Abstract
The ability of Trichoderma reesei, a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the β-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei. This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5_1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ββ, SS βO4 and GG β5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase (TrLOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by TrLOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.
Highlights
Agaricomycetes have been long studied for their ability to mineralize woody biomass.A number of representative white-rot fungi belonging to the Basidiomycetes, such as Phanerochaete chrysosporium, Trametes versicolor, and Pleurotus ostreatus, have in particular been analyzed because of their effectiveness in converting lignocellulosic material intoCO2, primarily due to the enzymes and small organic molecules that they secrete during growth on such biomass
Growth was greater in maltose-supplied medium, T. reesei was still able to grow on lignin alone with maximum growth reached after day 3 and the total quantified fungal genomic DNA was found to decrease overtime in both conditions (Figure S1C)
We report for the first time the enzymes produced by T. reesei during growth on High Pressure Size Exclusion chromatography method (HPSEC) analysis of the insoluble lignin fractions indicated the consumption of almost all the phenolic monomers (Figure 1B)
Summary
Mariane Daou 1,† , Alexandra Bisotto 1 , Mireille Haon 1 , Lydie Oliveira Correia 2 , Betty Cottyn 3 , Elodie Drula 1 , Soňa Garajová 1 , Emmanuel Bertrand 1 , Eric Record 1 , David Navarro 1,4 , Sana Raouche 1 , Stéphanie Baumberger 3 and Craig B. J. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Current address: Department of Biology, Khalifa University of Science and Technology, Abu Dhabi 127788, United Arab Emirates
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
