Open Access Highly Accessed Research article

Exploring laccase-like multicopper oxidase genes from the ascomycete Trichoderma reesei: a functional, phylogenetic and evolutionary study

Anthony Levasseur12*, Markku Saloheimo3, David Navarro12, Martina Andberg3, Pierre Pontarotti4, Kristiina Kruus3 and Eric Record12

Author Affiliations

1 UMR-1163, INRA de Biotechnologie des Champignons Filamenteux, IFR86-BAIM. Universités de Provence et de la Méditerranée, ESIL, 163 avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France

2 Universités Aix-Marseille 1 et 2, UMR-1163, 163 avenue de Luminy, CP925, 13288 Marseille Cedex 09, France

3 VTT Biotechnology: VTT Technical Research Centre of Finland, P.O. Box 1500, Tietotie 2, 02044 VTT Espoo Finland

4 Evolution Biologique et Modélisation: UMR 6639 CNRS Université de Aix Marseille, 3 place V. Hugo, 13331 Marseille, France

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BMC Biochemistry 2010, 11:32  doi:10.1186/1471-2091-11-32

Published: 24 August 2010



The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, Trichoderma reesei is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. T. reesei has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) to validate the function of a candidate LMCO gene from T. reesei, and ii) to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection.


After homologous overproduction of a candidate LMCO gene, extracellular laccase activity was detected when ABTS or SRG were used as substrates, and the recombinant protein was purified to homogeneity followed by biochemical characterization. The recombinant protein, called TrLAC1, has a molecular mass of 104 kDa. Optimal temperature and pH were respectively 40-45°C and 4, by using ABTS as substrate. TrLAC1 showed broad pH stability range of 3 to 7. Temperature stability revealed that TrLAC1 is not a thermostable enzyme, which was also confirmed by unfolding studies monitored by circular dichroism. Evolutionary studies were performed to shed light on the LMCO family, and the phylogenetic tree was reconstructed using maximum-likelihood method. LMCO and classical laccases were clearly divided into two distinct groups. Finally, Darwinian selection was tested, and the results showed that positive selection drove the evolution of sequences leading to well-known laccases involved in ligninolysis. Positively-selected sites were observed that could be used as targets for mutagenesis and functional studies between classical laccases and LMCO from T. reesei.


Homologous production and evolutionary studies of the first LMCO from the biomass-degrading fungus T. reesei gives new insights into the physicochemical parameters and biodiversity in this family.