Lignin-degrading White-rot Fungus Research Articles

Biotechnological Processing of Sugarcane Bagasse through Solid-State Fermentation with White Rot Fungi into Nutritionally Rich and Digestible Ruminant Feed

Sugarcane (Saccharum officinarum) bagasse (SCB) is one of the most widely produced lignocellulosic biomasses and has great potential to be recycled for sustainable food production as ruminant animal feed. However, due to severe lignification, i.e., lignin-(hemi)-cellulose complexes, ruminants can only ferment a minor fraction of the polysaccharides trapped in such recalcitrant lignocellulosic biomasses. This study was therefore designed to systematically evaluate the improvement in nutritional value, the in vitro dry matter digestibility (IVDMD), and the rate and extent of in vitro total gas (IVGP) and methane (CH4) production during the 72 h in vitro ruminal fermentation of SCB, bioprocessed with Agaricus bisporus, Pleurotus djamor, Calocybe indica and Pleurotus ostreatus under solid-state fermentation (SSF) for 0, 21 and 56 days. The contents of neutral detergent fiber, lignin, hemicellulose and CH4 production (% of IVGP) decreased (p < 0.05), whereas crude protein (CP), IVDMD and total IVGP increased (p < 0.05) after the treatment of SCB for 21 and 56 days with all white-rot fungi (WRF) species. The greatest (p < 0.05) improvement in CP (104.1%), IVDMD (38.8%) and IVGP (49.24%) and the greatest (p < 0.05) reduction in lignin (49.3%) and CH4 (23.2%) fractions in total IVGP were recorded for SCB treated with C. indica for 56 days. Notably, C. indica degraded more than (p < 0.05) lignin and caused greater (p < 0.05) improvement in IVDMD than those recorded for other WRF species after 56 days. The increase in IVGP was strongly associated with lignin degradation (R2 = 0.72) and a decrease in the lignin-to-cellulose ratio (R2 = 0.95) during the bioprocessing of SCB. Our results demonstrated that treatment of SCB with (selective) lignin-degrading WRF can improve the nutritional value and digestibility of SCB, and C. indica presents excellent prospects for the rapid, selective and more extensive degradation of lignin and, as such, for the improvement in nutritional value and digestibility of SCB for ruminant nutrition.

Fermenting and Lignin Degradability of a White-Rot Fungus Coriolopsis trogii Using Industrial Lignin as Substrate.

Bio-depolymerized the lignin macromolecules into low molecular lignin-derived aromatic compounds satisfies the requirement for carbon dioxide peaking and is also one of the important ways to realize lignin valorization. Coriolopsis trogii is a kind of less reported lignin-degrading white-rot fungus. The degradability of a self-isolated C. trogii TS01 on industrial lignins, including enzymatic hydrolysis lignin (EHL) and Kraft lignin (KL), was investigated in this paper. The results indicated that EHL could be used as an efficient carbon source to promote the cell growth and ligninolytic enzyme secretion of C. trogii TS01. Compared with using 2% glucose as carbon source, 1% EHL plus 1% glucose would increase the maximum cell dry weight, laccase activity, and manganese-dependent peroxidase activity of C. trogii TS01 by 24.8%, 164.1%, and 200%, respectively. However, the cell growth and ligninolytic enzyme secretion would be significantly inhibited in the case of 1% KL plus 1% glucose used as carbon source. As a result, at the 12th day of fermentation, the degradation rates of EHL and KL were 50.6% and 5.7%, respectively. The UV and FTIR analysis indicated that after been fermented by C. trogii TS01, S-unit content in EHL was decreased by 12.5% but G-unit content was increased by 53.7%. In conclusion, the research of this paper will provide a promising solution for the valorization of enzymatic hydrolysis lignin since the high biodegradation rate of lignin and high activity of ligninolytic enzymes could be achieved simultaneously.

Applicability of Recombinant Laccases From the White-Rot Fungus Obba rivulosa for Mediator-Promoted Oxidation of Biorefinery Lignin at Low pH.

Utilization of lignin-rich side streams has been a focus of intensive studies recently. Combining biocatalytic methods with chemical treatments is a promising approach for sustainable modification of lignocellulosic waste streams. Laccases are catalysts in lignin biodegradation with proven applicability in industrial scale. Laccases directly oxidize lignin phenolic components, and their functional range can be expanded using low-molecular-weight compounds as mediators to include non-phenolic lignin structures. In this work, we studied in detail recombinant laccases from the selectively lignin-degrading white-rot fungus Obba rivulosa for their properties and evaluated their potential as industrial biocatalysts for the modification of wood lignin and lignin-like compounds. We screened and optimized various laccase mediator systems (LMSs) using lignin model compounds and applied the optimized reaction conditions to biorefinery-sourced technical lignin. In the presence of both N–OH-type and phenolic mediators, the O. rivulosa laccases were shown to selectively oxidize lignin in acidic reaction conditions, where a cosolvent is needed to enhance lignin solubility. In comparison to catalytic iron(III)–(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation systems, the syringyl-type lignin units were preferred in mediated biocatalytic oxidation systems.

A novel and efficient fungal delignification strategy based on versatile peroxidase for lignocellulose bioconversion

BackgroundThe selective lignin-degrading white-rot fungi are regarded to be the best lignin degraders and have been widely used for reducing the saccharification recalcitrance of lignocellulose. However, the biological delignification and conversion of lignocellulose in biorefinery is still limited. It is necessary to develop novel and more efficient bio-delignification systems.ResultsPhysisporinus vitreus relies on a new versatile peroxidase (VP)-based delignification strategy to remove enzymatic recalcitrance of corn stover efficiently, so that saccharification of corn stover was significantly enhanced to 349.1 mg/g biomass (yield of glucose) and 91.5% (hydrolysis yield of cellulose) at 28 days, as high as levels reached by thermochemical treatment. Analysis of the lignin structure using pyrolysis–gas chromatography–mass spectrometry (Py–GC/MS) showed that the total abundance of lignin-derived compounds decreased by 54.0% and revealed a notable demethylation during lignin degradation by P. vitreus. Monomeric and dimeric lignin model compounds were used to confirm the ligninolytic capabilities of extracellular ligninases secreted by P. vitreus. The laccase (Lac) from P. vitreus could not oxidize nonphenolic lignin compounds and polymerized β-O-4 and 5-5′ dimers to precipitate which had a negative effect on the enzymatic hydrolysis of corn stover in vitro. However, the VP from P. vitreus could oxidize both phenolic and nonphenolic lignin model compounds as well as break the β-O-4 and 5-5′ dimers into monomeric compounds, which were measured by high-performance liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS). Moreover, we showed that addition of purified VP in vitro improved the enzymatic hydrolysis of corn stover by 14.1%.ConclusionsFrom the highly efficient system of enzymatic recalcitrance removal by new white-rot fungus, we identified a new delignification strategy based on VP which could oxidize both phenolic and nonphenolic lignin units and break different linkages in lignin. In addition, this is the first evidence that VP could break 5-5′ linkage efficiently in vitro. Moreover, VP improved the enzymatic hydrolysis of corn stover in vitro. The remarkable lignin-degradative potential makes VP attractive for biotechnological applications.

Delignification of Lignocellulose Biomasses by Alginate–Chitosan Immobilized Laccase Produced from Trametes versicolor IBL-04

An extracellular laccase was produced from a lignin-degrading white-rot fungus Trametes versicolor IBL-04 using the solid-state bioprocessing of corncobs. The enzyme thus produced was effectively immobilized on alginate–chitosan beads. Immobilized laccase furnished the highest immobilization yield (81.43 ± 2.98) under optimized working conditions of 3.0% (w/v) sodium alginate concentration, 2.4% CaCl2, 0.25% chitosan concentration following an immobilization time of 5 h. The immobilization process altered the optimum pH from 5.0 to 4.0 (an acidic shift), whereas the temperature optima were displaced from 40 to 50 °C (towards higher temperature). The Michaelis–Menten constant (K m) and the maximum reaction rate (V max) of the free and immobilized laccase were 0.14 mM, and 780 µmol/mL and 0.10 mM and 814 µmol/mL, respectively. The alginate–chitosan immobilized laccase was more stable and tolerant against the activity inhibition caused by the denaturants tested, concerning its soluble laccase. Delignification results revealed that the enzyme led to a marked reduction in lignin content of all the lignocellulosic wastes with the highest delignification of 78.29% in sugarcane bagasse, followed by wheat bran and corn stover after 15 h. It is demonstrated that the immobilized laccase could be a promising option as a green catalyst for the efficient delignification of lignocellulosic biomass residues.

Elimination and detoxification of fungicide miconazole and antidepressant sertraline by manganese peroxidase-dependent lipid peroxidation system

The imidazole fungicide miconazole (MCZ) and the antidepressant sertraline (SER) have the potential to adversely affect aquatic organisms when they are released into the environment. To assess if they could be broken down, MCZ and SER were treated with laccase and manganese peroxidase (MnP) from lignin-degrading white-rot fungi. The MnP-dependent lipid peroxidation system with Tween 80 containing unsaturated fatty acid was effective in eliminating MCZ and SER; the MnP-Tween 80 system helped eliminate 88% of MCZ and 85% of SER after 24 h of treatment. Furthermore, this system eliminated a metabolite of SER (desmethylsertraline or DSER), and no differences between the elimination rates of DSER and SER were observed throughout the treatment period. It was also confirmed that the MnP-Tween 80 system caused a complete loss of growth inhibition by MCZ of the gram-positive bacterium Bacillus subtilis and reduced the growth inhibition by SER of the green alga Pseudokirchneriella subcapitata by 78% after 24 h of treatment. On the other hand, no appreciable elimination of MCZ and SER was obtained by treatment with laccase, MnP, or the laccase-mediator system with 1-hydroxybenzotriazole.

Biochemical and molecular characterization of a novel laccase from selective lignin-degrading white-rot fungus Echinodontium taxodii 2538

A novel laccase was isolated and characterized from a new selective lignin-degrading white-rot fungus Echinodontium taxodii 2538, in which a high yield of laccase was obtained. No laccase isoenzyme was detected in the synthetic liquid media. The purified laccase (designated as EtL2538) had an apparent molecular mass of 56kDa, pI value of 3.1, and N-terminal amino acid sequence of GIGPVTDLHIVNAAV. EtL2538 showed optimum pH at 3.0 and optimum temperature at 60°C using ABTS as the substrate. EtL2538 revealed superior thermostability, and retained over 80% of its original activity after incubation for 2h at 50°C. The laccase gene, etl2538, was also cloned and sequenced. This gene encoded a mature laccase protein containing 499 amino acids (aa) preceded by a signal peptide of 21 aa, and the deduced protein sequence contained four copper-binding conserved domains of typical laccase protein. EtL2538 was further used in lignin oxidation and dye decolorization. Even without the existence of redox mediators, EtL2538 could cleave the methoxyl groups and β-O-4 ether linkages in lignin from bamboo, and significantly decolorize malachite green and RBBR. These novel properties of EtL2538 may render it as a potential biocatalyst for biotechnological and environmental applications.

Oxalate-metabolising genes of the white-rot fungus Dichomitus squalens are differentially induced on wood and at high proton concentration.

Oxalic acid is a prevalent fungal metabolite with versatile roles in growth and nutrition, including degradation of plant biomass. However, the toxicity of oxalic acid makes regulation of its intra- and extracellular concentration crucial. To increase the knowledge of fungal oxalate metabolism, a transcriptional level study on oxalate-catabolising genes was performed with an effective lignin-degrading white-rot fungus Dichomitus squalens, which has demonstrated particular abilities in production and degradation of oxalic acid. The expression of oxalic-acid decomposing oxalate decarboxylase (ODC) and formic-acid decomposing formate dehydrogenase (FDH) encoding genes was followed during the growth of D. squalens on its natural spruce wood substrate. The effect of high proton concentration on the regulation of the oxalate-catabolising genes was determined after addition of organic acid (oxalic acid) and inorganic acid (hydrochloric acid) to the liquid cultures of D. squalens. In order to evaluate the co-expression of oxalate-catabolising and manganese peroxidase (MnP) encoding genes, the expression of one MnP encoding gene, mnp1, of D. squalens was also surveyed in the solid state and liquid cultures. Sequential action of ODC and FDH encoding genes was detected in the studied cultivations. The odc1, fdh2 and fdh3 genes of D. squalens showed constitutive expression, whereas ODC2 and FHD1 most likely are the main responsible enzymes for detoxification of high concentrations of oxalic and formic acids. The results also confirmed the central role of ODC1 when D. squalens grows on coniferous wood. Phylogenetic analysis revealed that fungal ODCs have evolved from at least two gene copies whereas FDHs have a single ancestral gene. As a conclusion, the multiplicity of oxalate-catabolising genes and their differential regulation on wood and in acid-amended cultures of D. squalens point to divergent physiological roles for the corresponding enzymes.

Characterization of PIR1, a GATA family transcription factor involved in iron responses in the white-rot fungus Phanerochaete chrysosporium

Iron, although toxic in excess, is an essential element for biological systems. Therefore, its homeostasis is of critical importance and tight mechanisms participate in its acquisition by microbial organisms. Lately, the relevance of this metal for biomass conversion by wood-degrading fungi has been gaining increasing attention. Iron plays a critical role as cofactor of key enzymes such as lignin and manganese peroxidases in lignin-degrading white-rot fungi, while Fe(II) also serves a pivotal role in Fenton reactions that are central in cellulose depolymerization by brown-rotters. It has been hypothesized that multicopper oxidases with ferroxidase activity might participate in controlling the bioavailability of iron in the hyphal proximity, fine-tuning Fenton chemistry and balancing lignin versus cellulose degradation. In order to further explore the dynamics of iron regulation in the well known white-rot fungus Phanerochaete chrysosporium, we analyzed the mRNA levels of the multicopper oxidases genes (mcos) in response to iron supplementation, confirming down-regulation of their expression in response to this metal. To gain a better understanding on the transcriptional mechanisms mediating this effect, we searched for a gene encoding a GATA-type transcription factor with homology to URBS1, the major transcriptional regulator of iron homeostasis in Ustilago maydis. Due to the limitation of experimental tools in P. chrysosporium, the alleged Phanerochaete iron regulator (PIR1) was studied by complementation of a Neurospora SRE/URBS1-deficient strain, where phenotypic and molecular characteristics of this transcriptional regulator could be easily assessed. In addition, using a genome-wide in silico strategy, we searched for putative cis-acting iron-responsive elements in P. chrysosporium. Some of the identified genes showed reduced transcript levels after 30min in the presence of the metal, consistent with an SRE/URBS1-mediated mechanism, and suggesting a broad effect of iron on the regulation of several cellular processes.

Laccase: Microbial Sources, Production, Purification, and Potential Biotechnological Applications

Laccase belongs to the blue multicopper oxidases and participates in cross-linking of monomers, degradation of polymers, and ring cleavage of aromatic compounds. It is widely distributed in higher plants and fungi. It is present in Ascomycetes, Deuteromycetes and Basidiomycetes and abundant in lignin-degrading white-rot fungi. It is also used in the synthesis of organic substance, where typical substrates are amines and phenols, the reaction products are dimers and oligomers derived from the coupling of reactive radical intermediates. In the recent years, these enzymes have gained application in the field of textile, pulp and paper, and food industry. Recently, it is also used in the design of biosensors, biofuel cells, as a medical diagnostics tool and bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases have received attention of researchers in the last few decades due to their ability to oxidize both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. It has been identified as the principal enzyme associated with cuticular hardening in insects. Two main forms have been found: laccase-1 and laccase-2. This paper reviews the occurrence, mode of action, general properties, production, applications, and immobilization of laccases within different industrial fields.

Ultrahigh (0.93 Å) resolution structure of manganese peroxidase from Phanerochaete chrysosporium: Implications for the catalytic mechanism

Manganese peroxidase (MnP) is an extracellular heme enzyme produced by the lignin-degrading white-rot fungus Phanerochaete chrysosporium. MnP catalyzes the peroxide-dependent oxidation of Mn II to Mn III. The Mn III is released from the enzyme in complex with oxalate, enabling the oxalate–Mn III complex to serve as a diffusible redox mediator capable of oxidizing lignin, especially under the mediation of unsaturated fatty acids. One heme propionate and the side chains of Glu35, Glu39 and Asp179 have been identified as Mn II ligands in our previous crystal structures of native MnP. In our current work, new 0.93 Å and 1.05 Å crystal structures of MnP with and without bound Mn II, respectively, have been solved. This represents only the sixth structure of a protein of this size at 0.93 Å resolution. In addition, this is the first structure of a heme peroxidase from a eukaryotic organism at sub-Ångstrom resolution. These new structures reveal an ordering/disordering of the C-terminal loop, which is likely required for Mn binding and release. In addition, the catalytic Arg42 residue at the active site, normally thought to function only in the peroxide activation process, also undergoes ordering/disordering that is coupled to a transient H-bond with the Mn ligand, Glu39. Finally, these high-resolution structures also reveal the exact H atoms in several parts of the structure that are relevant to the catalytic mechanism.

Micromorphological characteristics and lignin distribution in bamboo (Phyllostachys pubescens) degraded by the white rot fungus Lentinus edodes

Abstract Little is known about the decay pattern of bamboo by wood decay fungi, and the information available on fungal degradation of wood from other plant taxa cannot form the basis for understanding wood decay in bamboo because of differences in lignin composition and distribution. The present work was undertaken to elucidate the degradation pattern of bamboo by a lignin-degrading white rot fungus Lentinus edodes using various microscopic techniques, with a particular focus on the relation of bamboo lignin composition to the decay patterns produced by this fungus. Small blocks of bamboo, Phyllostachys pubescens, were examined after 16 weeks of incubation with the white rot fungus L. edodes. The compound middle lamellae (CML), including the cell corner regions, were preferentially degraded at an early stage of decay, whereas the fiber secondary walls remained largely intact at this point. Bamboo fiber walls were also eroded from the cell lumen side but the extent of degradation was limited. The FT-IR bands assigned to lignin exhibited a significant decrease. Cellular ultraviolet microspectrophotometric investigation showed that CML and vessel walls were composed of not only guaiacyl and syringyl unit (GS lignin) but also grass lignin. In contrast, the secondary wall of fibers consisted mainly of the GS lignin. Even though the CML and vessel walls exhibited higher lignin concentration, these cell walls were nevertheless degraded. The preferential degradation of the CML over the fiber secondary walls strongly suggested the involvement of not only enzyme systems of the white rot fungus but also a relationship to physicochemical properties of bamboo cell walls, particularly the influence of lignin composition and distribution.

Structural and chemical properties of grass lignocelluloses related to conversion for biofuels

Grass lignocelluloses, such as those in corn and switchgrass, are a major resource in the emerging cellulose-to-ethanol strategy for biofuels. The potential bioconversion of carbohydrates in this potential resource, however, is limited by the associated aromatic constituents within the grass fiber. These aromatics include both lignins, which are phenylpropanoid units of various types, and low-molecular weight phenolic acids. Structural and chemical studies over the years have identified the location and limitation to fiber degradation imposed by a variety of these aromatic barriers. For example, coniferyl lignin appears to be the most effective limitation to biodegradation, existing in xylem cells of vascular tissues. On the other hand, cell walls with syringyl lignin, e.g., leaf sclerenchyma, are often less recalcitrant. Ferulic and p-coumaric acids that are esterified to hemicellulosic sugars constitute a major limitation to biodegradation in non-lignified cell walls in grass fibers, especially warm season species. Non-chemical methods to improve bioconversion of the lignocelluloses through modification of aromatics include: (1) use of lignin-degrading white rot fungi, (2) pretreatment with phenolic acid esterases, and (3) plant breeding to modify cell wall aromatics. In addition to increased availability of carbohydrates for fermentation, separation and collection of aromatics could provide value-added co-products to improve the economics of bioconversion.

Grass lignocellulose

Grass lignocelluloses are limited in bioconversion by aromatic constituents, which include both lignins and phenolic acids esters. Histochemistry, ultraviolet absorption microspectrophotometry, and response to microorganisms and specific enzymes have been used to determine the significance of aromatics toward recalcitrance. Coniferyl lignin appears to be the most effective limitation to biodegradation, existing in xylem cells of vascular tissues; cell walls with syringyl lignin, for example, leaf sclerenchyma, are less recalcitrant. Esterified phenolic acids, i.e., ferulic and p-coumaric acids, often constitute a major chemical limitation in nonlignified cell walls to biodegradation in grasses, especially warm-season species. Methods to improve biodegradation in grasses, especially warm-season species. Methods to improve biodegradability through modification of aromatics include: plant breeding, use of lignin-degrading white-rot fungi, and addition of esterases. Plant breeding for new cultivars has been especially effective for nutritionally improved forages, for example, bermudagrasses. In laboratory studies, selective white-rot fungi that lack cellulases delignified the lignocellulosic materials and improved fermentation of residual carbohydrates. Phenolic acid esterases released p-coumaric and ferulic acids for potential coproducts, improved the available sugars for fermentation, and improved biodegradation. The separation and removal of the aromatic components for coproducts, while enhancing the availability of sugars for bioconversion, could improve the economics of bioconversion.

Agrobacterium‐mediated delivery of marker genes to Phanerochaete chrysosporium mycelial pellets: a model transformation system for white‐rot fungi

The lack of an efficient transformation system in filamentous fungi is one of the major problems in carrying out cloning and expression of genes to produce a value-added product at the commercial level. Therefore an attempt has been made to develop an efficient, convenient and expeditious genetic transformation system for successful transfer of transferred DNA, carrying the genes coding for GUS (beta-glucuronidase) (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of Phanerochaete chrysosporium, a widely studied lignin-degrading white-rot fungus. The transformation of the marker gene was confirmed through GUS, PCR and Southern-blot hybridization. Transformation with acetosyringone, in the presence of light with partial bleaching for 1 h, were found to be a better combination for fungal transformation with 48% efficiency. The agro-transformation method to transform the modified mycelial pellets offers a practical means for exploiting transgenic approaches in genetic manipulation and improvement of P. chrysosporium, a model white-rot fungus. Moreover, it has the potential to overcome the technical hurdles in genetic manipulation to bio-technologically exploit many other filamentous fungi of industrial importance.

Wood stimulates the demethoxylation of [O14CH3]-labeled lignin model compounds by the white-rot fungi Phanerochaete chrysosporium and Phlebia radiata

Mineralization of polymeric wood lignin and its substructures is a result of complex reactions involving oxidizing and reducing enzymes and radicals. The degradation of methoxyl groups is an essential part of this process. The presence of wood greatly stimulates the demethoxylation of a non-phenolic lignin model compound (a [O(14)CH(3)]-labeled beta-O-4 dimer) by the lignin-degrading white-rot fungi Phlebia radiata and Phanerochaete chrysosporium. When grown on wood, both fungi produced up to 47 and 40% (14)CO(2) of the applied (14)C activity, respectively, under air and oxygen in 8 weeks. Without wood, the demethoxylation of the dimer by both fungi was lower, varying between 0.5 and 35%. Addition of nutrient nitrogen together with glucose decreased demethoxylation when the fungi were grown on spruce wood under air. Because the evolution of (14)CO(2) in the absence of wood was poor, the fungi may have preferably used wood as a carbon and nitrogen source. The amount of fungal mycelium, as determined by the ergosterol assay, did not show connection to demethoxylation. P. radiata also showed a high demethoxylation of [O(14)CH(3)]-labeled vanillic acid in the presence of birch wood. The degradation of lignin and lignin-related substances should be studied in the presence of wood, the natural substrate for white-rot fungi.

Degradation of Poly(acrylic acid)s with Phenolic Side-Chains by Manganese Peroxidase from White Rot Fungi

Poly(acrylic acid)s (PAAs) with various functional groups, such as phenolic hydroxyl, amino, and aldehyde groups, in the side-chains were treated with manganese peroxidase (MnP) prepared from the culture of lignin-degrading white rot fungi. While no change in the Mw of PAA without a functional group was observed after a 24-h MnP treatment, the Mw␣of␣PAA␣with p-aminophenol as side-chains decreased from 90,000 to 59,000, and that with␣o-aminophenol from 70,000 to 26,000. MnP treatment also decreased the Mw of PAA with a p-aminoaniline or aldehyde group. Furthermore, the MnP treatment generated a significant depolymerization of the cross-linked PAA with p-aminophenol from an insoluble polymer to water soluble products. These results suggest that functional groups generating radicals can act as elemental devices and induce degradation of the PAA main chain.

Production of organic acids and oxalate decarboxylase in lignin-degrading white rot fungi

Intracellular oxalate decarboxylase (ODC, EC 4.1.1.2) activity was screened in the mycelium of 12 white rot fungi. ODC activity was detected in the mycelial extracts of Dichomitus squalens, Phanerochaete sanguinea, Trametes ochracea, and Trametes versicolor (strain R/7) after addition of 5-mM oxalic acid to the liquid culture medium. In D. squalens, intracellular ODC activity increased six-fold with addition of oxalic acid. Production of extracellular organic acids by the four ODC-positive fungi was followed in liquid cultures and in solid state cultures of spruce wood chips by using HPLC and capillary zone electrophoresis (CZE). The four ODC-positive fungi secreted oxalic acid both in liquid and solid state cultures showing different production patterns until the end of growth (31 days). Upon cultivation on solid spruce wood chips, manganese peroxidase (MnP) activity peaked simultaneously in these fungi with the accumulation of extracellular oxalic acid. In addition to oxalic acid, glyoxylic and formic acids were detected in the cultures of D. squalens.

Proteasomal degradation pathways in Trametes versicolor and Phlebia radiata

Possible involvement of the non-lysosomal (proteasome-mediated) pathway in the regulation of ligninolytic activities was studied. Proteasome activity was detected in mycelial extracts of the efficient lignin-degrading white rot fungi, Trametes versicolor and Phlebia radiata, by monitoring cleavage of a fluorogenic (7-amido-4-methyl-coumarin-linked) peptide substrate (SucLLVY-MCA). One of the more potent peptide aldehyde inhibitors of the proteasome, MG132 (CbzLLLal) was used to define a role for the proteasome-mediated protein degradation in their metabolism. Proteasome activity was assayed in cultures of T. versicolor and P. radiata following a nutritional shift from primary growth (i.e. trophophase) to idiophase triggered by nitrogen or carbon starvation. In carbon-starved cultures, addition of MG132 decreased intracellular and extracellular laccase and peroxidase activities whereas in nitrogen-starved cultures, addition of this proteasome inhibitor increased intracellular activities of these enzymes by 1.5 to 2 fold and extracellular activities by 4 to 7 fold in T. versicolor. Similar but less dramatic changes were observed with P. radiata. The results indicate that proteasomes play a role in the metabolism of these organisms.

Fungal pyranose oxidases: occurrence, properties and biotechnical applications in carbohydrate chemistry.

Pyranose oxidases are widespread among lignin-degrading white rot fungi and are localized in the hyphal periplasmic space. They are relatively large flavoproteins which oxidize a number of common monosaccharides on carbon-2 in the presence of oxygen to yield the corresponding 2-keto sugars and hydrogen peroxide. The preferred substrate of pyranose oxidases is D-glucose which is converted to 2-keto-D-glucose. While hydrogen peroxide is a cosubstrate in ligninolytic reactions, 2-keto-D-glucose is the key intermediate of a secondary metabolic pathway leading to the antibiotic cortalcerone. The finding that 2-keto-D-glucose can serve as an intermediate in an industrial process for the conversion of D-glucose into D-fructose has stimulated research on the use of pyranose oxidases in biotechnical applications. Unique catalytic potentials of pyranose oxidases have been discovered which make these enzymes efficient tools in carbohydrate chemistry. Converting common sugars and sugar derivatives with pyranose oxidases provides a pool of sugar-derived intermediates for the synthesis of a variety of rare sugars, fine chemicals and drugs.