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Distribution of cellulases in the body of Coptotermes formosanus and the probability that the termite uses glucose as an energy and carbon sources
1997 - IRG/WP 97-10202
We assayed extracts of the digestive system and of the whole body of Coptotermes formosanus to determine where the various cellulases, glucose, and related substances were concentrated and to detect pyruvate dehydrogenase activity in the hindgut-removed body in order to verify its full cellulolytic system. About 20%, 18% and 36% of the total exo-1,4-ß-glucanase activity of C. formosanus were detected in the salivary glands, midgut, and hindgut, respectively. About a third of the total endo-1,4-ß-glucanase activity in the termite was detected in the salivary glands (34.5%) whereas the activities in the midgut and hindgut were 21.1% and 18.2%, respectively. About 75% of the total ß-D-glucosidase activity in the termite was detected in the midgut. Thus all the necessary cellulases for hydrolysis of natural cellulose to glucose were present in the region ranging from the salivary glands to the midgut in significant amounts. Most of the glucose and trehalose detected in the termite existed in the gutted-body. Most of the glucose detected in the gut existed in the midgut. Pyruvic acid was directly converted to acetyl-CoA in the presence of NAD+ by a crude extract of the gutted-body. These results suggest that natural cellulose ingested by the termite is hydrolyzed to oligosaccharides in the region of the foregut and midgut as well as in the hindgut, that oligosaccharides are hydrolyzed to glucose predominantly in the midgut, and that the resultant glucose is absorbed through the midgut wall into the tissues to be used as important energy and carbon sources.
S Itakura, H Tanaka, A Enoki


Physiologic response of Phanerochaete chrysosporium to exposure to triazole fungicides
1994 - IRG/WP 94-10066
Triazoles are increasingly important fungicides which are employed for a variety of applications included wood protection. Several recent studies suggest that white rot fungi are more tolerant of triazole compounds than other wood degrading fungi. Cultural studies using a white rot fungus, Phanerochaete chrysosporium, and 0.2 or 0.8 ppm of tebuconazole or propiconazole suggested that mycelial dry weight was most affected by the presence of triazoles. Extracellular carboxymethylcellulase, cellobiosidase and phenol oxidase activities were depressed but not inhibited by triazoles, while ß-glucosidase activity appeared to be slimulated by the presence of these biocides. The results suggest that white rot fungi may be less sensitive to triazoles and this diminished sensitivity may permit these fungi to become more important on wood treated with this biocide.
J J Morrell, R K Velicheti


Effect of light and ventilation condition on the rate of wood decay by the brown rot basidiomycete, Tyromyces palustris
1991 - IRG/WP 1517
Effect of light and the ventilation conditions of incubation jars on the wood decay by Tyromyces palustris (Berk. et Curt.) Murr. FFPRI 0507 was investigated. Under no irradiation of light, the ventilation conditions gave extensive effect on mass loss of the test pieces when the culturing was performed with culture medium designated in Japanese Industrial Standard (JIS) A 9302 (Medium A; glucose 4.0%, malt extract 1.5%, peptone 0.3%). On the other hand, no such kind of effect for ventilation conditions was recognized under light conditions, and the wood decay by the fungus accelerated by additional light irradiation of a considerably small intensity. Next, we investigated the relationship between light and the culture medium composition during the wood decay by the fungus. It was found that almost equivalent mass loss occurred after 60-90 days of cultures when the culturings were performed under light-shield conditions with medium A, the culture medium designated in Japan Wood Preserving Association (JWPA) standard No.1 (medium B; malt extract 2.0%, peptone 1.0%), and another culture medium diluted medium B by two times (medium C). Under the irradiation of light, the mass loss on the cultures in medium B and C was markedly less than that in the same media under no irradiation conditions. These results suggested that effect of light on the wood decay by Tyromyces palustris depended on the concentration of glucose in the culture medium. Further, we also investigated the activities of several extracellular and cell wall bound enzymes in wood meal medium contained medium A. From our experimental results, the activities of cellulase (b-1,4-glucan 4-glucanohydrolase, E.C. 3.2.1.4) and mannanase (b-1,4-mannan mannanohydrolase) depended on light irradiation during the wood decay and these enzyme activities may give extensive effect on the mass loss by Tyromyces palustris.
T Suzuki, M Higaki


Preliminary studies on cellulase production by selected Basidiomycetes and the effect of copper-chrome-arsenate on these enzymes
1980 - IRG/WP 1122
The growth of wood-destroying fungi on ligno-cellulosic materials depends on the production of many enzymes, of which probably the most important is the multi component cellulase system. Within this system, at least three different kinds of enzym are believed to be involved in crystalline cellulose decomposition. These are endo-1,4-glucanase, exo-1,4-ß glucanase and ß-glucosidase. Most of the recent research on cellulases has concerned isolation, purification and characterisation of the enzymes and their application in the utilisation of cellulosic waste. Information on the chemical inhibition of cellulases is available but there is little reference to the interaction of wood-attacking cellulases and the preservatives which are used to protect wood. The objective of this work is to study the production and activity of cellulases of selected basidiomycetes and to observe the effect of wood-preservatives on these enzymes. Preliminary studies with copper-chrome-arsenate (CCA) are reported here
O Collett


Preliminary studies of the decay mechanism of some brown-rot fungi
1989 - IRG/WP 1402
The importance of the enzymatic degradation of hemicellulose and cellulose by brown-rot fungi is still under discussion. Endo-ß-1,4-xylanase and endoglucanase activities of Coniophora puteana and Poria placenta cultures were measured by the increase in reducing groups. Enzymes were produced in liquid and solid sawdust based culture media. Enzyme activities were measured in two, four and eight week intervals from the beginning of the test. The ability of brown-rot fungi to degrade crystalline and noncrystalline cellulose by an enzymatic pathway was studied by measurement of total cellulase activities from Coniophora puteana and Serpula lacrymans. Low enzyme activities were noted. This result indicates strongly that these fungi possess a complete cellulose-degrading enzymatic pathway.
A-C Ritschkoff, H Viitanen


Biochemical aspects of white-rot and brown-rot decay
1987 - IRG/WP 1319
This paper presents an overview of the decomposition of wood by white- and brown-rot fungi - the most important and potent of known wood-decay fungi. These organisms are unique among cellulose destroyers because of their strong capability to enzymatically degrade lignified material. Special emphasis is given to the following aspects of wood decomposition by white- and brown-rot fungi: (1) effects on the chemical and physical properties of wood, (2) method of invasion and ultrastructural modification of wood, (3) nature and activities of extracellular degrating enzymes, (4) relationship of ultrastructural changes to the degradative enzyme systems, and (5) unique physiological features of the fungi that can be used to control decay.
T L Highley


Isolation of a putative endogenous endo-ß-1,4-glucanase (cellulase) from the midgut diverticulae of the wood-boring crustacean, Limnoria quadripunctata
2003 - IRG/WP 03-10494
Over the last 75 years, many workers have conducted research into the digestive biochemistry of Limnoria. Cellulolytic enzymes have been detected in crude extracts, although whether these are of endogenous or microbial origin has been in question. To elucidate the source of these enzymes, RNA was isolated from the midgut diverticulae of Limnoria quadripunctata. Reverse transcription polymerase chain reaction (RT-PCR) was performed using degenerate primers derived from conserved regions of known endoglucanases. This produced a fragment of cDNA that was extended by rapid amplification of complementary DNA ends (RACE). The overlapping cDNA fragments were cloned and sequenced, and the amino acid sequence of a putative protein was deduced. BLAST analysis was performed to determine the similarity between this sequence, and those of known proteins. The results indicated a conserved structural domain of a glycosyl hydrolase, and the greatest sequence homology was to endo-ß-1,4-glucanases from termite species. In situ hybridisation was performed using labelled sense and antisense probes. This indicated the presence of a cellulase-encoding mRNA in the tissues of the midgut diverticulae. These results provide the strongest evidence to date of endogenous endoglucanase production by Limnoria.
J Dymond, M J Guille, S M Cragg


A biochemical explanation for the observed patterns of fungal decay in timber
1980 - IRG/WP 1111
Experiments designed to compare the degree of localization of the cellulase enzymes of some white, brown and soft rot organisms are described. The site and nature of binding of the enzymes is discussed. The technique is ellution of mycelium grown in liquid culture with a variety: of agents including acetate buffer, carboxymethyl cellulose solution, borate/glycerol buffer and urea. The mycelium was assayed for cellulase activity before and after washing. Eluted protein was also assayed. The effect on retention of cellulases of treatment with a (1,3) ß glucanase was determined. Brown rot organisms showed a far lower retention of cellulases to the mycelium than the soft and white rot organismns. Carboxymethylcellulose solution was found to be only slightly effective as a protein eluent on the white and soft rot organisms indicating low substrate affinity. 8 M urea was found to be an effective protein eluting agent - possibly implying hydrogen bonding between cellulases and the fungus. Borate/glycerol buffer was also shown to be an effective agent for protein elution - however· less so than urea. This agent probably binds to carbohydrates, either glycoprotein enzymes or binding sites on the organism, thius displacing protein. (1,3) ß glucanase markedly decreased the retention of cellulase activity in soft and white rot organisms indicating binding to a (1,3) ß glucan. It is postulated that cellulase retention mechanisms found in soft and white rot organisms and absent from brown rots have a significant role in the production of the characteristic observed patterns of decay of the three types.
N B Green, D J Dickinson, J F Levy


The role of oxygen and oxygen radicals in one-electron oxidation reactions mediated by low-molecular weight chelators isolated from Gloeophyllum trabeum.markup
1994 - IRG/WP 94-10086
The KTBA assay for determination of one-electron oxidation activity was used to assay reactions of low-molecular weight chelators isolated from the brown rot fungus Gloeophyllum trabeum. The assay, performed either under air or nitrogen showed that molecular oxygen was an important factor in chelator-mediated oxidation reactions. A reduction in oxidative activity was observed when superoxide dismutase was introduced to the reaction, indicating that superoxide radicals also involved in the reaction and were scavenged by SOD. The KTBA assay showed, similarly to other assays in our laboratory, that the chelators could reduce Fe(III) to Fe(II). However, once chelators were 'oxidized' in this process they appeared to be redox inactive. Preliminary results indicate that chelator redox activity can only be regenerated in the presence of a reductant such as NADH or oxalate.
Jun Lu, B Goodell, Jiang Liu, A Enoki, J Jellison, H Tanaka, F Fekete


Genome-wide survey of cellulase related genes of white rot fungus,Pleurotus ostreatus
2007 - IRG/WP 07-10627
A white rot fungus, Pleurotus ostreatus, which is a important edible mushroom, has received much attention to apply to bioremediation and bioconversion because it has both cellulase and ligninase. To confirm copy number of cellulase-related genes encoded in P. ostreatus genome, we attempted to genomic Southern hybridization of P. ostreatus. The draft genome sequence and a large quantity of EST and cDNA information are now available for a white rot fungus, Phanerochaete crysosporium. To obtain sequence information of the cellulase-related genes in the white rot fungi, we also carried out public data base search. These genome-wide studies of P. ostreatus that consist of experimental and bioinformatical approach will provide meaningful advances to exploration of molecular mechanisms underlying wood decay.
T Tamenori, S Horisawa


Effect of wood polymers degradation during heat treatment on extracellular enzymatic activities involved in beech degradation by Trametes versicolor
2008 - IRG/WP 08-40392
Effect of heat treatment on extracellular enzymes involved in wood degradation by Trametes versicolor was investigated. Heat-treated and untreated beech blocks were exposed to T. versicolor on malt agar medium and extracellular enzymatic activities investigated. A strong ABTS oxidizing activity has been detected during the first stage of colonization in both cases, while cellulase activities are mainly detected in the case of untreated beech wood. Further investigations carried out on holocellulose, isolated using sodium chlorite delignification procedure, either on untreated beech wood or heat treated one, indicate that commercially available cellulases are able to hydrolyse totally holocellulose from untreated sawdust, while hollocellulose from heat treated one was only partially hydrolysed. CP/MAS 13C NMR analysis of heat treated beech wood but also its lignin and holocellulose fractions obtained after acidic hydrolysis of polysaccharides or delignification with sodium chlorite indicates an important modification of hollocellulose showing degradation of hemicelluloses as generally described in the literature, but also formation of carbonaceous materials within the wood structure. All these data suggest that chemical modifications of wood components during heat treatment disturb enzymatic system involved in wood degradation.
S Lekounougou, G Nguila Inari, M Pétrissans, S Dumarçay, J P Jacquot, E Gelhaye, P Gérardin


Protection mechanisms of modified wood against decay by white and brown rot fungi
2010 - IRG/WP 10-10713
The resistance of beech and pine wood blocks treated with 1,3-dimethylol-4,5-dihydroxyethylene urea (DMDHEU) against T. versicolor and C. puteana increased with increasing WPG. Full protection (mass loss below 3%) was reached at WPGs of approximately 15% (beech) and 10% (pine). Metabolic activity of the fungi in the wood blocks was assessed as heat or energy production determined by isothermal micro-calorimetry. Fungal activity in the wood decreased with increasing WPG. Still, activity was detected even in wood blocks of highest WPG and showed that the treatment was not toxic to the fungi. The infiltration of untreated and DMDHEU-treated wood blocks with nutrients and thiamine prior to fungal incubation did not result in an increased mass loss caused by the fungi. This shows that the destruction or removal of nutrients and vitamins during the modification process has no influence on fungal decay. In order to study the effect of cell wall bulking and increased surface area, the cell wall integrity was partly destroyed by milling and the decay of the fine wood flour was compared to that of wood mini-blocks. The mass losses caused by the fungi, however, also decreased with increasing WPG and showed comparable patterns like in the case of mini-blocks.To study the effect of the chemical change of cell wall polymers, cellulose was treated with DMDHEU and the product was subjected to hydrolysis by a cellulase preparation. The release of sugar during the incubation was clearly reduced as compared to untreated cellulose. Pre-treatment of modified cellulose with Fenton’s reagent increased the amount of released sugar due to the cellulase activity. Pine micro-veneers were subjected to Fenton’s reagents in acetate buffer over 48h. While untreated specimens and veneers treated with low DMDHEU concentration displayed strong and steady tensile strength loss, veneers treated to a higher WPG did hardly show tensile strength loss.
C Mai, P Verma, Yanjun Xie, J Dyckmans, H Militz


Measuring fungal cellulolytic enzyme activity in degrading wood
2011 - IRG/WP 11-10753
An enzyme assay which measures enzyme activity directly from wood will assist in the fundamental understanding of the enzyme components of the decay mechanism of rot fungi and any changes in the presence of wood protectants. At present, to measure fungal carbohydrate degrading enzyme activity in the presence of wood, two methods are used: either 1) the enzyme is measured in the growth media containing lignocellulosic material after the fungus has been allowed to utilize the cellulolytic substrate or 2) the fungus is grown on the lignocellulosic material and the enzymes are extracted using buffers after the growth period and the buffer is analyzed for enzyme activity. This research presents an enzyme assay where wood slivers are added to the assay mixture. The brown rot fungus Postia placenta was grown on wafers measuring 70mm by 23mm by 1.5mm for 12 days. The wafers were then sliced into 3mm by 1mm slivers and assayed using a micro assay using Azo-carbohydrates as substrates. The use of a 96-well microtiter plate allowed a large number of samples to be analyzed at one time with reduced reagent use. Using this assay method allows screening for enzyme activity along the length of the wafer. Our results showed detectable differences over the length of the wafer with a peak of endoglucanase activity closest to the colonization point of P.placenta. Small samples of decayed wood from the field were assayed for endoglucanase activity and the results indicate that this method has a potentially to be used to determine early decay. This assay will be useful in many research fields to gauge the presence and location of fungi and their carbohydrate degrading enzyme activity within a wood sample.
S M Duncan, J S Schilling


Postia placenta cellulase gene expression in modified wood during incipient decay
2013 - IRG/WP 13-40626
In optimization of modified wood, it is important to understand the mode of action of the wood modification and how the fungi response to it. The aim of this study was to investigate the expression of cellulases during the first two weeks of Postia placenta exposure in acetylated, DMDHEU-treated and thermally modified as well as in untreated wood. Using real-time PCR, the gene expression patterns of the P. placenta endoglucanase Ppl103675 and β-glucosidase Ppl112501 during 56 days of decay was analyzed. Preliminary data indicate that both genes are expressed at higher values in untreated wood at 56 days of exposure (56% mass loss) than at 14 days. We also saw high values for both genes at ten days of exposure for both untreated (11% mass loss) and modified woods (all 0% mass loss). We conclude that the high values at 10 and 56 days in untreated wood may be due to that monosaccharides and by-products from the cellulose degradation process, known to induce cellulase expression, have been released from the wood cell wall. Furthermore, the high values at ten days in the modified woods may be the result of an unknown regulatory mechanism. In addition, we found that the expression patterns for the P. placenta endoglucanase Ppl103675 and β-glucosidase Ppl112501 are very similar in the three modified woods investigated, but different from the patterns in untreated wood. This might be an indication that all the herein studied wood modifications affect fungi in the same way, i.e. they have the same mode of action.
R Ringman, A Pilgård, K Richter


Enhancing Our Understanding of Brown Rot Mechanisms through Catalytic Pretreatment and Cellulase Cocktail
2018 - IRG/WP 18-10909
A catalytic mechanism, described as the “chelator-mediated Fenton” (CMF) mechanism, is proposed to mimic the non-enzymatic action of brown rot fungi. A CMF treatment was used together with an enzymatic cocktail to study how wood was deconstructed and solubilized. This was done in-part to determine if the treatment mimicked the action of brown rot fungi, but also to explore improved treatment processes for bioprocessing of woody biomass. Our data suggest that the CMF mechanism is highly efficient in overcoming the lignin recalcitrance barrier to solubilize wood. Multiple pulses (up to 4 pulses) of CMF treatment were able to solubilize a majority of both the lignin and cellulose of wood at room temperature, using a hydrogen peroxide concentration of only 1%. Using a single pulse of the CMF system as a pretreatment allowed more wood residue to be retained, and enzymatic action on this pretreated wood was enhanced compared to control wood. In separate experiments, significantly greater solubilization of both sugars and lignin occurred when a single-pulse CMF pretreatment was used prior to enzymatic action than by enzymatic action alone on unmodified wood. This work suggests a key reason why the brown rot fungi have abandoned many of their extracellular enzymes to produce only a select suite of cellulases, which the fungus employs after prior modification of the cell wall using a CMF mechanism. This research further suggests that the CMF mechanism may have potential to be adapted for bioprocessing of woody biomass to produce sustainable fuels and bioproducts in the future.
S Tabor, L Orjuela, D Contreras, G Alfredsen, J Jellison, S Renneckar, B Goodell