Synthetic Lignin Mineralization by Ceriporiopsis subvermispora Is Inhibited by an Increase in the pH of the Cultures Resulting from Fungal Growth

(sup14)C-synthetic lignin mineralization by the basidiomycete Ceriporiopsis subvermispora occurs at the highest rate (about 30% after 29 days) in liquid cultures containing 1% glucose and a growth-limiting amount (1 mM) of ammonium tartrate. The titers of manganese peroxidase (MnP) and laccase are lower in these cultures than in cultures containing 1% glucose and 10 mM ammonium tartrate, where the extent of lignin mineralization in the same period is only about 15%. The inverse correlation between enzyme activity and lignin mineralization is also observed when ammonium tartrate is replaced by ammonium chloride or Casamino Acids as the source of nitrogen. This phenomenon can be explained by a gradual increase in the pH of the medium that takes place only in the cultures with high nitrogen concentrations. Supporting this finding, when cultures with 1 mM ammonium tartrate were grown at different pHs, (sup14)CO(inf2) evolved more rapidly from those with pH values near the optimum for MnP activity. On the other hand, (sup14)CO(inf2) evolution from cultures containing 1% glucose supplemented with 1 mM ammonium tartrate plus 9 mM sodium tartrate was as low as that from cultures with a high ammonium tartrate concentration. Since the changes in the pH of these cultures were not as pronounced as those in cultures containing high nitrogen concentrations, tartrate itself may also be contributing to limit the extent of lignin mineralization. Considering that pH instability seems to constitute a common feature of fungal cultures, precautions must be taken to avoid underestimation of their ligninolytic efficiencies.     

Manganese-Dependent Cleavage of Nonphenolic Lignin Structures by Ceriporiopsis subvermispora in the Absence of Lignin Peroxidase

Vol. 62, no. 10, p. 3684, column 2, line 16: "Tri(methylsilyl)" should read "Tri(trimethylsilyl)." Line 17: "Di(methylsilyl)" should read "Di(trimethylsilyl)." Line 20: "Tri(methylsilyl)" should read "Trimethylsilyl." Page 3686, column 1, reference 12: The journal should be Dokl. Akad. Nauk Belarusi. [This corrects the article on p. 3679 in vol. 62.].     

Manganese-Dependent Cleavage of Nonphenolic Lignin Structures by Ceriporiopsis subvermispora in the Absence of Lignin Peroxidase

Many ligninolytic fungi appear to lack lignin peroxidase (LiP), the enzyme generally thought to cleave the major, recalcitrant, nonphenolic structures in lignin. At least one such fungus, Ceriporiopsis subvermispora, is nevertheless able to degrade these nonphenolic structures. Experiments showed that wood block cultures and defined liquid medium cultures of C. subvermispora rapidly depolymerized and mineralized a (sup14)C-labeled, polyethylene glycol-linked, high-molecular-weight (beta)-O-4 lignin model compound (model I) that represents the major nonphenolic structure of lignin. The fungus cleaved model I between C(inf(alpha)) and C(inf(beta)) to release benzylic fragments, which were shown in isotope trapping experiments to be major products of model I metabolism. The C(inf(alpha))-C(inf(beta)) cleavage of (beta)-O-4 lignin structures to release benzylic fragments is characteristic of LiP catalysis, but assays of C. subvermispora liquid cultures that were metabolizing model I confirmed that the fungus produced no detectable LiP activity. Three results pointed, instead, to the participation of a different enzyme, manganese peroxidase (MnP), in the degradation of nonphenolic lignin structures by C. subvermispora. (i) The degradation of model I and of exhaustively methylated (nonphenolic), (sup14)C-labeled, synthetic lignin by the fungus in liquid cultures was almost completely inhibited when the Mn concentration of the medium was decreased from 35 (mu)M to approximately 5 (mu)M. (ii) The fungus degraded model I and methylated lignin significantly faster in the presence of Tween 80, a source of unsaturated fatty acids, than it did in the presence of Tween 20, which contains only saturated fatty acids. Previous work has shown that nonphenolic lignin structures are degraded during the MnP-mediated peroxidation of unsaturated lipids. (iii) In experiments with MnP, Mn(II), and unsaturated lipid in vitro, this system mimicked intact C. subvermispora cultures in that it cleaved nonphenolic (beta)-O-4 lignin model compounds between C(inf(alpha)) and C(inf(beta)) to release a benzylic fragment.     

Evidence That Ceriporiopsis subvermispora Degrades Nonphenolic Lignin Structures by a One-Electron-Oxidation Mechanism

The white-rot fungus Ceriporiopsis subvermispora is able to degrade nonphenolic lignin structures but appears to lack lignin peroxidase (LiP), which is generally thought to be responsible for these reactions. It is well established that LiP-producing fungi such as Phanerochaete chrysosporium degrade nonphenolic lignin via one-electron oxidation of its aromatic moieties, but little is known about ligninolytic mechanisms in apparent nonproducers of LiP such as C. subvermispora. To address this question, C. subvermispora and P. chrysosporium were grown on cellulose blocks and given two high-molecular-weight, polyethylene glycol-linked model compounds that represent the major nonphenolic arylglycerol-(beta)-aryl ether structure of lignin. The model compounds were designed so that their cleavage via one-electron oxidation would leave diagnostic fragments attached to the polyethylene glycol. One model compound was labeled with (sup13)C at C(inf(alpha)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(alpha))-C(inf(beta)) cleavage after one-electron oxidation. The other model compound was labeled with (sup13)C at C(inf(beta)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(beta))-O-aryl cleavage after one-electron oxidation. To assess fungal degradation of the models, the high-molecular-weight metabolites derived from them were recovered from the cultures and analyzed by (sup13)C nuclear magnetic resonance spectrometry. The results showed that both C. subvermispora and P. chrysosporium degraded the models by routes indicative of one-electron oxidation. Therefore, the ligninolytic mechanisms of these two fungi are similar. C. subvermispora might use a cryptic LiP to catalyze these C(inf(alpha))-C(inf(beta)) and C(inf(beta))-O-aryl cleavage reactions, but the data are also consistent with the involvement of some other one-electron oxidant.     

Structural Characterization of Lignin during Pinus taeda Wood Treatment with Ceriporiopsis subvermispora

Pinus taeda wood chips were biotreated with Ceriporiopsis subvermispora under solid-state fermentation for periods varying from 15 to 90 days. Milled wood lignins extracted from sound and biotreated wood samples were characterized by wet-chemical and spectroscopic techniques. Treatment of the lignins by derivatization followed by reductive cleavage (DFRC) made it possible to detect DFRC monomers and dimers that are diagnostic of the occurrence of arylglycerol-β-O-aryl and β-β, β-5, β-1, and 4-O-5 units in the lignin structure. Quantification of these DFRC products indicated that β-O-aryl cleavage was a significant route for lignin biodegradation but that β-β, β-5, β-1, and 4-O-5 linkages were more resistant to the biological attack. The amount of aromatic hydroxyls did not increase with the split of β-O-4 linkages, suggesting that the β-O-4 cleavage products remain as quinone-type structures as detected by UV and visible spectroscopy. Nuclear magnetic resonance techniques also indicated the formation of new substructures containing nonoxygenated, saturated aliphatic carbons (CH₂ and CH₃) in the side chains of lignins extracted from biotreated wood samples.     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.     

Degradation of wood and enzyme production by Ceriporiopsis subvermispora

The degradation of the components of Japanese beech and Japanese cedar wood was measured over time in cultures of the white-rot fungus Ceriporiopsis subvermispora. Although there was no initial degradation of cedar wood, after 12 weeks the mass loss of both cedar and beech wood was 15–20%. The mass losses of filter paper in beech wood-containing cultures and glucose cultures after 12 weeks were 87% and 70%, respectively. The ratio of lignin loss to mass loss of both beech and cedar wood cultures approached 2.0. Although the cellulose loss in cedar wood was very low throughout the 12-week incubation, C. subvermispora degraded the hemicellulose in Japanese cedar much more effectively than that in Japanese beech. These results confirm that C. subvermispora is a selective lignin degrader. 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 Mn-dependent peroxidase activity peaking at 10 weeks. Lignin peroxidase and carboxymethylcellulase activities peaked after 3 weeks of incubation. Avicelase activity was present throughout the incubation period, although the activity was very low. The low-molecular-mass fraction of the extracellular medium, which catalyzes a redox reaction between O₂ and electron donors to produce hydroxyl radical, may act synergistically with the enzymes to degrade wood cell walls.     

Epoxy ceriporic acid produced by selective lignin-degrading fungus Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a selective white rot basidiomycete which degrades lignin in wood at a distance far from enzymes. Low molecular mass metabolites play a central role in the oxidative degradation of lignin. To understand the unique wood-decaying mechanism, we surveyed the oxidized derivatives of ceriporic acids (alk(en)ylitaconic acids) produced by C. subvermispora using high-resolution liquid chromatography multiple-stage mass spectrometry (HR-LC/MSⁿ). The analysis of the precursor and product ions from the extract suggested that an epoxidized derivative of ceriporic acid is produced by the fungus. To identify the new metabolite, an authentic compound of ceriporic acid epoxide was synthesized in vitro by reacting (R)-3-[(Z)-hexadec-7-enyl]-itaconic acid (ceriporic acid C) with m-chloroperbenzoic acid. The precursor and product ions from the natural metabolite and authentic epoxide were identical and distinguishable from those of hydroxy and hydroperoxy derivatives after reduction with NaBD₄. Feeding experiments with [U-¹³C]-glucose, 99% and the subsequent analyses of the first and second generation product ions demonstrated that the oxidized ceriporic acid was (R)-3-(7,8-epoxy-hexadecyl)-itaconic acid. To our knowledge, this study is the first to report that natural alkylitaconic acid bears an epoxy group on its side chain.     

Variable optimization for biopulping of agricultural residues by Ceriporiopsis subvermispora

Ceriporiopsis subvermispora was used for biochemical pulping of agricultural residues and the results were compared with chemical pulping. Independent variables were screened by Plackett-Burman and optimized by full factorial experimental designs. Biological treatment of rice, wheat and barley straw samples resulted in decrease of the kappa number of these straws by 34%, 21% and 19%, respectively, as compared with controlled samples. The tensile strength and burst factor of hand sheets produced from rice straw were increased by 51% and 33% as compared with the control straws. The tensile strength and burst factor of hand sheets produced from wheat straws were improved by 67% and 36%, these variables for barely straws were 36.7% and 45%, respectively. Although the delignification of wheat and barley straws are not as efficient as chemical process, but the quality of papers produced by biochemical pulping of straws were excellent.     

Oxalate oxidase from Ceriporiopsis subvermispora: biochemical and cytochemical studies.

The enzyme oxalate oxidase was identified in mycelial extracts of the basidiomycete Ceriporiopsis subvermispora and thereafter purified to homogeneity. The purification procedure included only three steps: Q-Sepharose chromatography, precipitation at pH 3.0, and phosphocellulose chromatography. The enzyme is a 400-kDa homohexamer, as determined by gel permeation in Sephadex G-200 and SDS-polyacrylamide gel electrophoresis. Isoelectrofocusing revealed a pI of 4.2. Optimal activity was obtained at pH 3.5 and at 45 degrees C. The purified enzyme has Km and kcat values of 0.1 mM and 88 s-1, respectively. It is highly specific for oxalate, although it is inhibited at concentrations of this substrate above 2.5 mM. Hystochemistry studies conducted over mycelium slices showed reactions products in both endocellular and periplasmic associated elements. A possible connection between the intracellular metabolism of oxalate and the extracellular ligninolytic activity of the fungus is proposed.     

Effectiveness of microbial pretreatment by Ceriporiopsis subvermispora on different biomass feedstocks

Different types of feedstocks, including corn stover, wheat straw, soybean straw, switchgrass, and hardwood, were tested to evaluate the effectiveness of fungal pretreatment by Ceriporiopsis subvermispora. After 18-d pretreatment, corn stover, switchgrass, and hardwood were effectively delignified by the fungus through manganese peroxidase and laccase. Correspondingly, glucose yields during enzymatic hydrolysis reached 56.50%, 37.15%, and 24.21%, respectively, which were a 2 to 3-fold increase over those of the raw materials. A further 10-30% increase in glucose yields was observed when pretreatment time extended to 35 d. In contrast, cellulose digestibility of wheat straw and soybean straw was not significantly improved by fungal pretreatment. When external carbon sources and enzyme inducers were added during fungal pretreatment of wheat straw and soybean straw, only glucose and malt extract addition improved cellulose digestibility of wheat straw. The cellulose digestibility of soybean straw was not improved.     

Influence of cultivation conditions on production of lignocellulolytic enzymes by Ceriporiopsis subvermispora

The aim of this work was to make a survey describing factors that influence the production of extracellular enzymes by white-rot fungus Ceriporiopsis subvermispora responsible for the degradation of lignocellulolytic materials. These factors were: carbon sources (glucose, cellulose, hemicellulose, lignin, maltose and starch), nitrogen sources (ammonium sulphate, potassium nitrate, urea, albumin and peptone), pH, temperature and addition of three different concentrations of Cu²⁺ and Mn²⁺. The cellulase and xylanase activities were similar in medium with different carbon sources and the highest cellulase and xylanase activities were measured in medium with urea and potassium nitrate as nitrogen sources, respectively. The highest laccase activity was observed in medium with lignin and peptone as carbon and nitrogen sources. In other experiments, time course of production of lignocellulolytic enzymes by white-rot fungus C. subvermispora in medium with lignin or glucose as carbon sources was observed.     

Mineralization of C-Ring-Labeled Synthetic Lignin Correlates with the Production of Lignin Peroxidase, not of Manganese Peroxidase or Laccase

Recently, Mn(II) has been shown to induce manganese peroxidases (MnPs) and repress lignin peroxidases (LiPs) in defined liquid cultures of several white rot organisms. The present work shows that laccase is also regulated by Mn(II). We therefore used Mn(II) to regulate production of LiP, MnP, and laccase activities while determining the effects of Mn(II) on mineralization of ring-labeled synthetic lignin. At a low Mn(II) level, Phanerochaete chrysosporium and Phlebia brevispora produced relatively high titers of LiPs but only low titers of MnPs. At a high Mn(II) level, MnP titers increased 12- to 20-fold, but LiPs were not detected in crude broths. P. brevispora formed much less LiP than P. chrysosporium, but it also produced laccase activity that increased more than sevenfold at the high Mn(II) level. The rates of synthetic lignin mineralization by these organisms were similar and were almost seven times higher at low than at high Mn(II). Increased synthetic lignin mineralization therefore correlated with increased LiP, not with increased MnP or laccase activities.     

Ceriporic acid C, a hexadecenylitaconate produced by a lignin-degrading fungus, Ceriporiopsis subvermispora

A lignin-degrading basidiomycete, Ceriporiopsis subvermispora produces a series of alkyl- and alkenylitaconates (ceriporic acids). Previously, two alkylitaconic acids with tetradecyl and hexadecyl side chains were isolated and identified as 1-heptadecene-2,3-dicarboxylic acid (ceriporic acid A) and 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B). In the present study, one hexadecenylitaconate (ceriporic acid C) was isolated and its chemical structure was analyzed by glycolation and subsequent (1) trimethylsilation, or (2) acetalation with acetone and acetone-d6. Analyses of the isolated metabolite demonstrated that the hexadecenylitaconic acid was (Z)-1,10-nonadecadiene-2,3-dicarboxylic acid. The structure of the side chain in ceriporic acid C was the same as that of hexadecenylcitraconate, chaetomellic acid B. Thus, it was found that ceriporic acids share close structural similarity with alk(en)yl citraconate derivatives, chaetomellic acids and other lichen lactones, protolichesterinic, lichesterinic, and murolic acids.     

Mineralization of 14C-Ring-Labeled Synthetic Lignin Correlates with the Production of Lignin Peroxidase, not of Manganese Peroxidase or Laccase

Recently, Mn(II) has been shown to induce manganese peroxidases (MnPs) and repress lignin peroxidases (LiPs) in defined liquid cultures of several white rot organisms. The present work shows that laccase is also regulated by Mn(II). We therefore used Mn(II) to regulate production of LiP, MnP, and laccase activities while determining the effects of Mn(II) on mineralization of ring-labeled synthetic lignin. At a low Mn(II) level, Phanerochaete chrysosporium and Phlebia brevispora produced relatively high titers of LiPs but only low titers of MnPs. At a high Mn(II) level, MnP titers increased 12- to 20-fold, but LiPs were not detected in crude broths. P. brevispora formed much less LiP than P. chrysosporium, but it also produced laccase activity that increased more than sevenfold at the high Mn(II) level. The rates of synthetic lignin mineralization by these organisms were similar and were almost seven times higher at low than at high Mn(II). Increased synthetic lignin mineralization therefore correlated with increased LiP, not with increased MnP or laccase activities.     

Secretion of Ligninolytic Enzymes and Mineralization of C-Ring-Labelled Synthetic Lignin by Three Phlebia tremellosa Strains

Production of ligninolytic enzymes by three strains of the white rot fungus Phlebia tremellosa (syn. Merulius tremellosus) was studied in bioreactor cultivation under nitrogen-limiting conditions. The Mn(II) concentration of the growth medium strongly affected the secretion patterns of lignin peroxidase and laccase. Two major lignin peroxidase isoenzymes were expressed in all strains. In addition, laccase and glyoxal oxidase were purified and characterized in one strain of P. tremellosa. In contrast, manganese peroxidase was not found in fast protein liquid chromatography profiles of extracellular proteins under either low (2.4 muM) or elevated (24 and 120 muM) Mn(II) concentrations. However, H(2)O(2)- and Mn-dependent phenol red-oxidizing activity was detected in cultures supplemented with higher Mn(II) levels. Mineralization rates of C-ring-labelled synthetic lignin (i.e., dehydrogenation polymerizate) by all strains under a low basal Mn(II) level were similar to those obtained for Phanerochaete chrysosporium and Phlebia radiata. A high manganese concentration repressed the evolution of CO(2) even when a chelating agent, sodium malonate, was included in the medium.     

Using Ceriporiopsis subvermispora CZ-3 laccase for indigo carmine decolourization and denim bleaching

In this study, the production of laccase by Ceriporiopsis subvermispora CZ-3 has been studied under semi solid-state conditions using natural waste as solid support materials. Different concentrations of xylidine derivatives were also investigated as inducer affecting laccase production. Melon peel having the lowest C/N ratio in comparison to other supports led to the highest activity levels, reaching maximum values of about 3000 UL⁻¹ for C. subvermispora CZ-3 in the presence of 2,4-xylidine. Laccase produced by this fungus was partially purified by ammonium sulphate precipitation and Sephacryl S-100 HR size exclusion chromatography. Several kinetic parameters of enzyme were also determined with 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) as a substrate. On the other hand, several compounds were investigated with respect to mediator effect for decolourization of indigo carmine and denim bleaching by this enzyme. It was observed that most of the compounds have mediator effect for decolourization of indigo carmine whereas 1-hydroxybenzotriazol is an appropriate compound for indigo dyed denim bleaching.     

Characterization of white zones produced on Pinus radiata wood chips by Ganoderma australe and Ceriporiopsis subvermispora

White zones produced on biodegraded Pinus radiata wood chips were characterized by micro-localized-FTIR (Fourier Transformed Infra Red) spectroscopy and scanning electron microscopy. Both techniques permitted assignment of the white zones to a selective lignin removal process. Although both fungi studied have degraded lignin selectively in these restricted superficial areas, chemical analysis of the wood chips indicated that Ganoderma australe removed 16% of the initial amount of glucan at the 20% weight loss level. Ceriporiopsis subvermispora did not remove glucan at weight loss values below 17%. Prolonged biodegradation resulted in reduction of white zones by G. australe, and increased white zones from C. subvermispora decayed samples. This revised version was published online in July 2006 with corrections to the Cover Date.     

Secretion of Ligninolytic Enzymes and Mineralization of 14C-Ring-Labelled Synthetic Lignin by Three Phlebia tremellosa Strains

Production of ligninolytic enzymes by three strains of the white rot fungus Phlebia tremellosa (syn. Merulius tremellosus) was studied in bioreactor cultivation under nitrogen-limiting conditions. The Mn(II) concentration of the growth medium strongly affected the secretion patterns of lignin peroxidase and laccase. Two major lignin peroxidase isoenzymes were expressed in all strains. In addition, laccase and glyoxal oxidase were purified and characterized in one strain of P. tremellosa. In contrast, manganese peroxidase was not found in fast protein liquid chromatography profiles of extracellular proteins under either low (2.4 μM) or elevated (24 and 120 μM) Mn(II) concentrations. However, H₂O₂- and Mn-dependent phenol red-oxidizing activity was detected in cultures supplemented with higher Mn(II) levels. Mineralization rates of ¹⁴C-ring-labelled synthetic lignin (i.e., dehydrogenation polymerizate) by all strains under a low basal Mn(II) level were similar to those obtained for Phanerochaete chrysosporium and Phlebia radiata. A high manganese concentration repressed the evolution of ¹⁴CO₂ even when a chelating agent, sodium malonate, was included in the medium.