acid and lowering of the local pH, often to about pH 2. Modelling of Fe(II) speciation in the presence of oxalate has revealed that Fe(II) oxalate complexes are important at pH 4-5, but at pH 2 almost all Fe(II) is uncomplexed. The uncomplexed Fe(II) reacts very slowly with dioxygen. Diffusion of Fe(II) away from the hyphae will promote conversion to Fe(II)-oxalate and autoxidation with H2O2 as product. Thus the critical Fe(II)/H2O2 combination is formed at a distance.
S M Hyde, P M Wood

Isolation and characterization of hydroxyl-radical-producing glycopeptide genes from the white-rot basidiomycete Phanerochaete chrysosporium
2006 - IRG/WP 06-10588
During wood decay, the white-rot basidiomycete Phanerochaete chrysosporium secretes low-molecular-weight glycopeptides that catalyze a redox reaction between O2 and electron donors to produce hydroxyl radical. This reaction accounts for most of the hydroxyl radical produced in wood-degrading cultures of P. chrysosporium. In combination with phenol oxidases, hydroxyl radical is believed to play a role in lignin degradation. Low-molecular-weight fractions with high one-electron oxidation activity were isolated from wood-containing cultures of P. chrysosporium. They were composed of protein (55% w/w), neutral carbohydrate (25% w/w), and Fe(II) (0.04% w/w). A partially-purified glycopeptide was separated by tricine-SDS-PAGE and the bands were excised and digested with endoproteinase Asp-N. The N-terminal and four internal fragments were sequenced. BLAST and FASTA searches revealed no homologies between these amino-acid sequences and known proteins. However 70-100% similarities were found when the P. chrysosporium genome database was searched for the amino-acid sequences. cDNAs and two putative genes encoding these glycopeptides, glp1 and glp2, were isolated and sequenced. The 875-bp glp1 and 864-bp glp2 are located on scaffold 2 of the P. chrysosporium genome. These presumptive genes each consist of seven introns and eight exons. The latter encode a predicted mature peptide of 138 amino acids and a 22-amino-acid signal sequence for secretion. The predicted peptide sequences are nearly identical to N-terminal and internal sequences obtained from the partially-purified glycopeptide. The molecular weights of the deduced mature proteins coincide with the molecular weight of the glycopeptide as determined by tricine-SDS-PAGE. Finally, the amino-acid composition of the glycopeptide is nearly identical to the amino-acid compositions deduced from the glp1 and glp2 sequences. It appears that glp1 and glp2 encode the partially-purified hydroxyl-radical-producing glycopeptide Glp.
H Tanaka, G Yoshida, Y Baba, K Matsumura, S Itakura, A Enoki

Phenol oxidase activity and one-electron oxidation activity in wood degradation by soft-rot deuteromycetes
2007 - IRG/WP 07-10615
Wood degradation, one-electron oxidation activity as assayed by ethylene generation from 2-keto-4-thiomethylbutyric acid (KTBA), and phenol oxidase activity were measured in cultures of six deuteromyce fungi, with glucose or wood as the carbon source. The four fungi that degraded Japanese beech wood had higher one-electron oxidation activities in wood-containing cultures than in glucose-containing cultures. These four fungi also had measurable phenol oxidase activity in wood-containing cultures, but not in glucose-containing cultures. The two mould fungi that did not significantly degrade wood had no phenol oxidase activity in either wood- or glucose-containing cultures. The one-electron oxidation activity in intact cultures of the soft-rot deuteromycetes was roughly related with the rate of mass loss during wood degradation in those cultures. However, there was no clear relationship between phenol oxidase activity and either one-electron oxidation activity or the rate of wood mass loss, either over time, or in total. Most of the one-electron oxidation activity resulted from phenol oxidase and hydroxyl radical. Most of the phenol oxidase activity resulted from laccase. Furthermore, the mechanism of wood degradation by one of these deuteromycete fungi, Graphium sp., was investigated. Most of the phenol oxidase activity appeared to derive from laccase. Most of the ethylene generation from KTBA was attributed to hydroxyl radicals, produced by a low-molecular-mass substance in the extracellular media. This substance was composed of protein, carbohydrates, and Fe(II), and catalyzed redox reactions between O2 and unidentified electron donors, to produce hydroxyl radicals via H2O2. It is suggested that hydroxyl radicals may produce new phenolic substructures on the lignin polymer, making it susceptible to attack by laccase. Thus, one-electron oxidation acitivity and laccase activity are both important in wood degradation by Graphium sp.
H Tanaka, M Yamakawa, S Itakura, A Enoki

The involvement of hydroxyl-radical-producing glycoprotein from the white-rot basidiomycete Ceriporiopsis subvermispora in wood decay
2009 - IRG/WP 09-10688
White-rot decay can be divided into two subtypes. One type involves the simultaneous degradation of all wood components through the formation of erosion troughs with a progressive thinning of wood cell walls. This type of decay is consistent with a model in which several polymer-degrading enzymes act on the exposed surfaces of the wood cell walls, producing progressive erosion from the lumen to the middle lamella. The second type of white rot is selective lignin degradation, carried out by a relatively small number of fungi. In this case, lignin and non-cellulosic polysaccharide are removed without progressive thinning of the wood cell walls. Ceriporiopsis subvermispora is one of the best examples of a selective lignin degrader. Although the reason why this fungus selectively degrades lignin is still unclear, the lack of a complete cellulolytic enzymatic complex has been considered as one of the motives for inexpressive cellulose degradation. Moreover, it has been reported that this fungus secretes a number of low-molecular-mass compounds including oxalic acid during solid-state fermentation of wheat straw [1] and several fatty acids during solid-state fermentation of wood [2,3]. Independent of their origins, low-molecular-mass compounds may play key roles in the overall wood biodegradation process. During wood decay, the white-rot basidiomycete Phanerochaete chrysosporium secretes low-molecular-mass glycoproteins that catalyze a redox reaction between O2 and electron donors to produce hydroxyl radical (•OH). This reaction accounts for most of the •OH produced in wood-degrading cultures of P. chrysosporium. In combination with phenol oxidases, •OH is believed to play a role in lignin degradation [4,5]. In our previous study [6], cellulose and lignin depolymerization, as well as the production of the hydrolytic enzymes, carboxymethylcellulase (CMCase) and Avicelase (exo-1,4-β-D-cellobiohydrolase), and the ligninolytic enzymes, laccase, manganese peroxidase (MnP), and lignin peroxidase (LiP), have been determined during the wood-decay process of C. subvermispora. During the 12-week incubation with Japanese beech wood, C. subvermispora continuously produced at least one of three phenol oxidases: laccase was produced initially, followed by Mn-independent peroxidase activity peaking at 6 weeks and MnP activity peaking at 10 weeks. Lignin peroxidase and CMCase activities peaked after 3 weeks of incubation. Avicelase activity was present throughout the incubation period, although the activity was very low. Furthermore, it was also shown that the low-molecular-mass fraction of the extracellular medium, which catalyzes a redox reaction between O2 and electron donors to produce •OH, may act synergistically with the enzymes to degrade wood cell walls. A. low molecular-weight, extracellular substance with one-electron oxidizing activity was isolated from C. subvermispora. The substance was partially purified by ammonium sulfate precipitation, Sephadex G-50 gel filtration, and DEAE-Sepharose ion-exchange chromatography. The partially purified material was a glycoprotein composed of 32% protein and 46 % neutral carbohydrate, containing 0.05% Fe(II) by weight. Tricine-SDS-PAGE showed 2 bands with a molecular mass of around 13000. One mg of the partially purified glycoprotein reduced 1.3 μmol of Fe(III) to Fe(II) and contained at least 0.4 μmol of α-hydroxyketone or endiol groups. Most of the α-hydroxyketone groups were 1-amino-2-ketose produced by the condensation of side-chain amino groups and carbohydrates. We have reported that the white-rot fungi Irpex lacteus [7], P. chrysosporium [4,5], and Trametes versicolor [8] produce at least one phenol-oxidizing enzyme, as well as •OH produced by agents other than phenol oxidase. Herein we demonstrate that C. subvermispora produces laccase, MnP, LiP, and a low-molecular-mass fraction which can generate ethylene from 2-keto-4-thiomethylbutyric acid due to the oxidizing activity of •OH. This low-molecular-mass fraction described above has very similar properties to the •OH-producing glycoprotein from the other white-rot fungi [4,5,8]. These glycoproteins catalyze a redox reaction between O2 and electron donors to produce •OH. The Fenton system [Fe(II) and H2O2], which is known to depolymerize cellulose [9], can be generated in white-rot-fungal cultures, since H2O2 is present and Fe(II) can be formed by the activity of enzymes, such as cellobiose dehydrogenase [10] or MnP [11], and also by the Fe(III)-reducing activity of fungal-produced hydroxyaromatic carboxylic acids [12]. The glycoprotein reduced O2 to H2O2 and Fe(III) to Fe(II), and thus could generate •OH via a Fenton reaction. Thus the •OH-producing glycoprotein found in white-rot fungi [4,5,8] could also be involved. We propose that during wood degradation by C. subvermispora, laccase and MnP preferentially degrade lignin, in concert with a system that produces •OH. In addition, this fungus’s incomplete cellulase system and its •OH-generating system may act synergistically to degrade and metabolize cellulose.
H Tanaka, Y Inoue, T Morikawa, S Itakura, A Enoki

Accessibility of Wood Cell Walls to Well-defined Platinum Nanoparticles
2012 - IRG/WP 12-20494
Copper nanoparticles are found in the walls of parenchyma cells in southern pine sapwood treated with a micronised wood preservative, but they are absent from tracheid walls. Hence, we hypothesized that small nanoparticles can penetrate the walls of unlignified parenchyma cells, but are excluded from lignified tracheid walls. This paper tests this hypothesis by treating pine sapwood with an aqueous emulsion of coated, inert, platinum nanoparticles (2-4 nm). A focused ion beam was used to make ultra-thin sections of the cell wall layers of earlywood tracheid and ray parenchyma cells excised from treated southern pine sapwood. High resolution transmission electron microscopy and high-angle annular dark-field scanning transmission electron microscopy in combination with energy dispersive analysis of x-rays were used to examine the penetration of cell walls by platinum nanoparticles. Platinum nanoparticles were only deposited on the wall adjacent to the cell lumen of tracheids and were not detected in the cell wall. In contrast, platinum nanoparticles penetrated ray parenchyma cell walls. These particles were identified as crystalline (metallic) platinum by lattice image analysis in high resolution transmission electron microscopy. Therefore we conclude that small nanoparticles (2-4 nm) are able to penetrate ray parenchyma cell walls, but are excluded from lignified tracheid walls.
H Matsunaga, Y Kataoka, M Kiguchi, P D Evans