Characterization of the cellulolytic enzyme system from the brown-rot fungus Coniophora puteana

Summary The cellulolytic enzymes of various strains of the brown-rot fungus Coniophora puteana were studied. The organism was grown in an air-lift fermentor in mineral medium containing glucose, cellobiose or amorphous cellulose. The specific growth rate varied between 0.082 and 0.062 h^–1. On amorphous cellulose as sole carbon source, the organism secreted various proteins, some of which were characterized. The mixture contained inter alia four endocellulases, two exo-cellobiohydrolases and a cellobiose dehydrogenase. Three endocellulases (named type I) were active on soluble cellulose derivatives but inactive on p-nitrophenyllactoside (p-NPL), whereas a fourth endocellulase (named type II) was active on both. The two exo-cellobiohydrolases released cellobiose from amorphous cellulose; they were inactive on soluble cellulose derivatives but hydrolyzed p-NPL with strong cellobiose inhibition. A cellobiose dehydrogenase having spectral characteristics compatible with a flavo b-cytochrome was also identified. Neither the exo-cellobiohydrolase nor the type II endocellulase were secreted during growth on cellobiose whereas type I endocellulases and cellobiose dehydrogenase were formed at a reduced rate. No formation of cellulolytic enzymes was observed during growth on glucose alone. Correspondence to: G. Canevascini     

Evaluating biopulping as an alternative application on oil palm trunk using the white-rot fungus Trametes versicolor

The objective of the research was to investigate the suitability of the white-rot fungus Trametes versicolor for biopulping using oil palm biomass as the substrate. Fungi are grown on solid-state cultures Kirk's enrichment media to determine the lignocellulolytic activities. Samples subjected to fungal pretreatment for periods of 1, 2, 3, and 4 weeks were investigated and compared to the untreated control. The crude enzyme extracts were assayed using specific substrates and enzyme activities were calculated. The highest level of laccase activity was 218.66 U/L; the peak activity of manganese peroxidase was 162.10 U/L, and lignin peroxidase is 42.56 U/L. The activity levels of cellulase and hemicellulase were insignificant in all extracts (53.30 and 1.50 U/L, respectively). When the chips were pulped mechanically the Kappa number, pulp yield, and screened pulp yield decreased significantly and paper strength increased marginally with the exposure time. Hand sheet properties were also improved significantly by fungal treatment. Weight loss, lignin loss, cellulose, and holocellulose loss were 8.45%, 9.35%, 4.58%, and 7.2%, respectively. Images from SEM seem to indicate a simultaneous type of decay pattern involving cell wall breakdown combined with lignin modification. Considering all its pulping and papermaking properties, the performance of T. versicolor is good and has potential for use in large-scale biotechnological processes.     

Effect of aromatic monomers on production of carbohydrate-degrading enzymes by white-rot and brown-rot fungi

Abstract The effects were studied of 12 monomeric aromatic compounds on the production of six carbohydrate-degrading enzymes of two brown-rot fungi, Postia placenta and Gloeophyllum trabeum , and one white-rot fungus, Coriolus versicolor . Most compounds were inhibitory to growth of the decay fungi at concentrations of 0.05%. At lower concentrations, the phenolics often stimulated growth. Relatively high concentrations (> 0.1%) of aromatic monomers were required to inhibit xylanase of P. placenta in situ. The effects of the monomers incorporated in liquid media on enzyme production varied depending on compound and fungal species. Some compounds were quite inhibitory to production of enzyme activities at very low concentrations. For example, catechol and vanillin (50 ppm) caused 100% inhibition of xylanase and β-1,4-endoglucanase production by P. placenta . However, no aromatic monomer was strongly inhibitory to production of all enzyme activities of all fungi.     

Degradation of chlorinated lignin compounds in a bleach plant effluent by the white-rot fungus Trametes versicolor

Summary Chlorinated lignin derivatives in a combined bleach plant effluent from sulphite pulping were degraded by several white-rot fungi among which Trametes versicolor (Coriolus versicolor) strains were the most efficient. With glucose as co-substrate, about 90% colour reduction was achieved within 3 days. Simultaneously, the concentration of chloro-organic compounds measured as adsorbable organic halogens decreased by about 45%. As shown by gel chromatography, the high-molecular-weight fraction in the effluent was completely depolymerized while over 50% of total aromatic compounds were degraded. The presence of a co-substrate was necessary for all these activities of the fungus. The residue obtained after degradation was extremely recalcitrant and not further degradable. Offprint requests to: M. Bergbauer     

Oxidation of aromatic compounds in organic solvents with laccase from Trametes versicolor

Summary Laccase purified from Trametes versicolor oxidizes 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine in hydrophobic solvents presaturated with water, and in hydrophilic organic solvents provided that a sufficient amount of water is added. Ease of performance of the laccase test in organic solvents is improved after immobilization of the enzyme by entrapping in Sepharose CL-6B during enzyme filtration through the gel beads. The gel-enzyme association has been shown to be stable in water-presaturated solvents. Efficiency of the immobilized laccase in organic solvents containing 7% water was 10%–20% of that in potassium-citrate buffer. Immobilized laccase in organic solvents showed good stability and high tolerance to elevated temperatures.     

Changes in oxidative enzyme activity during interspecific mycelial interactions involving the white-rot fungus Trametes versicolor

Interspecific fungal antagonism leads to biochemical changes in competing mycelia, including up-regulation of oxidative enzymes. Laccase, manganese peroxidase (MnP), manganese-repressed peroxidase (MRP) and lignin peroxidase (LiP) gene expression and enzyme activity were compared during agar interactions between Trametes versicolor and five other wood decay fungi resulting in a range of interaction outcomes from deadlock to replacement of one fungus by another. Increased laccase and Mn-oxidising activities were detected at all interaction zones, but there were few changes in activity in regions away from the interaction zone in T. versicolor mycelia compared to self-pairings. Whilst no LiP activity was detected in any pairing, low level LiP gene expression was detected. MnP activity was detected but not expression of MnP genes; instead, MRP could explain the observed activity. No relationship was found between extent of enzyme activity increase and interaction outcome. Similarities between patterns of gene expression and enzyme activity are discussed.     

Characterization of a lignin peroxidase gene from the white-rot fungus Trametes versicolor.

A genomic library of the white-rot fungus Trametes versicolor has been constructed and a gene coding for a lignin peroxidase has been isolated and sequenced. The gene, which contains 6 introns, encodes a protein of 346 amino acid residues, preceded by a tentative 26-residue signal peptide. The deduced amino-terminal sequence agrees with the amino-terminal end of a lignin peroxidase isozyme previously isolated from carbon-limited cultures of T versicolor.     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

Cloning and characterization of a lignin peroxidase gene from the white-rot fungus Trametes versicolor

Six putative lignin peroxidase (LIP) genes were isolated from a lambda EMBL3 phage library of the white-rot fungus, Trametes versicolor, using the Phanerochaete chrysosporium LIP cDNA CLG5 as the probe. Sequence analysis of one of the genes, VLG1, showed that its coding region is interrupted by six small introns (49-64 bp) and that it encodes a mature LIP protein (341 aa; Mr: 36,714) that is preceded by a 25 aa signal sequence. This protein has a relatively high degree of aa homology to the N-termini of the LIP proteins purified from T. versicolor and has an aa homology of 55-60% to the LIP proteins of P. chrysosporium, which is comparable to that found between P. chrysosporium and Phlebia radiata LIP proteins.     

Effect of onion-type multilamellar liposomes on Trametes versicolor laccase activity and stability

Trametes versicolor laccase was encapsulated into onion-type, lipid-based multilamellar vesicles (MLVs). When encapsulated, laccase was isolated from the assay medium but was still active once freed from its capsule. The encapsulation efficiency was larger than 65% at 25 °C and 37 °C and decreased to 55% by introducing 140 mM NaCl into the buffered medium (pH = 4.5). MLVs were shown to drastically improve both laccase stability and activity. At 25 °C, laccase activity was doubled in the presence of MLVs. At 37 °C in the salt-free medium, the half-life time of laccase was increased from 2hr 30-65 h without and with MLVs, respectively. This effect was even more pronounced in the salted medium where laccase activity was unchanged for 6 days in the presence of MLVs. These beneficial effects were attributed to the immobilization of laccase onto MLV surface. Laccase activity as well as stability was notably shown to be directly correlated to MLV stability.     

Metabolite secretion,Fe3+-reducing activity and wood degradation by the white-rot fungus Trametes versicolor ATCC 20869

Trametes versicolor is a promising white-rot fungus for the biological pretreatment of lignocellulosic biomass. In the present work, T. versicolor ATCC 20869 was grown on Pinus taeda wood chips under solid-state fermentation conditions to examine the wood-degrading mechanisms employed by this fungus. Samples that were subjected to fungal pretreatment for one-, two- and four-week periods were investigated. The average mass loss ranged from 5 % to 8 % (m m⁻¹). The polysaccharides were preferentially degraded: hemicellulose and glucan losses reached 13.4 % and 6.9 % (m m⁻¹) after four weeks of cultivation, respectively. Crude enzyme extracts were obtained and assayed using specific substrates and their enzymatic activities were measured. Xylanases were the predominant enzymes, while cellobiohydrolase activities were marginally detected. Endoglucanase activity, β-glucosidase activity, and wood glucan losses increased up to the second week of biodegradation and remained constant after that time. Although no lignin-degrading enzyme activity was detected, the lignin loss reached 7.5 % (m m⁻¹). Soluble oxalic acid was detected in trace quantities. After the first week of biodegradation, the Fe³⁺-reducing activity steadily increased with time, but the activity levels were always lower than those observed in the undecayed wood. The progressive wood polymer degradation appeared related to the secretion of hydrolytic enzymes, as well as to Fe³⁺-reducing activity, which was restored in the cultures after the first week of biodegradation.     

Decoloration of Amaranth by the white-rot fungus Trametes versicolor. Part II. Verification study

The involvement of lignin peroxidase (LiP) in the decoloration of the mono-azo substituted napthalenic dye Amaranth was investigated with pure enzymes and whole cultures of Trametes versicolor. The verification study confirmed that LiP has a direct influence on the initial decoloration rate and showed that another enzyme, which does not need hydrogen peroxide to function and is not a laccase, also plays a role during decoloration. These results confirm the results of a previous statistical analysis. Furthermore, the fungal mycelium affects the performance of the decoloration process.     

Decoloration of Amaranth by the white-rot fungus Trametes versicolor. Part I. Statistical analysis

The white-rot fungus Trametes versicolor decolorized the mono-azo-substituted naphthalenic dye Amaranth. The relationship between the amount of enzymes present in the system and the efficiency of the decoloration process was investigated. The two responses used to quantify the process of decoloration (i.e., initial decoloration rate, v0, and the percent concentration of dye decolorized in 1 h, %c) were correlated with the amount of three enzymes considered for the study (lignin peroxidase, manganese peroxidase, and laccase) and analyzed through stepwise regression analysis (forward, backward, and mixed). The results of the correlation analysis and those of the regression analysis indicated that lignin peroxidase is the enzyme having the greatest influence on the two responses.     

Uptake of cadmium ions in white-rot fungus Trametes versicolor: effect of cd (II) ions on the activity of laccase

The effects of cadmium Cd (II) ions on the physiology and biological activity of Trametes versicolor, a strain belonging to white-rotting Basidiomycetes, were examined. Cd (II) ions were added to 10-day-old cultures grown on a liquid medium, or at the time of inoculation. Our experiments showed that T. versicolor is a good cadmium biosorbent from aqueous solution, this strain removing almost all the Cd (ll) ions over the first 2h of incubation by what appears to be a rapid, energy-independent surface binding phenomenon, at the rate of approximately 2mg Cd per g mycelial dry weight. An additional slower and energy-dependent transport mechanism was also present, taking in approximately 0.3mg Cd (II) perg dry weight. It is also shown that these Cd (II) ions significantly stimulate the activity of extracellular laccase when added to 10-day-old cultures.     

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.     

Effect of aromatic compounds on the production of laccase and manganese peroxidase by white-rot basidiomycetes

Three white-rot fungi displayed a wide diversity in their response to supplemented aromatic compounds. Pyrogallol stimulated Cerrena unicolor laccase and manganese peroxidase (MnP) synthesis in synthetic medium 2.5- and 2-fold, respectively, whereas 2,4,6-trinitrotoluene (TNT) brought about a 2.8-fold increase in laccase yield by Trametes versicolor in submerged fermentation of ethanol production residue. No effect of the tested aromatic compounds on enzyme secretion by Ganoderma lucidum in mannitol-containing medium was detected. Nevertheless, G. lucidum is a potent producer of laccase in submerged fermentation of wheat bran and enzyme synthesis can be further increased by supplementation of medium with an appropriate inducer. The structure and the concentration of aromatic compounds play an important role in the regulation of enzyme synthesis. The supplementation of synthetic medium with 0.03–0.3 mM TNT or hydroquinone increased the differential rate of laccase synthesis by C. unicolor from 1,267 to 3,125–8,630 U mg biomass^?1 day^?1. Moreover, the same aromatic compound may function as either an inducer or a repressor, depending on the fungus and enzyme studied. Thus, hydroquinone increased 3-fold T. versicolor laccase activity decreasing 2- and 8-fold the yields of MnP and endoglucanase, respectively.     

Stability of a polyphenol oxidase from the white-rot fungus Trametes versicolor in the presence of canola meal

The stability of a polyphenol oxidase (PPO) preparation from the white-rot fungus Trametes versicolor during a process for the enzymatic decrease of the phenolic content of commercial canola meal (CM) was investigated. The effects of temperature, pH, protein origin and concentration, and meal particles were considered. The results showed that the thermal stability of the enzyme preparation was significantly increased in the presence of CM. The half-life times for the enzyme preparation, pre-incubated with CM at 50, 60, 70 and 75°C, were 45, 10.5, 3.5 and 1.5 hours, respectively; this represents an increase in the thermal stability of the enzyme preparation of up to four times in the presence of CM compared to the stability in the absence of CM. This effect was caused by the protective actions of both the CM particles and CM proteins, with the former responsible for 90% of the observed effect. The thermal stability of the enzyme in the presence of CM, from which 20% of the extractable proteins was extracted, was 5% lower compared to the stability in the presence of untreated CM. Changes in pH level from 5.0 to 3.2 resulted in a loss of stability comparable to that observed when the pre-incubation temperature was increased from 50 to 70°C. A semi-empirical model describing the changes in the concentration of the active enzyme pre-incubated in the presence and absence of CM at various incubation temperatures was proposed. A very good agreement between the model and experimental data was obtained. The proposed model, together with a general set of model parameters, can be used as a tool for the optimization of a process for the upgrade of CM by enzymatically decreasing the meal's phenolic content.     

Effect of ionic liquids activation on laccase from Trametes versicolor: Enzymatic stability and activity

The stability and activity of laccase from Trametes versicolor in two water‐soluble ionic liquids (ILs), namely 1‐butyl‐3‐methylimidazolium methyl sulfate, [bmim][MeSO₄] and 1,3‐dimethylimidazolium methyl sulfate, [mmim][MeSO₄], were investigated in this study. Thermal inactivation of laccase was characterized in the presence of these both ILs and as expected first‐order kinetics was followed. Inactivation rate constant (k), half‐life time (t_1/2), and energy of activation (Ea) were determined. Kinetics of 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) oxidation by laccase in the presence of these ILs was studied and Michaelis–Menten parameters were calculated. There is no enzymatic inactivation since the maximum reaction rate remained constant for IL concentrations up to 25%, and surprisingly, it was found that laccase was activated for concentrations of 35% of ILs, since the reaction rate increased 1.7 times.     

Synthesis and antifungal activity of oxygenated cholesterol derivatives

A series of oxygenated cholesterol derivatives were prepared from new synthetic methods and evaluated for their in vitro antimicrobial properties against human pathogens. The activity was highly dependent on the structure of the different compounds involved. The best results were obtained with hydroxy ketones 2, 4 and 5 and diketone 7 exhibiting activities against S. cerevisiae (ATCC 28383) and Candida albicans (CIP 1663-86). For example, compound 2 exhibited high activities against C. albicans (CIP 1663-86) and Amphotericine B and miconazole resistant strain C. albicans (CIP 1180-79) at a concentration of 1.5 microg/mL.     

Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications

Improvement of the catalytic properties of fungal laccases is a current challenge for the efficient bioremediation of natural media polluted by xenobiotics. We developed the heterologous expression of a laccase from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica as a first step for enzyme evolution. The full-length cDNA consisted of a 1,561-bp open reading frame encoding lacIIIb, a 499-amino-acid protein and a 21-amino-acid signal peptide. Native and yeast secretion signals were used to direct the secretion of the enzyme, with the native signal yielding higher enzyme activity in the culture medium. The level of laccase activity secreted by the transformed yeast was similar to that observed for the non-induced wild-type strain of T. versicolor. The identity of the recombinant enzyme was checked by Western blot and matrix-assisted laser desorption/ionization time-of-flight analysis. Electrophoresis separation in native conditions indicated a molecular mass of the recombinant protein slightly higher (5 kDa) than that of the mature T. versicolor laccase IIIb, suggesting a limited excess of glycosylation. The laccase production level reached 2.5 mg/l (0.23 units/ml), which is suitable for engineering purpose.The two first authors have contributed equally to this work.