A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe²⁺, with a K_m close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.     

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.     

Phenol Oxidase Activity of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 Mutants with Modified Motility and <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 Phase Variants with Different Plasmid Composition

Herein, we reveal the alteration in phenol oxidase enzymes complex production from Azospirillum brasilense Sp245 omegon mutants with polar and lateral flagella dysfunction and from A. brasilense Sp7 phase variants with different plasmid composition. The enzymatic activities for various laccases, tyrosinases, Mnperoxidases, and lignin peroxidases as well as the isomorphic composition of intracellular laccases and tyrosinases were estimated for the studied variants and the parent strains. It was noted that various genetic events correlating with phenotypic heterogeneity in A. brasilense populations affect their phenol oxidase activity level.     

Activity of tocopherol oxidase in Phaseolus coccineus seedlings

In the present study, we have demonstrated that membrane-free extracts of etiolated shoots of Phaseolus coccineus seedlings show tocopherol oxidase activity. For this reaction, presence of membrane lipids, such as lecithin and mixture of plant lipids was required. The rate of the reaction was the highest for α-tocopherol and decreased in the order α ? β > γ > δ tocopherols. In the case of α-tocopherol, the main oxidation product was α-tocopherolquinone, while for the other tocopherol homologues the dominant products were other derivatives. When the enzyme activity was measured in leaves, hypocotyls and roots of etiolated seedlings of P. coccineus, the oxidase activity was the highest in extracts of leaves and decreased towards the roots where no activity was detected. The effect of hydrogen peroxide and of different inhibitors on the reaction suggest that tocopherol oxidase does not belong to peroxidases or flavin oxidases but rather to multi-copper oxidases, such as polyphenol oxidases or laccases. On the other hand, catechol, the well-known substrate of polyphenol oxidases and laccases, was not oxidized by the enzyme, indicating a high substrate specificity of the tocopherol oxidase.     

Lindane degradation by Candida VITJzN04, a newly isolated yeast strain from contaminated soil: kinetic study,enzyme analysis and biodegradation pathway

A new yeast strain was isolated from sugarcane cultivation field which was able to utilize lindane as sole carbon source for growth in mineral medium. The yeast was identified and named as Candida sp. VITJzN04 based on a polyphasic approach using morphological, biochemical and 18S rDNA, D1/D2 and ITS sequence analysis. The isolated yeast strain efficiently degraded 600 mg L^?1 of lindane within 6 days in mineral medium under the optimal conditions (pH 7; temperature 30 °C and inoculum dosage 0.06 g L^?1) with the least half-life of 1.17 days and degradation constant of 0.588 per day. Lindane degradation was tested with various kinetic models and results revealed that the reaction could be described best by first-order and pseudo first-order models. In addition, involvement of the enzymes viz. dechlorinase, dehalogenase, dichlorohydroquinone reductive dechlorinase, lignin peroxidase and manganese peroxidase was noted during lindane degradation. Addition of H₂O₂ in the mineral medium showed 32 % enhancement of lindane degradation within 3 days. Based on the metabolites identified by GC–MS and FTIR analysis, sequential process of lindane degradation by Candida VITJzN04 was proposed. To the best of our knowledge, this is the first report of isolation and characterization of lindane-degrading Candida sp. and elucidation of enzyme systems during the degradation process.     

Evaluation of holocellulase production by plant-degrading fungi grown on agro-industrial residues

Agaricus brasiliensis CS1, Pleurotus ostreatus H1 and Aspergillus flavus produced holocellulases when grown in solid and submerged liquid cultures containing agro-industrial residues, including sugar cane bagasse and dirty cotton residue, as substrates. These isolates proved to be efficient producers of holocellulases under the conditions used in this screening. Bromatological analysis of agro-industrial residues showed differences in protein, fiber, hemicellulose, cellulose and lignin content. Maximal holocellulase activity (hemicellulase, cellulase and pectinase) was obtained using solid-state cultivation with 10% substrate concentration. In this case, remarkably high levels of xylanase and polygalacturonase activity (4,008 and 4,548 IU/l, respectively) were produced by A. flavus when grown in media containing corn residue, followed by P. ostreatus H1 with IU/l values of 1,900 and 3,965 when cultivated on 5% and 10% sugar cane bagasse, respectively. A. brasiliensis CS1 showed the highest reducing sugar yield (11.640 mg/ml) when grown on medium containing sugar cane bagasse. A. brasiliensis was also the most efficient producer of protein, except when cultivated on dirty cotton residue, which induced maximal production in A. flavus. Comparison of enzymatic hydrolysis of sugar cane bagasse and dirty cotton residue by crude extracts of A. brasiliensis CS1, P. ostreatus H1 and A. flavus showed that the best reducing sugar yield was achieved using sugar cane bagasse as a substrate.     

Protein mapping of Chaetomium globosum,a potential biological control agent through proteomics approach

Chaetomium globosum is a ubiquitous filamentous fungus having biological control properties. The potential isolates mycoparasitize the pathogen and produce antifungal metabolites which suppress the growth of pathogenic fungi. A proteomics approach was undertaken to separate and identify proteins from a mycoparasitic strain Cg1 of C. globosum under normal and heat shock conditions in order to identify differentially expressed proteins. We developed and standardized the procedure for extraction of total proteins and 2D gel electrophoresis, which resulted in profiling of more than 100 protein spots. 48 proteins were identified by a combination of matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF) and liquid chromatography mass spectrometry (LCMS/MS). Out of total proteins identified, 79 % were hypothetical proteins and 21 % proteins were functionally characterized. Out of total 79 % hypothetical proteins 24 % proteins matched with C. globosum while 18 % proteins matched with Aspergillus spp., 13 % with Coprinopsis cinerea, 10 % with Giberrella zaea, 8 % with Magnaporthe grisea and 5 % with Neurospora crassa and Lodderomyces elonisporus. Some of the functionally characterized proteins included MAP kinase, maltose permease, GTP binding protein, dyenin heavy chain, HET- C2, vacuolar Dig A protein, polyketide synthase, peptide prolyl cis trans isomerase and translation elongation factor. This study has generated a protein reference map for Chaetomium globosum, and being the first report on proteomics studies would greatly help to unravel biocontrol mechanism and its survival under heat stress conditions.     

Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation

Laccase, widely distributed in bacteria, fungi, and plants, catalyzes the oxidation of wide range of compounds. With regards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesis while fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual function of laccases using in-silico molecular docking and dynamics simulation approaches. Modeling and superimposition analyses of one each representative of plant and fungal laccases, namely, Populus trichocarpa and Trametes versicolor, respectively, revealed low level of similarity in the folding of two laccases at 3D levels. Docking analyses revealed significantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as compared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites were found to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analyses for the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetrameric lignin model compound remains attached to the active site of fungal laccase throughout the simulation period, while it protrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basis of binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than that of plant. The overall data suggested a situation favorable for the degradation of lignin polymer by fungal laccase while its synthesis by plant laccase.     

Potential of selected fungal species to degrade wheat straw,the most abundant plant raw material in Europe

Background

Structural component of plant biomass, lignocellulose, is the most abundant renewable resource in nature. Lignin is the most recalcitrant natural aromatic polymer and its degradation presents great challenge. Nowadays, the special attention is given to biological delignification, the process where white-rot fungi take the crucial place owing to strong ligninolytic enzyme system. However, fungal species, even strains, differ in potential to produce high active ligninolytic enzymes and consequently to delignify plant biomass. Therefore, the goals of the study were characterization of Mn-oxidizing peroxidases and laccases of numerous mushrooms as well as determination of their potential to delignify wheat straw, the plant raw material that, according to annual yield, takes the first place in Europe and the second one in the world.

Results

During wheat straw fermentation, Lentinus edodes HAI 858 produced the most active Mn-dependent and Mn-independent peroxidases (1443.2 U L⁻¹ and 1045.5 U L⁻¹, respectively), while Pleurotus eryngii HAI 711 was the best laccase producer (7804.3 U L⁻¹). Visualized bends on zymogram confirmed these activities and demonstrated that laccases were the dominant ligninolytic enzymes in the studied species. Ganoderma lucidum BEOFB 435 showed considerable ability to degrade lignin (58.5%) and especially hemicellulose (74.8%), while the cellulose remained almost intact (0.7%). Remarkable selectivity in lignocellulose degradation was also noted in Pleurotus pulmonarius HAI 573 where degraded amounts of lignin, hemicellulose and cellulose were in ratio of 50.4%:15.3%:3.8%.

Conclusions

According to the presented results, it can be concluded that white-rot fungi, due to ligninolytic enzymes features and degradation potential, could be important participants in various biotechnological processes including biotransformation of lignocellulose residues/wastes in food, feed, paper and biofuels.

     

Characterization of Clostridium thermocellum Isolates Grown on Cellulose and Sugarcane Bagasse

Although plant cell walls may be degraded by microbial free enzymes, many bacteria degrade cellulose via enzyme complexes called cellulosomes. The study of the structures and mechanisms of these large macromolecular complexes is an active and ongoing research topic, with the goal of developing methods to improve lignocellulosic biomass conversion using cellulosomes. The aim of the present work was to evaluate and characterize the holocellulolytic activities produced by two new isolates (ISO1 and ISO2) of the spore-forming thermophilic anaerobic bacterium Clostridium thermocellum, during growth on crystalline cellulose and sugarcane bagasse, in comparison with activities obtained from the C. thermocellum strain CthJW. The pH and temperature values for optimal growth of the isolates were pH 7 and 60 °C, respectively. The isolates produced cellulolytic, xylanolytic, and pectinolytic activities when cultured on crystalline cellulose or sugarcane bagasse, which have never been used previously as the sole carbon source for these bacteria. The profiles of secreted proteins for these isolates, ISO1 and ISO2, were quite different from those obtained for the standard strain CthJW and from each other, as shown by 2D gel electrophoresis maps, and these profiles also depend on the carbon source used. Different protein isoforms were also detected in the maps for all growth conditions and bacterial strains. MALDI-TOF mass spectrometry was used to identify the differentially expressed proteins for ISO1 and ISO2 under growth in the presence of cellulose as carbon source. Twenty-five differentially expressed spots were identified and grouped into 8 functional categories: metabolism (20 %), motor function (20 %), protein synthesis (12 %), oxidative stress (16 %), secretory pathway (12 %), cellulose hydrolysis (4 %), protein folding (4 %), and defense (12 %). Spots 200 and 197, identified as a glycosyl hydrolase family member 9 and as a chaperone GroEL, respectively, were detected for all isolates and are potentially related to cellulosome architecture.     

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.     

Production of Fermentable Sugars and Hydrogen-Rich Gas from <Emphasis Type="Italic">Agave tequilana</Emphasis> Biomass

The Mexican tequila industry annually processes approximately 1 × 10⁶ Agave tequilana plants, generating approximately 1.78 × 10⁸ kg of bagasse per year. This biomass is considered an attractive alternative to fossil fuels as an energy source and to produce biofuels and/or chemical products because it is produced and used without adversely affecting the environment. The first aim of the present work was to determine the effect of temperature, the concentration of H₂SO_4, and reaction time on the hydrolysis of agave bagasse to maximize the fermentable sugars using a steam explosion. This step process generated 71.11 g/L of reducible sugars in the supernatant (59.29 % glucose, 29.05 % xylose, and 11.66 % fructose) and unconverted organic matter of enzymatic hydrolysis bagasse (35.4 % α-cellulose, 7.33 % hemicellulose, 49.91 % lignin, and 7.31 % ashes). A mathematical surface response analysis of the hydrolysis was used for process optimization. The second aim involves the study of the thermodynamics of the reforming of unconverted organic matter from enzymatic hydrolysis of Agave tequilana bagasse (ATB) evaluated by the Gibbs free energy minimization method for hydrogen production. The effect of the parameters on the system performance measures, such as reaction temperature (T), Water/Biomass ratio (WBR), and pressure (P), were also investigated. The maximum H₂ production obtained was 23.2 mol of H₂/271.5 g ATB with a WBR ≥ 11 and a temperature of 740 °C. These findings indicate that the temperature and WBR are essential factors in the production of H₂, which was reflected in the efficiency of the process.     

Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose

The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.     

Influence of cultivation conditions on pyrene degradation by the fungus Pleurotus Ostreatus D1

For the first time the dependence of completeness of pyrene degradation by the white-rot fungus Pleurotus ostreatus D1 on cultivation conditions was found. In Kirk’s medium about 65.6 ± 0.9% of the initial pyrene was metabolized after 3 weeks, with pyrene-4,5-dihydrodiol accumulating. This process was accompanied by laccase production only. In basidiomycetes rich medium, P. ostreatus D1 metabolized up to 89.8 ± 2.3% of pyrene within 3 weeks without pyrene-4,5-dihydrodiol accumulation throughout the time of cultivation. Phenanthrene and phthalic acid were identified as the metabolites produced from pyrene degradation under these conditions. Accumulation of phenanthrene with its subsequent disappearance was observed. One more metabolite probably was the product of phenanthrene degradation. Pyrene metabolism in basidiomycetes rich medium was accompanied first by laccase and tyrosinase production and later by versatile peroxidase production. The cell-associated activities of laccase, tyrosinase, and versatile peroxidase were found. The data obtained indicate that both enzymes (laccase and versatile peroxidase) are necessary for complete degradation of pyrene. Furthermore, both cell-associated and extracellular laccases can catalyse the first stages of pyrene degradation, and versatile peroxidase can be necessary for oxidation of the resulting metabolites.     

Combinatorial evaluation of laccase-mediator system in the oxidation of veratryl alcohol

Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both commercially prepared and crude extracts, for their ability to oxidize veratryl alcohol in the presence of various solvents and mediators. Screening revealed complete conversion of veratryl alcohol to veratraldehyde catalyzed by a crude preparation of the laccase from Trametes versicolor ATCC 11235 and the mediator TEMPO in 20 % (v/v) tert-butanol.     

Proteomic Changes in the Stem of Wild <Emphasis Type="Italic">Pteridium aquilinum</Emphasis> During Development

Lignification is one of the most crucial factors affecting the edible value of the stem of wild Pteridium aquilinum. To investigate the probable mechanism of lignification, the changes in protein profiles in the stem of wild P. aquilinum during its development were investigated by means of two-dimensional electrophoresis technology. The two-dimensional electrophoresis results revealed that there were twenty-seven differential proteins, twenty-four proteins of which were identified by MALDI-TOF/TOF. We classified these twenty-four proteins into six functional categories: photosynthesis (8, 33.3 %); respiratory metabolism (4, 16.7 %); stress response and defence (6, 25.0 %); cell structure (1, 4.2 %); phenylpropanoid metabolism (4, 16.6 %) and unclassified protein (1, 4.2 %). According to the functional analysis of these differentially expressed proteins, we concluded that photosynthesis was enhanced during P. aquilinum’s development and sugars generated from photosynthesis were partially metabolized through the glycolysis pathway and phosphopentose pathway, respectively, thus producing the precursors for lignin biosynthesis. The up-regulation of caffeoyl-CoA-O-methyl-transferase and SAM synthetase in abundance and the down-regulation of chalcone synthase can be directly responsible for lignification during stem development. This experiment is useful for understanding the biochemical mechanisms of the lignification process of P. aquilinum during its development.     

Characteristics of Lignin Extracted from Different Lignocellulosic Materials via Organosolv Fractionation

Among biomass-derived compounds, lignin is an underused component with potential for conversion to industrial-needed products in biorefinery. In this study, organosolv fractionation of four lignocellulosic materials including bagasse (BG), pararubber wood sawdust (PS), palm fiber (PF), and cassava fiber (CF) was studied using a ternary solvent mixture comprising methyl isobutyl ketone (MIBK), ethanol, and water in the presence of H₂SO₄ to separate high-purity lignin. The fractionation reaction was performed at 160 °C for 40 min with MIBK/ethanol/water proportion of 0.25/0.42/0.33 and 0.025 M of H₂SO₄, which led to the highest lignin removal efficiency of 88.2, 70.6, 67.3, and 71.7% (w/w) from BG, PS, PF, and CF, respectively. Physicochemical characteristics of the fractionated lignin were determined for Klason lignin and by X-ray fluorescence spectroscopy, organic elemental analysis, ¹H nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. The lignin samples were thermally depolymerized in MIBK to determine the content of specific lignin-derived chemicals. The main phenolic derivatives from BG-lignin were 4-ethylphenol and 4-vinylguaiacol, whereas those from PS-lignin were syringaldehyde and cis-isoeugenol. Phenol and bis(2-ethylhexyl) phthalate were mainly produced from depolymerization of PF-lignin while trans-isoeugenol and hexadecanoic acid were the major products from CF-lignin. This work demonstrates the potential of the fractionated lignin for production of valuable chemicals in biorefineries.     

Quantitative proteomics of pomegranate varieties with contrasting seed hardness during seed development stages

In pomegranate (Punica granatum), seed hardness is an important trait directly affecting fruit marketability. However, seed formation in pomegranate has not been well studied. We investigated the genetic mechanism underlying pomegranate seed hardness by comparing protein expression profiles between soft- and hard-seeded varieties 60 and 120 days after flowering. We identified 1940 proteins, of which 399 were differentially expressed. Most of the differentially expressed proteins were involved in posttranslational modification and carbohydrate metabolism. Cell wall biosynthesis, which showed positive correlations with seed hardness, was selected as the candidate pathway. The mRNA levels of 14 proteins involved in cell wall biosynthesis were further analyzed by qPCR. Lignin biosynthesis-related differentially expressed proteins showed lower expression at protein and gene levels in a soft-seeded variety at the early stages. Moreover, cellulose biosynthesis-related differentially expressed proteins showed higher expression levels in the soft-seeded variety at 60 days after flowering. Thus, the soft-seeded variety showed lower lignin but higher cellulose biosynthesis at the early fruit developmental stage, suggesting that lignin and cellulose play opposing roles in cell wall formation in pomegranate seeds. Moreover, differentially expressed proteins involved in cell wall degradation showed higher expression levels in the soft-seeded variety at both developmental stages. These results suggested that differences in seed hardness between soft- and hard-seeded pomegranates might result from cell wall biosynthesis and also be affected by cell wall degradation. The present proteome-wide profiling of pomegranate genotypes with contrasting seed hardness adds to the current knowledge base of the molecular basis of seed hardness development.