Monday, May 5, 2008 - 4:30 PM
6-06
Exploration of Trichoderma reesei enzymatic pools and their hydrolysis potential using proteomic and liquid chromatography tools
Antoine Margeot1, Nicolas Lopes Ferreira1, Isabelle Herpoël-Gimbert2, Hugues Mathis1, Audrey Mascle1, Alain Dolla3, Senta Blanquet1, Marcel Asther2, and Frederic Monot1. (1) Biotechnology Dpt., IFP, 1 & 4 Avenue de Bois-Préau, Rueil-Malmaison, 92852, France, (2) UMR 1163 de Biotechnologie des Champignons Filamenteux, 163 Avenue de Luminy, case postale 925, Marseille, 13288, France, (3) UPR 9036 bioénergétique et ingénierie des protéines, Institut de Biologie Structurale et Microbiologie, 31, chemin joseph Aiguier cedex 20, Marseille, 13402, France
Trichoderma reesei is a well-known producer of cellulolytic enzymes, both for fundamental research and industrial production. Sequencing of its genome has revealed an unexpected profusion of different cellulolytic enzymes. Both regulation and role of these enzymes are not well characterized. Using 2D-electrophoresis coupled with mass spectrometry, we systematically identified major spots (more than sixty), leading to a very detailed T. reesei secretome map (between 84% and 95% spot volume). Using this map, we determined the composition of the secretomes of two T. reesei strains, RutC30 and CL847 (an industrial strain), produced on different carbon sources and inducers. Some of these enzymes were tested for hydrolysis of steam-exploded wheat straw. This strategy revealed information on regulation of cellulases and hemicellulases produced in T. reesei, such as variation in inducibility of xylanases XYN1 and XYN4, of so-called minor endoglucanases EG3, EG4 and EG6 and arabinofuranosidases ABF1, ABF2 and ABF3 or on the role of the carbon source used for T. reesei cell growth.
In another approach, we purified the major T. reesei enzymes CBH1, CBH2, EG1, EG2 by FPLC and used them for reconstitution of enzymatic pools in miniaturized hydrolysis assays. This convenient tool contributes to clarify the role of each enzyme in substrate degradation. In addition, testing a more extended range of enzymes obtained after fractionation of secretomes will allow determination of the factors lacking for an efficient hydrolysis of biomass. Comparison with secretomes characterized using 2D-electrophoresis will help fine-tuning the best fermentation conditions for production of this "ideal" enzymatic pool.
In another approach, we purified the major T. reesei enzymes CBH1, CBH2, EG1, EG2 by FPLC and used them for reconstitution of enzymatic pools in miniaturized hydrolysis assays. This convenient tool contributes to clarify the role of each enzyme in substrate degradation. In addition, testing a more extended range of enzymes obtained after fractionation of secretomes will allow determination of the factors lacking for an efficient hydrolysis of biomass. Comparison with secretomes characterized using 2D-electrophoresis will help fine-tuning the best fermentation conditions for production of this "ideal" enzymatic pool.
See more of Advances in Enzyme Science and Technology 1
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See more of The 30th Symposium on Biotechnology for Fuels and Chemicals (May 4 -- 7, 2008)
See more of General Submissions
See more of The 30th Symposium on Biotechnology for Fuels and Chemicals (May 4 -- 7, 2008)