Purification and characterization of two laccase isoenzymes from a ligninolytic fungus Trametes gallica

Constant laccase activities were detected in culture supernatant of newly isolated basidiomycete Trametes gallica. Tryptone and glucose have great effects on the production of laccase. Two laccase isoenzymes (Lac I and Lac II) produced by T. gallica were purified to homogeneity (51- and 50-fold, respectively) by gel filtration chromatography, anion exchange chromatography, and improved native PAGE, with an overall yield of 24.8%. Lac I and Lac II from this fungus are glycoproteins with 3.6% and 4% carbohydrate content, the same molecular masses (by SDS-PAGE) of 60 kDa, and the pI of 3.1 and 3.0, respectively. Native gel electrophoresis indicates that the two laccases have different migration ratios. Lac I and Lac II have the same optimal pH of 3.0 on 2,6-dimethoxyphenol (DMP), pH 2.2 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and of pH 4.0 on guaiacol. The highest rate of ABTS oxidation for both laccases was reached at 70 degrees C. Both laccases are stable from pH 6 to 9, retaining 88-90% activity after 24 hr incubation, and show good stability when incubated at temperatures lower than 40 degrees C. The Km values of Lac I for ABTS, DMP, and guaiacol are 0.118 x 10(-2), 0.420, and 0.405 mM, respectively; the Km values of Lac II for ABTS, DMP, and guaiacol are 0.086 x 10(-2), 0.41, and 0.40 mM, respectively. Their N-terminal sequences are determined and show strong similarity with those from other basidiomycetes. Graphite-furnace atomic absorption analysis revealed that both laccases have four copper atoms per protein molecule, but they have no type I copper signal at around 600 nm and a type III copper signal near 330 nm. Cyanide, azide, and halides completely inhibit the enzyme activity, whereas EDTA has less inhibition.     

Studies of the production and characterization of laccase activity in the basidiomycete Coriolopsis gallica, an efficient decolorizer of alkaline effluents

The basidiomycete Coriolopsis gallica decolorizes alkaline paper effluents efficiently. In this work, we found that C. gallica produces laccase during this decolorization process. This enzymatic activity was produced in all media studied; however, the highest enzymatic activity was obtained in a medium containing paper effluent, where laccase was detected on the 2nd day of the experiment. The laccase activity of C. gallica was purified and characterized. The amino-terminal sequence of this protein showed the highest similarity with the laccase I of the basidiomycete PM1 and with Coriolus hirsutus laccase. Received: 20 April 1998 / Accepted: 21 September 1998     

Characterization and immobilization of the laccase from Pleurotus ostreatus and its use for the continuous elimination of phenolic pollutants

A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity and characterized. The enzyme is a monomeric protein with a molecular weight of 67 000 Da and an isoelectric point of 3.6. Type I and type III Cu(2+) centers were identified by spectrophotometry. With syringaldazine as substrate laccase showed the highest oxidation rates at pH 5.8, 50 degrees C, and in 40 mM phosphate buffer. Among the tested stabilization parameters laccase retained most of its activity in high ionic buffer, pH 10, -20 degrees C, in the presence of 10 mM benzoic acid and with 35% ethylene glycol respectively. Crude laccase was covalently immobilized to Eupergit((R))C. Benzoate was found to stabilize the enzyme during the immobilization process. The activity loss of laccase during 10 days at 25 degrees C storage was 2% on average. Continuous elimination of 2,6-dimethoxyphenol by immobilized laccase was carried out in a packed bed reactor followed by filtration of the formed precipitate. The solubility of the polymerisates of oxidized syringaldazine, o-dianisidine, and 2,6-dimethoxyphenol with respect to temperature, pH-value and organic solvents were examined. The precipitates were found to be insoluble under non-extreme environmental conditions.     

Removal of estrogenic activity from endocrine-disrupting chemicals by purified laccase of Phlebia tremellosa

A white-rot basidiomycete, Phlebia tremellosa, produced a laccase that showed increased activity during degradation of phthalates. A laccase was purified through the ion exchange chromatography and preparative gel electrophoresis, and the estimated molecular weight was 75 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 degrees C, respectively. The K(m) value of the enzyme was 55.7 microM, and the V(max) was 0.0541 OD min(-1) U(-1) for o-tolidine. Purified laccase reduced the estrogenic activity of four different endocrine-disrupting chemicals. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was involved in the removal of estrogenic activity.     

Biochemical and molecular characterization of a laccase from Marasmius quercophilus

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak (Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the "blue copper oxidase" family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75 degrees C, and the K(m) was 7.1 microM. The structural gene, lac1, was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.     

Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum

Ganoderma lucidum, a medicinal white-rot basidiomycete, produces many laccase isozymes in liquid culture. Three laccase isozymes (GaLc 1, 2, 3) have been purified 32.4-fold from the crude enzyme protein through anion exchange chromatography, preparative gel electrophoresis, and electroelution. Their estimated molecular weights are 65-68 kDa, and they contain 7-10% N-linked carbohydrates. The three isozymes have identical N-terminal amino acid sequences: G-I-G-P-T. The optimum pH and temperature both for each isozyme singly and the isozyme mixture are pH 3.5 and 20 degrees C, respectively. One isozyme (GaLc 3) is quite stable at pH 4.0-10.0, and shows good stability when incubated at temperatures lower than 40 degrees C. The Km values of GaLc 3 for o-tolidine and 2,2'-azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) are 401.6 microM and 3.7 microM respectively, and the Vmax of GaLc 3 for these substrates is 0.0198 OD min(-1) unit(-1) and 0.0142 OD min(-1) unit(-1), respectively.     

Purification and characterization of extracellular laccase fromPleurotus ostreatus

Laccase (EC 1.10.3.2) from the culture filtrate of a strain of white rot basidiomycetePleurotus ostreatus was purified using DEAE-Toyopearl 650M and butyl-Toyopearl 650M column chromatographies and Superdex 75 HR 10/30 fast protein liquid chromatography. Molecular weight of the purified laccase was about 55,000, and the isoelectric point was 3.0. The optimum pH for enzyme activity was 6.5, and the optimum temperature was 50°C. This enzyme contained 7.4% sugar and two copper atoms per molecule. The substrate specificity was similar to those of other fungal laccases. Comparison of the N-terminal amino acid sequence of theP. ostreatus laccase with those fromPleurotus ostreatus Florida,Coriolus hirsutus, Phlebia radiata, basidiomycete PM1 (CECT 2971),Trametes villosa, Pycnoporus cinnabarinus, Ceriporiopsis subvermispora, andAgaricus bisporus showed 95, 65, 60, 55, 55, 55, 50, and 35% similarity, respectively, in the first 20 residues. No similarity in this region was detected with laccases fromNeurospora crassa, Aspergillus nidulans, andCryptococcus neoformans.     

A homodimeric laccase with unique characteristics from the yellow mushroom Cantharellus cibarius

The aim of the present study was to isolate a laccase from fruiting bodies of the yellow mushroom Cantharellus cibarius. The fruiting body extract was subjected to a purification protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and Con A-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on Con A-Sepharose. The laccase was composed of two identical subunits each with a molecular mass of 46 kDa. The laccase exhibited a temperature-dependent rise in activity over the temperature range 20-50 degrees C. When the temperature was raised above 60 degrees C there was a fall in enzyme activity. The enzyme manifested maximal activity at pH 4. At and above pH 6 there was a dramatic reduction in activity. The unique features of this fruiting body laccase compared with previously reported mycelial laccases include homodimeric nature, a distinctive N-terminal sequence, a higher optimal pH, and adsorption on only ConA-Sepharose among the various chromatographic media tested.     

Copper induction of lignin-modifying enzymes in the white-rot fungus Trametes trogii

Trametes trogii, a white rot basidiomycete involved in wood decay worldwide, produces several ligninolytic enzymes, laccase being the dominant one, with higher titers than those reported for most other white rot fungi studied up to date. The effect of copper on in vitro production of extracellular ligninolytic activities was studied. CuSO(4)·5H(2)O concentrations from 1.6 μM to 1.5 mM were tested in a synthetic medium with glucose 20 g/L and asparagine 3 g/L. The addition of copper (up to 1 mM) did not affect growth but strongly stimulated ligninolytic enzyme production; faster decolorization of the polymeric dye Poly R-478 was observed as well. Maximal production of manganese peroxidase, laccase, and glyoxal oxidase [1.28 U/mL, 93.8 U/mL (with a specific activity of 720 U/mg protein), and 0.46 U/mL respectively] was attained with 1 mM CuSO(4)·5H(2)O. However, higher copper concentrations inhibited growth and notably decreased manganese peroxidase production, although they did not affect laccase secretion. Laccase activity in the culture filtrate was maximal at 50 C and pH 3.4, and the enzyme was completely stable at pH 4.4 and above, and at 30 C for up to 5 d. Denaturing polyacrylamide gel electrophoresis of extracellular culture fluids showed two laccase activity bands (mol wt 38 and 60 kDa respectively). The pattern of isoenzyme production was not affected by medium composition but differed with culture age.     

Electrochemical characterization of purified Rhus vernicifera laccase: voltammetric evidence for a sequential four-electron transfer

Rhus vernicifera (Rv) laccase was purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A comprehensive study of the direct electrochemistry of Rv laccase covalently immobilized at a gold electrode using alkanethiol monolayers was undertaken. The observed midpoint potential was 410 mV versus the normal hydrogen electrode (NHE), consistent with reduction potentials obtained by potentiometric titration for the T1 copper site. Evidence is presented for a concerted 4-electron reversible process at slow scan rates (v) on the basis of peak current ratios (i(pa)/i(pc)). Catalytic currents were observed in the presence of the biological substrate oxygen, indicating that laccase activity is retained throughout the immobilization process. Electrochemical characteristics of the immobilized laccase were essentially invariant across the pH range 5.5-8.5 and the temperature range 5-35 degrees C. The purified enzyme displayed a pH optimum of 9.0, when assayed spectrophotometrically with syringaldazine as a substrate. Inhibition of the laccase activity with azide or fluoride showed an I(50)(NaN(3)) of 2.5 mM and an I(50)(NaF) of 18.5 mM. Electrochemistry in the presence of azide reduces the anodic current by ca. one-half, consistent with the 4-electron process decreasing to a 2-electron process. However, fluoride has no effect on anaerobic electrochemistry. These electrochemical results suggest that the pH dependence of laccase activity is related to the effects of pH on the structure or binding of the substrate.     

Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity

Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft. In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain. This protein is representative of a new family of enzymes--the two-domain laccases. Disruption of the corresponding gene abrogates laccase activity in the growth media. We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it. The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact. We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family. The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment. The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH. SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules.     

Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces

A novel laccase from the ascomycete Melanocarpus albomyces was purified and characterised. The enzyme was purified using anion exchange chromatography, hydrophobic interaction chromatography and gel filtration, and the purified laccase was biochemically characterised. It had activity towards typical substrates of laccases including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate), dimethoxyphenol, guaiacol, and syringaldazine. The laccase showed good thermostability and it had a pH optimum at neutral pH, both unusual properties for most known fungal laccases. The activity of the laccase from M. albomyces was highest at 60-70 degrees C. With guaiacol and syringaldazine the pH optima were rather broad: 5-7.5 and 6-7, respectively. It retained 50% of its activity after 5 h incubation at 60 degrees C. The molecular weight of the laccase was about 80 kDa and the isoelectric point 4.0. The ultraviolet-visible absorption and electron paramagnetic resonance spectra of the purified laccase indicated that the typical three types of copper were present.     

Biochemical characterization and molecular evidence of a laccase from the bird's nest fungus Cyathus bulleri

Cyathus bulleri, a bird's nest fungus, known to decolorize polymeric dye Poly R-478, was found to produce 8 U ml(-1) of laccase in malt extract broth. Laccase activity appeared as a single band on non-denaturing gel. Laccase was purified to homogeneity by anion exchange chromatography and gel filtration. The enzyme was a monomer with an apparent molecular mass of 60 kD, pI of 3.7 and was stable in the pH range of 2-6 with an optimum pH of 5.2. The optimal reaction temperature was 45 degrees C and the enzyme lost its activity above 70 degrees C. Enzyme could oxidize a broad range of various phenolic substrates. K(m) values for ABTS, 2,6-dimethoxyphenol, guaiacol, and ferulic acid were found to be 48.6, 56, 22, and 14 mM while K(cat) values were 204, 180, 95.6, and 5.2, respectively. It was completely inhibited by KCN, NaN(3), beta-mercaptoethanol, HgCl(2), and SDS, while EDTA had no effect on enzyme activity. The N-terminal amino acid sequence of C. bulleri laccase showed close homology to N-terminal sequences of laccase from other white-rot fungi. A 150 bp gene sequence encoding copper-binding domains I and II was most similar to the sequence encoding a laccase from Pycnoporus cinnabarinus with 74.8% level of similarity.     

Random mutants of a Pleurotus ostreatus laccase as new biocatalysts for industrial effluents bioremediation

Aims: To select better performing laccase variants among the 2300 randomly mutated variants of Pleurotus ostreatus POXA1b laccase to develop improved laccase‐based biocatalysts. Methods and Results: Screening of collections of 2300 randomly mutated variants of POXA1b was performed by assaying activity towards the phenolic substrate 2,6‐dimethoxyphenol. Two new variants endowed with higher enzyme activity than the wild‐type laccase were characterized, and their ability to decolourize industrial dyes with complex trisazo‐, polyazo‐ and stilbene‐type structures, in the absence of mediators, was demonstrated. One of the mutants (2L4A) was also proved to be highly stable at both acidic and alkaline pH values (displaying a half‐life of around 1 month at the pH levels of both 5 and 10). Conclusions: In comparison with the wild‐type laccase, the new selected 2L4A mutant shows a significant increase in stability at acidic pH, whilst storing its high stability at alkaline pH. This variant also represents a more versatile enzyme with respect to both the variety of xenobiotics degraded and the operative conditions. Significance and Impact of the Study: This work represents the first example of improvement of a basidiomycete laccase for industrial effluents bioremediation by directed evolution.     

The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques

The specific activities of crude and purified Coprinus cinereus laccase preparations could be enhanced by a factor of 10-12 by activation with copper ions. The copper to protein contents of purified non-activated laccase were 2.3 ± 0.1 compared to 3.3 ± 0.1 in purified activated laccase indicating that only a fraction of the laccase can be activated. Purified laccase not activated with copper ions shows in isoelectric focusing four bands in order of decreasing pI in a ratio 1/5/3/1 where only bands I and II had laccase activity. Purified activated laccase showed only three bands (I, II and III) in the ratio 5/4/1 all with some laccase activity. The pH profile of the activity for activated and non-activated laccase showed identical behavior indicating that the active forms were the same. The change in UV-Vis around 330 nm following the depletion and reconstitution of the enzyme combined with activity measurements supports the reversibility of the selective removal and insertion of copper ions at the type 2 site. The circular dichroism spectrum of activated purified laccase has characteristic changes around 350 nm relative to non-activated laccase indicative of changes at the type 2/type 3 sites. The difference between the electron paramagnetic resonance spectra of non-activated and activated C. cinereus laccase indicates that a fraction of the non-activated purified laccase contained a copper(II) signal with a coupling constant between a type 1 and a type 2 copper(II). This electron paramagnetic resonance signal could be explained by an induced asymmetry in the type 3 site due to a missing type 2 copper ion.     

Purification and characterization of laccase from basidiomycete Fomitella fraxinea

A laccase was isolated from the culture filtrate of basidiomycete Fomitella fraxinea. The enzyme was purified to electrophoretical homogeneity using ammonium sulfate precipitation, anion-exchange chromatography, and gel-filtration chromatography. The enzyme was identified a monomeric protein with a molecular mass of 47 kDa sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography, and had an isoelectric point of 3.8. The N-terminal amino acid sequence for the enzyme was ATXSNXKTLAAD, which had a very low similarity to the sequences previously reported for laccases from other basidiomycetes. The optimum and temperature for 2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulfonate) (ABTS) were 3.0 and 70 degrees C, respectively. The enzyme also showed a much higher level of specific activity for ABTS and 2,6-dimethoxyphenol (DMP), where the values of the enzyme for ABTS and 2,6-DMP were 270 and 426 microM, respectively, and the Vmax values were 876 and 433.3 microM/min, respectively. The laccase activity was completely inhibited by L-cysteine, dithiothreitol (DTT), and sodium azide, significantly inhibited by Ni+, Mn+ and Ba+2, and slightly stimulated by K+ and Ca+2.     

Degradation of bisphenol A by purified laccase from Trametes villosa

Degradation of bisphenol A (BPA), an endocrine-disrupting chemical, was studied with a purified laccase from the basidiomycete Trametes villosa. SDS--polyacrylamide gel electrophoresis of the purified laccase gave one single band with a mobility corresponding to MW 65 kDa. The absorption spectrum showed the characteristics of a blue copper protein with a maximum peak at 600 nm. HPLC analysis revealed that 2.2 micromol BPA were degraded by incubation with 1.5 units of the purified laccase in a total volume of 1.0 ml at pH 6.0 and 60 degrees C for 3 h. The enzyme reaction proceeded rapidly without requirement of mediators for the electron transfer. Isolation and identification of several reaction products are in progress, in which one product was identified as 4-isopropenylphenol by a gas chromatography--mass spectrophotometer.     

Biochemical and Molecular Characterization of a Laccase from Marasmius quercophilus

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak (Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the “blue copper oxidase” family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75°C, and the K_m was 7.1 μM. The structural gene, lac1, was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.