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This presentation demonstrates that a laccase from the thermophilic fungus Melanocarpus albomyces binds effectively to lignocellulose, Avicel and bacterial microcrystalline cellulose (BMCC). Binding to cellulose has previously been demonstrated for enzymes involved in the modification of lignocellulose, however, the adsorption of a laccase on pure cellulose has not been reported before. Binding on lignin was also studied but no adsorption on alkali lignin was observed. A binding isotherm was established with bacterial microcrystalline cellulose. The data points fitted the Langmuir type one-site binding model well, and the binding parameters for maximum binding capacity (Bmax) and relative partition coefficient (Kp) were calculated from the isotherm. The Bmax of 1.94 mol/g was relatively low, as compared to the values for various cellulases, whereas the Kp of 320 1/g, which describes the binding affinity, was clearly higher than the values reported for cellulases. The combination of high affinity and quite low capacity of binding suggests that M. albomyces laccase is able to bind very effectively to BMCC, but only on relatively few binding sites. Furthermore, the binding was shown to be reversible and not influenced by non-specific, protein or 0.1-0.5 M Na2SO4. The binding of T. hirsuta and Mauginiella sp. laccases was also studied. No adsorption was detected on BMCC or softwood, suggesting that adsorption onto cellulose is not a general feature among laccases.

Previous studies onMelanocarpus albomyceslaccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungusTrichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin–laccase fusion protein. Both proteins were expressed inT. reeseiunder thecbh1promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l−1). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin–laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l−1. The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.

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 α-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.

We have crystallized the ascomycete laccase from Melanocarpus albomyces with all four coppers present and determined the crystal structure at 2.4 Å resolution. The enzyme is heavily glycosylated and consists of three cupredoxin-like domains, similar to those found in the Cu-depleted basidiomycete laccase from Coprinus cinereus. However, there are significant differences in the loops forming the substrate-binding pocket. In addition, the crystal structure of the M. albomyces laccase revealed elongated electron density between all three coppers in the trinuclear copper site, suggesting that an oxygen molecule binds with a novel geometry. This oxygen, required in the reaction, may enter the trinuclear site through the tunnel, which is open in the structure of the C. cinereus laccase. In contrast, the C-terminus on the M. albomyces laccase forms a plug that blocks this access.

Aims: To discover novel laccases potential for industrial applications. Methods and Results: Fungi were cultivated on solid media containing indicator compounds that enabled the detection of laccases as specific colour reactions. The indicators used were Remazol Brilliant Blue R (RBBR), Poly R-478, guaiacol and tannic acid. The screening work resulted in isolation of 26 positive fungal strains. Liquid cultivations of positive strains confirmed that four efficient laccase producers were found in the screening. Biochemical characteristics of the four novel laccases were typical for fungal laccases in terms of molecular weight, pH optima and pI. The laccases showed good thermal stability at 60°C. Conclusions: Plate-test screening based on polymeric dye compounds, guaiacol and tannic acid is an efficient way to discover novel laccase producers. The results indicated that screening for laccase activity can be performed with guaiacol and RBBR or Poly R-478. Significance and Impact of the Study: Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp and effluent detoxification. It is essential to find novel, efficient enzymes to further develop these applications. This study showed that relatively simple plate test screening method can be used for discovery of novel laccases.

A novel laccase from the ascomycete Melanocarpus albomyces was isolated, purified and characterized. The ultraviolet-visible absorption and electron paramagnetic resonance spectra indicated that the three types of coppers were present. Redox potential of the T1 copper of M. albomyces laccase was determined to be 0.46±0.01 V. The enzyme had very interesting pH and temperature behaviour. It had a pH optimum measured with guaiacol at neutral and slightly alkalic pH and substantial activity still at pH 8. The laccase showed very good thermostability, retaining full activity for two hours at 60°C. The gene encoding the M. albomyces laccase was isolated and sequenced. The length of the open reading frame of the M. albomyces laccase was 623 amino acid residues. The copper binding residues were well conserved and the amino acid sequence had high homology to other ascomycete laccases. Interestingly the secreted laccase was processed both from the amino and carboxy terminus. The laccase was also crystallized with all four coppers present.

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.