Increased production of extracellular laccase by the white rot fungus Coriolus versicolor MTCC 138

Summary The white rot fungus Coriolus versicolor MTCC 138 has been identified as an excellent producer of the industrially important enzyme laccase. The basal medium containing glucose gave laccase activity of 155 U/ml. Screening of different media components and their effects on laccase production by Coriolus versicolor was studied using one factor at a time and L₉ (3⁴) orthogonal array method. A two-fold increase (305 U/ml) in laccase production was observed using a combination of glucose and starch as carbon source and yeast extract as nitrogen source. This activity is very high as compared to most of the reported strains. Hence this strain was explored for enhancement in laccase. The formation of extracellular laccase could be considerably stimulated by the addition of copper in the optimized medium. Addition of 1 mM copper enhanced laccase activity to 460 U/ml. Laccase production was further enhanced using different aromatic inducers. Among different inducers used 2,5-xylidine was found to be the best, and gave maximum laccase activity of 820 U/ml with 1 mM concentration. Thus, this strain could be an efficient and attractive source for laccase production.     

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.     

Laccase component of the Ceriporiopsis subvermispora lignin-degrading system.

Laccase activity in the lignin-degrading fungus Ceriporiopsis subvermispora was associated with several proteins in the broth of cultures grown in a defined medium. Activity was not increased significantly by adding 2,5-xylidine or supplemental copper to the medium. Higher activity, associated with two major isoenzymes, developed in cultures grown on a wheat bran medium. These two isoenzymes were purified to homogeneity. L1 and L2 had isoelectric points of 3.4 and 4.8, molecular masses of 71 and 68 kDa, and approximate carbohydrate contents of 15 and 10%, respectively. Data indicated 4 copper atoms per mol. L1 and L2 had overlapping pH optima in the range of 3 to 5, depending on the substrate, and exhibited half-lives of 120 and 50 min at 60 degrees C. They were strongly inhibited by sodium azide and thioglycolic acid but not by hydroxylamine or EDTA. The isoenzymes oxidized 1,2,4,5-tetramethoxybenzene but not other methoxybenzene congeners. A variety of usual laccase substrates, including lignin-related phenols and ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)], were also oxidized. Kinetic parameters were similar to those of the laccases of Coriolus versicolor. The N-terminal amino acid sequence (20 residues for L1) showed significant homology to those of laccases of other white rot basidiomycetes but not to those of the laccases of Agaricus bisporus or Neurospora crassa.     

Enhanced production of laccase fromTrametes sp. by combination of various inducers

In this study, we have attempted to determine the optimum concentration of inducers responsible for efficient laccase production by the white-rot fungus,Trametes sp. Variations in laccase activity were investigated with changing concentrations of 2,5-xylidine, syringaldazine, ABTS, and guaiacol. Enhancement of peak laccase activity was achieved via the combination of 2,5-xylidine with ABTS, syringaldazine, or guaiacol, resulting in increases of up to 359, 313, and 340%, respectively, as compared to control values. Among the tested inducers, the addition of 0.1 mM of ABTS coupled with 1.0 mM of 2,5-xylidine in the medium after 24 h of cultivation proved optimal with regard to laccase enzyme production.     

Laccase induction in fungi and laccase/N-OH mediator systems applied in paper mill effluent

There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent.     

Docking Simulation and Competitive Experiments Validate the Interaction Between the 2,5-Xylidine Inhibitor and Rigidoporus lignosus Laccase

Abstract

Laccases are polyphenol oxidases which oxidize a broad range of reducing substrates, preferably phenolic compounds, and their use in biotechnological applications is increasing.

Recently, the first X-ray structure of active laccase from white rot fungus Rigidoporus lignosus has been reported containing a full complement of copper ions. Comparison among selected fungal laccases of known 3D structure has shown that the Rigidoporus lignosus laccase has a very high similarity with the Trametes versicolor laccase that, being co-crystallized with 2,5-xylidine, shows a well defined binding pocket for the substrate. Global sequence alignment between Rigidoporus lignosus and Trametes versicolor laccases shows 73% of identity but, surprisingly, there is no identity and neither conservative substitutions between the residues composing the loops directly contacting the 2,5-xylidine. Moreover the structural alignment of these two enzymes identifies in these loops a striking structural similarity proposing the question if 2,5-xylidine may bind in same enzyme pocket.

Here we report the protein-ligand docking simulation of 3D structure of Rigidoporus lignosus laccase and 2,5-xylidine. Docking simulation analyses show that spatial conformation of the two 2,5-xylidine binding pockets, despite differences in the residues directly contacting the ligand, may arrange a similar pocket that allows a comparable accommodation of the inhibitor. To validate these results the binding of 2,5-xylidine in the substrate cavity has been confirmed by kinetic competitive experiments.     

Oligomeric compounds formed from 2,5-xylidine (2,5-dimethylaniline) are potent enhancers of laccase production in Trametes versicolor ATCC 32745

Numerous chemicals, including the xenobiotic 2,5-xylidine, are known to induce laccase production in fungi. The present study was conducted to determine whether the metabolites formed from 2,5-xylidine by fungi could enhance laccase activity. We used purified laccases to transform the chemical and then we separated the metabolites, identified their chemical structure and assayed their effect on enzyme activity in liquid cultures of Trametes. versicolor. We identified 13 oligomers formed from 2,5-xylidine. (4E)-4-(2,5-dimethylphenylimino)-2,5-dimethylcyclohexa-2,5-dienone at 1.25×10^–5 M was an efficient inducer, resulting in a nine-fold increase of laccase activity after 3 days of culture. Easily synthesized in one step (67% yield), this compound could be used in fungal bioreactors to obtain a great amount of laccases for biochemical or biotechnological purposes, with a low amount of inducer.     

Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum

Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (M(r) of 40,000 and 66, 000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity ( approximately 100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.     

Demonstration of Laccase in the White Rot Basidiomycete Phanerochaete chrysosporium BKM-F1767

It has been widely reported that the white rot basidiomycete Phanerochaete chrysosporium, unlike most other white rot fungi, does not produce laccase, an enzyme implicated in lignin biodegradation. Our results showed that P. chrysosporium BKM-F1767 produces extracellular laccase in a defined culture medium containing cellulose (10 g/liter) and either 2.4 or 24 mM ammonium tartrate. Laccase activity was demonstrated in the concentrated extracellular culture fluids of this organism as determined by a laccase plate assay as well as a spectrophotometric assay with ABTS [2,2(prm1)-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] as the substrate. Laccase activity was observed even after addition of excess catalase to the extracellular culture fluid to destroy the endogenously produced hydrogen peroxide, indicating that the observed activity is not due to a peroxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by activity staining with ABTS revealed the presence of a laccase band with an estimated M(infr) of 46,500.     

Lignin-Modifying Enzymes of the White Rot Basidiomycete Ganoderma lucidum

Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (M_r of 40,000 and 66,000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity (~100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.     

The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase.

The white rot fungus Pycnoporus cinnabarinus was characterized with respect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with low amounts of an unusual peroxidase. Neither lignin peroxidase nor manganese peroxidase was detected. Laccase was produced constitutively during primary metabolism. Addition of the most effective inducer, 2,5-xylidine, enhanced laccase production ninefold without altering the isoenzyme pattern of the enzyme. Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,000 Da, as determined by calibrated gel filtration chromatography, and a carbohydrate content of 9%. The enzyme displayed an unusual behavior on isoelectric focusing gels; the activity was split into one major band (pI, 3.7) and several minor bands of decreasing intensity which appeared at regular, closely spaced intervals toward the alkaline end of the gel. Repeated electrophoresis of the major band under identical conditions produced the same pattern, suggesting that the laccase was secreted as a single acidic isoform with a pI of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This appeared to be confirmed by N-terminal amino acid sequencing of the purified enzyme, which yielded a single sequence for the first 21 residues. Spectroscopic analysis indicated a typical laccase active site in the P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indicated the enzyme to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus laccase showed close homology to the N-terminal sequences determined for laccases from Trametes versicolor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The principal features of the P. cinnabarinus enzyme system, a single predominant laccase and a lack of lignin- or manganese-type peroxidase, make this organism an interesting model for further studies of possible alternative pathways of lignin degradation by white rot fungi.     

Phlebia radiata laccase forms induced by veratric acid and xylidine in relation to lignin peroxidase and manganese-dependent peroxidase

White-rot basidiomycete Phlebia radiata grown in the nitrogen-poor liquid medium was tested for the inducing effect of 2,5-xylidine and veratric acid on laccase production and properties of its preparations. Also lignin peroxidase and manganese-dependent peroxidase were assayed under the same conditions. The maximum of laccase activity for veratric acid as a stimulator was done much earlier than that of xylidine. It was very sharp and disappeared quickly. At that time only weak lignin peroxidase and manganese-dependent peroxidase activities were noticed. The maximum of laccase induced by xylidine was observed much later and kept longer. Both laccase preparations showed the same properties. For biotechnological reasons, the production of laccase induced by the nontoxic veratric acid is much more economic and better acceptable than that induced by xylidine.     

Characterisation of the diol dehydratase pdu operon of Lactobacillus collinoides

Addition of copper (0.5-5 mM) or cadmium (1-5 mM) to the white rot fungus Pleurotus ostreatus cultivated in liquid nitrogen-limited medium for 12 days increased the activity of laccase. The addition of 2 mM Cd led to an 18.5-fold increase of activity, 1 mM Cu increased the activity eight-fold. When added earlier than 12 days, the activation of laccase was delayed (Cu) or decreased (Cd). Ag, Hg, Pb, Zn, and H(2)O(2) decreased laccase activity. To study the effect on native enzymes, purified laccase was incubated with Cd, Cu, and Hg. The addition of Hg decreased the activity of laccase immediately and reduced the temporal stability of the enzyme, while the addition of Cu (0.05-50 mM) increased both enzyme activity and stability. Laccase extracted at different stages of straw colonisation differed in its response to heavy metals.     

The role of the ubiquitin-proteasome system in the response of the ligninolytic fungus Trametes versicolor to nitrogen deprivation

The white rot fungus Trametes versicolor is an efficient lignin degrader with ecological significance and industrial applications. Lignin-modifying enzymes of white rot fungi are mainly produced during secondary metabolism triggered in these microorganisms by nutrient deprivation. Selective ubiquitin/proteasome-mediated proteolysis is known to play a crucial role in the response of cells to various stresses such as nutrient limitation, heat shock, and heavy metal exposure. Previous studies from our laboratory demonstrated that proteasomal degradation of intracellular proteins is involved in the regulation of laccase, a major ligninolytic enzyme of T. versicolor, in response to cadmium. In the present study, it was found that the 6-h nitrogen starvation leads to depletion of intracellular free ubiquitin pool in T. versicolor. The difference in the intracellular level of free monomeric ubiquitin observed between the mycelium extract from the nitrogen-deprived and that from the nitrogen-sufficient culture was accompanied by the different pattern of ubiquitin-dependent degradation. Furthermore, it was found that nitrogen deprivation affected 26S proteasome activities of T. versicolor. Proteasome inhibition by lactacystin beta-lactone, a highly specific agent, increased laccase activity in nitrogen-deprived cultures, but not in nitrogen-sufficient cultures. The present study implicates the ubiquitin/proteasome-mediated proteolytic pathway in the response of T. versicolor to nitrogen deprivation.     

Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha

The hard wood-colonizing ascomycete Xylaria polymorpha, that is seemingly lacking peroxidases, produces laccase as sole ligninolytic oxidoreductase. The fungus secreted the enzyme preferably during the growth in complex media based on tomato juice. Addition of 2,5-xylidine considerably stimulated laccase production (up to 14,000 U l⁻¹). The enzyme was purified to homogeneity by anion exchange and size exclusion chromatography and characterized by biochemical and molecular methods. Xylaria laccase has a molecular mass of 67 kDa, a pI of 3.1 and an absorption maximum at 605 nm that is characteristic for blue copper proteins. It oxidized all typical laccase substrates including ABTS, 2,6-dimethoxyphenol, guaiacol as well as syringaldazine (catalytic efficiencies 3 × 10³ to 7 × 10⁴ M⁻¹ s⁻¹). The deduced amino acid sequence of one amplified laccase gene sequence between the copper binding regions 1 and 3 showed a high level of identity to some other laccases from ascomycetes. Furthermore, the sequence of an internal peptide fragment of the purified laccase was identical with an amino acid sequence deduced from the nucleotide sequence of the laccase gene. Xylaria laccase was found to oxidize a non-phenolic β-O-4 lignin model compound in presence of 1-hydroxybenzotriazole into the corresponding keto-form. The results of this study show that – in addition to ligninolytic basidiomycetes – also wood-dwelling ascomycetes can produce high titers of laccase that may be involved in the oxidation of lignin.     

Extracellular Enzyme Production and Synthetic Lignin Mineralization by Ceriporiopsis subvermispora

The ability of the white rot fungus Ceriporiopsis subvermispora to mineralize ¹⁴C-synthetic lignin was studied under different culture conditions, and the levels of two extracellular enzymes were monitored. The highest mineralization rates (28% after 28 days) were obtained in cultures containing a growth-limiting amount of nitrogen source (1.0 mM ammonium tartrate); under this condition, the levels of manganese peroxidase (MnP) and laccase present in the culture supernatant solutions were very low compared with cultures containing 10 mM of the nitrogen source. In contrast, cultures containing a limiting concentration of the carbon source (0.1% glucose) showed low levels of both enzymes and also very low mineralization rates compared with cultures containing 1% glucose. Cultures containing 11 ppm of Mn(II) showed a higher rate of mineralization than those containing 0.3 or 40 ppm of this cation. Levels of MnP and laccase were higher when 40 ppm of Mn(II) was used. Mineralization rates were slightly higher in cultures flushed daily with oxygen, whereas laccase levels were lower and MnP levels were approximately the same as in cultures maintained under an air atmosphere. The presence of 0.4 mM veratryl alcohol reduced both mineralization rates and MnP levels, without affecting laccase levels. Lignin peroxidase activity was not detected under any condition. Addition of purified lignin peroxidase to the cultures in the presence or absence of veratryl alcohol did not enhance mineralization rates significantly.     

Controversial role of fungal laccases in decreasing the antibacterial effect of olive mill waste-waters

Antibacterial diffusion tests (against Bacillus megaterium) detected both bacterial growth-promoting and growth-inhibiting components in olive mill waste-water (OMW). Mixtures of OMW aromatic components showed antibacterial effects that did not show antibacterial activity when tested as individual compounds. Strains of white rot fungi (WRF) producing different patterns of lignin modifying enzymes (LMEs) have been evaluated for OMW remediation under nutritional conditions affecting the LMEs produced. The decrease of both the content in OMW phenols and in the OMW antibacterial activity was compared with fungal growth and LMEs production. OMW addition to the cultures increased fungal growth and laccase activity irrespectively of the nutritional conditions of the cultures. Laccase was the sole LME activity that increased after OMW addition to the cultures. Neither the increased growth of WRF in OMW-containing cultures, their content in laccase nor the amount of OMW phenols were direct indications of a greater decrease in OMW antibacterial effect. The higher decrease in OMW antibacterial activity was obtained in cultures of Phanerochaete flavido-alba in an N-rich media.