Sunday, April 29, 2007

Characterization of different enzyme cocktails secreted by Trichoderma reesei using 2D-electrophoresis

Antoine Margeot1, Rezak Boubrit1, Isabelle Herpoël-Gimbert2, Hugues Mathis1, Nicolas Lopes Ferreira1, Alain Dolla3, Marcel Asther2, Senta Blanquet1, 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 one of the most widely used filamentous fungi for the production of cellulolytic and hemicellulolytic enzymes because of its capacity to secrete high levels of an efficient hydrolytic cocktail. However, knowledge of the detailed composition of the enzyme cocktail is still too incomplete to be able to draw any relationship between the secretome composition and its hydrolytic efficiency. Such a knowledge is useful to determine differences between T. reesei strains and to optimize T. reesei culture conditions. That is the reason why we developed a  methodology for characterizing Trichoderma reesei secretome composition in one step, using 2D-electrophoresis .
First, using systematic spot picking and MALDI-TOF mass spectrometry, we were able to detect the main enzymes secreted by T. reesei. More than 50 spots, corresponding to 24 proteins species and representing more than 80% of enzyme composition in weight, were identified. An annotated secretome map was obtained including several previously unpublished proteins.
The possibility to "fingerprint" T. reesei secreted proteins was used to compare various fermentation conditions, especially culture medium composition. Significant differences in protein expression were observed, both on a global level and on a single-enzyme basis. Moreover, the results can lead to predictions regarding the function of some unknown protein by completing sequence information with secretion patterns on different substrates.
Relating secretion patterns using 2D-electrophoresis and hydrolysis properties of enzyme cocktails is an invaluable tool for optimisation of fermentation conditions and a guide for genetic engineering of industrial T. reesei strains.