Abstract
Due to its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum (T. harzianum) has considerable potential in biomass hydrolysis application. Cellulases from Trichoderma reesei have been widely used in studies of cellulose breakdown. However, cellulases from T. harzianum are less-studied enzymes that have not been characterized biophysically and biochemically as yet. Here, we examined the effects of pH and temperature on the secondary and tertiary structures, compactness, and enzymatic activity of cellobiohydrolase Cel7A from T. harzianum (Th Cel7A) using a number of biophysical and biochemical techniques. Our results show that pH and temperature perturbations affect Th Cel7A stability by two different mechanisms. Variations in pH modify protonation of the enzyme residues, directly affecting its activity, while leading to structural destabilization only at extreme pH limits. Temperature, on the other hand, has direct influence on mobility, fold, and compactness of the enzyme, causing unfolding of Th Cel7A just above the optimum temperature limit. Finally, we demonstrated that incubation with cellobiose, the product of the reaction and a competitive inhibitor, significantly increased the thermal stability of Th Cel7A. Our studies might provide insights into understanding, at a molecular level, the interplay between structure and activity of Th Cel7A at different pH and temperature conditions.
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Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) via grant numbers 2008/56255-9, 2009/54035-4, 2010/08370-3, 2010/16542-9; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) via grant number 482166/2010-0; and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We also thank Laboratório Nacional de Luz Sincrotron (LNLS) for beam time and help with X-ray data collection.
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Francieli Colussi and Wanius Garcia contributed equally to this work.
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Colussi, F., Garcia, W., Rosseto, F.R. et al. Effect of pH and temperature on the global compactness, structure, and activity of cellobiohydrolase Cel7A from Trichoderma harzianum . Eur Biophys J 41, 89–98 (2012). https://doi.org/10.1007/s00249-011-0762-8
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DOI: https://doi.org/10.1007/s00249-011-0762-8