Shuji Itakura

Hydroxyl radical generation by an extracellular low-molecular-weight substance and phenol oxidase activity during wood degradation by the white-rot basidiomycete Trametes versicolor

One-electron oxidation activity, as measured by ethylene generation from 2-keto-4-thiomethylbutyric acid, phenol oxidase activity, and the generation of hydroxyl radical were examined in cultures of the lignin-degrading white-rot basidiomycete fungus, Trametes (Coriolus) versicolor. The activity levels of specific lignin-degrading enzymes and cellulases, as well as the rate of wood degradation, also were examined. The fungus secreted a low-molecular-weight substance (M(r) 1000-5000) that catalyzed a redox reaction between molecular oxygen and an electron donor, to produce the hydroxyl radical via hydrogen peroxide. During wood decay, T. versicolor also produced significant amounts of laccase and lignin peroxidase, carboxymethyl cellulase, and Avicelase. The roles of the hydroxyl radical, phenol oxidases, and cellulases in wood degradation by white-rot fungi are discussed. That the hydroxyl radical produced by the low-molecular-weight substance secreted by T. versicolor results in new phenolic substructures on the lignin polymer, making it susceptible to attack by laccase or manganese peroxidase is suggested.

The involvement of extracelluar substances for reducing molecular oxygen to hydroxyl radical and ferric iron to ferrous iron in wood degradation by wood decay fungi

Abstract Extracelluar substances that had single-electron oxidation activity were isolated from cultures of brown-rot and white-rot. The molecular weights of the substances estimated by gel-filtration were very small, in a range of 1000–5000. The substances catalyzed a redox reaction between electron donors and O2 to produce H2O2 via O2− and reduced H2O2 to HO·. Furthermore, the substances reduced Fe(III) to Fe(II) and strongly adsorbed Fe(II). The production of HO· in cultures of brown-rot fungi was directly proportional to the rates in the degradation of wood, crystalline cellulose and lignin-related model compounds in the cultures. Most of the HO· was produced by a redox reaction between O2 and certain electron donors catalyzed by the low molecular weight substance in Tyromyces palustris cultures. The extent of one-electron oxidation activity of the low molecular weight substance from white-rot basidiomycetes was also correlated with that of the wood-degrading activity, but the phenol oxidase activity was not.

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.

An extracellular substance from the white-rot basidiomycete Phanerochaete chrysosporium for reducing molecular oxygen and ferric iron

An extracellular substance that had single-electron oxidation activity was isolated from wood-containing cultures of the white-rot basidiomycete Phanerochaete chrysosporium. The substance was partially purified by acetone precipitation, gel filtration chromatography on Sephadex G-50 and G-25, and DEAE Affi-Gel Blue gel chromatography. It contained about 18% protein, 20% neutral carbohydrate and 0.28% Fe(II) by weight. This substance was low molecular weight and could catalyze redox reactions between an electron donor, such as NADH, and O 2 to produce H 2 O 2 and to reduce H 2 O 2 to OH. It reduced Fe(III) to Fe(II) and strongly absorbed Fe(II). This extracellular substance seems to be involved in the initial degradation of cellulose and lignin in wood by the fungus.

Phenol oxidase activity and one-electron oxidation activity in wood degradation by soft-rot deuteromycetes.

Summary Wood degradation, one-electron oxidation activity as assayed by ethylene production from 2-keto-4-thiomethylbutyric acid, and phenol oxidase activity were measured in cultures of six deuteromycete 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 weight 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 weight 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.

Occurrence and metabolic role of the pyruvate dehydrogenase complex in the lower termite Coptotermes formosanus (Shiraki)

Abstract The activities of the pyruvate dehydrogenase complex in extracts of the gutted body, head, foregut/midgut and hindgut (hindgut epithelium and microorganisms) tissues of the lower termite Coptotermes formosanus (Shiraki) were determined by measuring the [ 14 C]-acetyl-CoA produced from [2- 14 C]-pyruvate and the 14 CO 2 produced from [1- 14 C]-pyruvate. The activities of pyruvate dehydrogenase, l -lactate dehydrogenase, acetyl-CoA synthetase, malate dehydrogenase (decarboxylating), and acetate kinase in the termite tissues and the hindgut also were determined. The sum (7.1 nmol/termite/h) of the pyruvate dehydrogenase complex activities in the termite tissues other than the hindgut was five times higher than the activity in the hindgut. Significant amounts of l -lactate dehydrogenase activity were found in all of the tissues. All of the tissues other than the hindgut had significant amounts of acetyl-CoA synthetase activity. Malate dehydrogenase (decarboxylating) activity was about ten times higher in the hindgut extract than in the gutted body and head extracts and the activity in the foregut/midgut extract was very low. These results indicate that acetyl-CoA for the TCA cycle is produced effectively in the tissues of the termite itself, both from pyruvate by the pyruvate dehydrogenase complex and from acetate by acetyl-CoA synthetase.

Effect of Norharmane in vitro on Juvenile Hormone Epoxide Hydrolase Activity in the Lower Termite, Reticulitermes speratus

Abstract The aromatic &bgr;-carboline norharmane was determined in workers, nymphs, and ergatoids of Reticulitermes speratus (Kolbe) (Isoptera: Rhinotermitidae) by gas chromatography/mass spectrometry. Norharmane levels in workers, nymphs, and ergatoids collected in May (∼4.3 ng/termite) were higher than those in November (∼0.2 ng/termite). Fluorescence of norharmane was observed in histological sections in whole animals and in the fat body. Norharmane, at a final concentration of 1 mM, stimulated juvenile hormone epoxide hydrolase (JHEH) activity of enzyme extract from ergatoids, but inhibited JHEH activity at higher concentrations. The elevated JHEH activity stimulated by norharmane should accelerate juvenile hormone metabolism in R. speratus.

Pyruvate and acetate metabolism in termite mitochondria

Intact mitochondria have been successfully prepared from body tissues from the termites Nasutitermes walkeri and Coptotermes formosanus. This is the first report of the successful isolation of mitochondria from termites (Isoptera: Termitidae). Using an oxygen electrode, oxygen consumption by the mitochondria during the oxidation of various respiratory substrates was determined and their properties measured in terms of respiratory control index and ADP/O. ADP/O was as expected for substrates such as pyruvate, acetylcarnitine and acetyl-CoA and carnitine. Pyruvate and acetate were the major respiratory substrates in both species. The total activity of the pyruvate dehydrogenase complex (PDHc) in the mitochondria from N. walkeri and C. formosanus was determined to be 72.87+/-8.98 and 8.29+/-0.42 nmol/termite/h, respectively. Mitochondria isolated in the presence of inhibitors of PDHc interconversion were used to determine that about 60% of the PDHc was maintained in the active form in both N. walkeri and C. formosanus. The sufficient PDHc activity and high rate of pyruvate oxidation in mitochondria from N. walkeri suggest that pyruvate is rapidly metabolised, whereas the low mitochondrial PDHc activity of C. formosanus suggests that in this species more pyruvate is produced than can be oxidised in the termite tissues.

Identification of Two Subterranean Termite Species (Isoptera: Rhinotermitidae) using Cellulase Genes

Abstract To identify subterranean termite species, we designed a pair of common primers that amplified 381-bp fragments from cDNAs encoding the endo-β-1,4-glucanases (EGases) of Coptotermes formosanus Shiraki and Reticulitermes speratus (Kolbe). cDNAs from C. formosanus and R. speratus, and genomic DNA from R. speratus, were amplified by polymerase chain reaction (PCR) by using this primer pair and then cloned and sequenced. Sequences amplified from C. formosanus cDNA displayed 97–99% identity to cDNA encoding the EGase of C. formosanus (CfEG), and 92–94% identity to cDNA encoding the EGase of R. speratus (RsEG). By contrast, cDNA from R. speratus displayed 99–100% identity to RsEG cDNA and 93–94% identity to CfEG cDNA. CfEG and RsEG cDNAs can therefore be used as markers for the identification of these termite species. This is the first report of the successful identification of termite species by using cDNA and genomic DNA sequences of termite origin.

Presence of Extracellular NAD+ and NADH in Cultures of Wood-Degrading Fungi

Our previous studies indicated that extracellular glycoproteins produced by some white-rot and brown-rot basidiomycetous fungi reduce Fe(III) to Fe(II) and O2 to H2O2 and produce hydroxyl radicals. The continuous generation of hydroxyl radicals requires a constant supply of O2 and an electron donor for the reduction of oxidized forms of the glycoproteins to the reduced forms. However, electron donors for this reaction, such as NADH, have not been identified. In this study, the amounts of the extracellular pyridine coenzymes, NAD(＋) and NADH, were measured in agar cultures of four white-rot fungi, one brown-rot fungus, and three soft-rot fungi. The sums of NAD(＋) and NADH detected in wood-containing cultures of all five basidiomycetes were greater than those in glucose cultures. The amounts of NAD(＋) were higher than those of NADH in all wood-containing cultures except that of Irpex lacteus, and NAD(＋) was greater than NADH in all glucose cultures except that of Fomitopsis palustris. Significant amounts of pyridine coenzymes were present in glucose and wood-containing cultures of the three soft-rot fungi. The non-wood-degrading fungus, Penicillium funiculosum, did not produce NAD(＋) or NADH in either glucose or wood-containing cultures. The extracellular pyridine coenzyme levels were relatively high compared to the rates of extracellular hydroxyl radical generation in wood-degrading fungal cultures. Thus, white-, brown-, and soft-rot fungi produce pyridine coenzymes that could serve as electron donors for the production of hydroxyl radicals during wood degradation.