Cloning and expression of a cDNA encoding the laccase from Schizophyllum commune

We cloned and analyzed the nucleotide sequence of a cDNA that encodes polyphenol oxidase (laccase) from the white-rot basidiomycete Schizophyllum commune. The nucleotide sequence of the full-length cDNA contained a 1554-base open reading frame that encoded a polypeptide of 518 amino acid residues, including a putative signal peptide of 16 residues. It contained four highly similar regions that are conserved in the deduced amino acid sequences of other laccases, including the region thought to be involved in copper binding. Aspergillus sojae strain 1860 (which has low protease levels) was transformed with the plasmid lacAL/pTPT, which contained the laccase gene under the control of the tannase promoter from Aspergillus oryzae. Laccase was secreted into the medium when transformants A1 and A2 were cultured in tannic acid-containing medium.     

Degradation of endocrine disrupting chemicals by genetic transformants with two lignin degrading enzymes in <Emphasis Type="Italic">Phlebia tremellosa</Emphasis>

A white rot fungus Phlebia tremellosa produced lignin degrading enzymes, which showed degrading activity against various recalcitrant compounds. However, manganese peroxidase (MnP) activity, one of lignin degrading enzymes, was very low in this fungus under various culture conditions. An expression vector that carried both the laccase and MnP genes was constructed using laccase genomic DNA of P. tremellosa and MnP cDNA from Polyporus brumalis. P. tremellosa was genetically transformed using the expression vector to obtain fungal transformants showing increased laccase and MnP activity. Many transformants showed highly increased laccase and MnP activity at the same time in liquid medium, and three of them were used to degrade endocrine disrupting chemicals. The transformant not only degraded bisphenol A and nonylphenol more rapidly but also removed the estrogenic activities of the chemicals faster than the wild type strain.     

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.     

Biodegradation of endocrine-disrupting bisphenol A by white rot fungus Irpex lacteus

Biodegradation of endocrine-disrupting bisphenol A was investigated with several white rot fungi (Irpex lacteus, Trametes versicolor, Ganoderma lucidum, Polyporellus brumalis, Pleurotus eryngii, Schizophyllum commune) isolated in Korea and two transformants of T versicolor (strains MrP 1 and MrP 13). I. lacteus degraded 99.4% of 50 mg/l bisphenol A in 3 h incubation and 100% in 12 h incubation. which was the highest degradation rate among the fungal strains tested. T. versicolor degraded 98.2% of 50 mg/l bisphenol A in 12 h incubation. Unexpectedly, the transformant of the Mn-repressed peroxidase gene of T. versicolor, strain MrP 1, degraded 76.5% of 50 mg/l bisphenol A in 12 h incubation, which was a lower degradation rate than wild-type T. versicolor. The removal of bisphenol A by I. lacteus occurred mainly by biodegradation rather than adsorption. Optimum carbon sources for biodegradation of bisphenol A by I. lacteus were glucose and starch, and optimum nitrogen sources were yeast extract and tryptone in a minimal salts medium; however, bisphenol A degradation was higher in nutrient-rich YMG medium than that in a minimal salts medium. The initial degradation of endocrine disruptors was accompanied by the activities of manganese peroxidase and laccase in the culture     

Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the ascomycete Melanocarpus albomyces

The lac1 gene encoding an extracellular laccase was isolated from the thermophilic fungus Melanocarpus albomyces. This gene has five introns, and it encodes a protein consisting of 623 amino acids. The deduced amino acid sequence of the laccase was shown to have high homology with laccases from other ascomycetes. In addition to removal of a putative 22-amino-acid signal sequence and a 28-residue propeptide, maturation of the translation product of lac1 was shown to involve cleavage of a C-terminal 14-amino-acid extension. M. albomyces lac1 cDNA was expressed in Saccharomyces cerevisiae under the inducible GAL1 promoter. Extremely low production was obtained with the expression construct containing laccase cDNA with its own signal and propeptide sequences. The activity levels were significantly improved by replacing these sequences with the prepro sequence of the S. cerevisiae alpha-factor gene. The role of the C-terminal extension in laccase production in S. cerevisiae was also studied. Laccase production was increased sixfold with the modified cDNA that had a stop codon after the native processing site at the C terminus.     

Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-A resolution containing a full complement of coppers

Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccases oxidize a broad range of substrates, preferably phenolic compounds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Until now, only one crystal structure of a laccase in an inactive, type-2 copper-depleted form has been reported. We present here the first crystal structure of an active laccase containing a full complement of coppers, the complete polypeptide chain together with seven carbohydrate moieties. Despite the presence of all coppers in the new structure, the folds of the two laccases are quite similar. The coordination of the type-3 coppers, however, is distinctly different. The geometry of the trinuclear copper cluster in the Trametes versicolor laccase is similar to that found in the ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for the copper oxidation and the O(2) reduction. In contrast to most blue copper proteins, the type-1 copper in the T. versicolor laccase has no axial ligand and is only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.     

Oxidation of Anthracene and Benzo[a]pyrene by Laccases from Trametes versicolor

The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of <=7.45 eV, is catalyzed by laccases from Trametes versicolor. Crude laccase preparations were able to oxidize both anthracene and the potent carcinogen benzo[a]pyrene. Oxidation of benzo[a]pyrene was enhanced by the addition of the cooxidant 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), while an increased anthracene oxidizing ability was observed in the presence of the low-molecular-weight culture fluid ultrafiltrate. Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. Oxidation of anthracene by the purified isozymes was enhanced in the presence of ABTS, while ABTS was essential for the oxidation of benzo[a]pyrene. In all cases anthraquinone was identified as the major end product of anthracene oxidation. These findings indicate that laccases may have a role in the oxidation of polycyclic aromatic hydrocarbons by white rot fungi.     

Degradation of endocrine disrupting chemicals by genetic transformants in Irpex lacteus with an inducible laccase gene of Phlebia tremellosa

Irpex lacteus was genetically transformed using an laccase expression vector to get increased laccase producing strains. Stable integration of the vector was confirmed by PCR using the vector-specific primers, and the transformants showed increased laccase activities. When the transformants were grown with several endocrine disrupting chemicals, laccase activity of each transformant was induced up to six times higher than that of the wild type. They showed increased degrading activities against EDCs as well as increased removal rates of estrogenic activities generated by the EDCs than the wild type, and the laccase expression was increased during the degradations of the EDCs.     

Cloning and sequencing of a second laccase gene from the white-rot fungus Coriolus versicolor

A second laccase gene, CVLG1, was isolated from Coriolus versicolor. CVLG1 encodes a precursor protein of 526 amino acids which contains a 23-amino acid signal sequence, and the coding region is interrupted by 11 introns. The number of potential N-glycosylation sites in this product is 12 and the greatest among that of polyporales laccases. Moreover, this protein shares about 70% homology with other polyporales laccases. Genomic Southern analysis showed that C. versicolor laccases are encoded by more than four genes including CVLG1 and a transposed allele of this gene.     

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR.

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases.     

Preparation and characterization of cross-linked laccase aggregates and their application to the elimination of endocrine disrupting chemicals

Laccase from the white rot fungus Coriolopsis polyzona was immobilized for the first time through the formation of cross-linked enzyme aggregates (CLEAs). Laccase CLEAs were produced by using 1000g of polyethylene glycol per liter of enzyme solution as precipitant and 200muM of glutaraldehyde as a cross-linking agent. These CLEAs had a laccase activity of 148Ug(-1) and an activity recovery of 60.2% when using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate. CLEAs formed by co-aggregation with bovine serum albumin (BSA) as a stabilizer showed lower laccase activity and affinity for ABTS than those without BSA. The CLEAs co-aggregated with BSA showed higher residual activity against a protease, NaN(3), EDTA, methanol and acetone. The thermoresistance was higher for CLEAs than for free laccase and also higher for CLEAs co-aggregated with BSA than for simple CLEAs when tested at a pH of 3 and a temperature of 40 degrees C. Finally, laccase CLEAs were tested for their capacity to eliminate the known or suspected endocrine disrupting chemicals (EDCs) nonylphenol, bisphenol A and triclosan in a fluidized bed reactor. A 100-ml reactor with 0.5mg of laccase CLEAs operated continuously at a hydraulic retention time of 150min at room temperature and pH 5 could remove all three EDCs from a 5mgl(-1) solution.     

Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus.

The gene (pox1) encoding a phenol oxidase from Pleurotus ostreatus, a lignin-degrading basidiomycete, was cloned and sequenced, and the corresponding pox1 cDNA was also synthesized and sequenced. The isolated gene consists of 2,592 bp, with the coding sequence being interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA consensus sequences could be identified at positions -174 and -84, respectively. The isolation of a second cDNA (pox2 cDNA), showing 84% similarity, and of the corresponding truncated genomic clones demonstrated the existence of a multigene family coding for isoforms of laccase in P. ostreatus. PCR amplifications of specific regions on the DNA of isolated monokaryons proved that the two genes are not allelic forms. The POX1 amino acid sequence deduced was compared with those of other known laccases from different fungi.     

Phylogenetic and biochemical characterisation of a recombinant laccase from Trametes versicolor

Laccases are important enzymes for bioremediation and the best-characterised are from the fungus Trametes versicolor. Here, we describe the cloning and characterisation of a new variant of laccase from T. versicolor and its expression in Saccharomyces cerevisiae. We have performed a sequence-based analysis of Trametes laccases that leads to a proposal for a new nomenclature of this fungus laccases according to their phylogenetic relationships since their nomenclature based on IPs is ambiguous. We also describe the kinetic properties of the recombinant enzyme.     

Purification of a novel extracellular laccase from solid-state culture of the edible mushroom <Emphasis Type="Italic">Lentinula edodes</Emphasis>

The laccases (EC 1.10.3.2) secreted into solid-state culture by Lentinula edodes were analyzed. The fungus secreted at least two laccases in the solid-state culture. One laccase was purified to a homogeneous preparation using anion-exchange, hydrophobic, and size-exclusion chromatography. SDS-PAGE analysis showed that the purified laccase, Lcc6, was a monomeric protein of 58.5 kDa. The optimum pH for enzyme activity was about 3.5, and the laccase was most active at 40°C. The N-terminal amino acid sequence of Lcc6 did not correspond to the sequence of Lcc1, which was previously purified from L. edodes. Lcc6 had decolorization activity to some chemical dyes.