Characterization and heterologous production of a novel laccase from Melanocarpus albomyces: Dissertation

Laccases (EC 1.10.3.2) are multicopper oxidases that catalyze oxidation of various substituted phenolic compounds, aromatic amines and even certain inorganic compounds by using molecular oxygen as the electron acceptor. Their substrate versatility makes laccases highly interesting for various applications, including textile dye bleaching, pulp bleaching and bioremediation, where enzymatic catalysis could serve as a more environmentally benign alternative than the currently used chemical processes. However, most laccases studied thus far are not well-suited for the applications because of their low stability at high temperatures or pH values. This work focused on identifying and characterizing novel fungal laccases having potential for the applications as well as on development of efficient production methods for laccases. Laccase-producing fungi were screened from various environmental samples by plate tests using the indicator compounds guaiacol, tannic acid and the polymeric dyes Remazol brilliant blue R and Poly R-478. A total of 26 positive fungal strains were isolated, and their ability to produce laccase was studied in liquid media. Four fungal strains produced significant amounts of laccase, and these enzymes were preliminarily characterized. The novel laccases were found to be rather typical basidiomycete laccases, although they had notably high thermostabilities as compared to other fungal laccases. A novel laccase from the ascomycete Melanocarpus albomyces was purified and biochemically characterized. The substrate specificity and susceptibility towards inhibitors were shown to be typical for laccases. Spectral data measured for the purified laccase indicated that the characteristic three types of copper were present. Interestingly, M. albomyces laccase showed good thermostability and it had a pH optimum at neutral pH with phenolic substrates. Both of these are unusual properties for fungal laccases. The crystal structure of M. albomyces laccase containing all four copper atoms was resolved at 2.4 Å resolution. The overall structure was shown to consist of three cupredoxin-like domains, similarly to other blue copper oxidases. Surprisingly, elongated electron density was observed in the trinuclear center, indicating binding of a dioxygen molecule with a novel geometry. In addition, an exceptional C-terminal end, which protrudes into the active site of the enzyme, was detected. The gene encoding M. albomyces laccase was isolated and it was shown to encode a protein of 623 amino acids. The level of homology of the laccase was about 60-70% with laccases from other ascomycetes and about 30% with basidiomycete laccases. Maturation of M. albomyces laccase was shown to consist of the removal of a putative signal sequence, a propeptide and a C-terminal extension. M. albomyces laccase cDNA was expressed in Saccharomyces cerevisiae under the inducible GAL1 promoter. Very low laccase production was detected with the expression construct containing laccase cDNA with its own signal and propeptide sequences. The production was significantly improved by replacing these with the prepro-sequence of the S. cerevisiae -factor gene. Further six-fold improvement in the production level was obtained by introducing a stop codon into the cDNA after the native C-terminal processing site. These results suggested that correct post-translational processing was essential for efficient production of M. albomyces laccase in S. cerevisiae. M. albomyces laccase was also expressed in the filamentous fungus Trichoderma reesei. The laccase was expressed as a non-fused laccase and as a fusion protein that contained the T. reesei hydrophobin I protein at the N-terminus. About five times higher activity levels were obtained with the non-fused laccase than with the fusion protein in shake flask cultures. Analyses of transformants from both expression constructs indicated that production of the fusion protein was limited at the post-transcriptional level by proteolytic degradation and inefficient secretion. No induction of the unfolded response pathway by laccase production was detected in the transformants. The unmodified recombinant M. albomyces laccase was produced in batch and fed-batch fermentations and the production level of 920 mg l-1 in the fed-batch cultivation was the highest heterologous laccase production level hitherto reported. Recombinant M. albomyces laccase was purified and biochemically characterized and it was shown to be very similar to the native laccase. This work also showed for the first time that a laccase can adsorb on cellulose, as M. albomyces laccase was shown to bind to lignocellulose and purified cellulose. The binding isotherm obtained with bacterial microcrystalline cellulose fitted well the Langmuir type one-site binding model. The adsorption parameters obtained from the model indicated that M. albomyces laccase binds to cellulose very efficiently but with a relatively low binding capacity. The binding was shown to be reversible and not influenced by non-specific protein or the presence of salt. No binding was detected with laccases from Trametes hirsuta or Mauginiella sp., which suggests that binding to cellulose is not a common feature among laccases.