Jessica Kelly Moisan, Fatma Meddeb-Mouelhi, David M. Charbonneau and Marc Beauregard* Pages 561 - 572 ( 12 )
Background: Understanding the relationship between enzymatic stability and the amino acid sequence encoding carboxylesterases is of utmost importance.Objectives: Here we thoroughly characterized the behavior of the carboxylesterase EstGtA2 from Geobacillus thermodenitrificans during thermal denaturation at different pH with various salt concentrations. Method: EstGtA2 was characterized by circular dichroism regarding conformation and thermal stability, by dynamic light scattering for detection of association/aggregation, by enzymatic assays for activity and by monitoring the impact of heat treatments on activity. Results: Our investigation revealed a particular dependence between aggregation/association and preservation of secondary structures upon heating in EstGtA2. At pH 7, 8 and 9, depending on salt concentration, a folded but non-native associated state characterised by an apparent particle size of 300 nm resisted secondary structure unfolding up to 95°C. Conclusion: The paths leading to various aggregative states were found to be controlled by pH (depending on proximity to pI) and to a lesser extent, ionic strength, suggesting that ionic interactions at the surface of the protein are responsible for behavior of EstGtA2. The various paths available to EstGtA2 could be important for protection of Geobacillus termodenitrificans when exposed to heat stress. The understanding and/or control of these paths would allow for optimal use of EstGtA2 in industrial processes.
Thermal denaturation, circular dichroism, dynamic light scattering, protein aggregation, protein association, Est- GtA2.
Centre de Recherche sur les Matériaux Lignocellulosiques, Université du Québec à Trois-Rivières, Trois-Rivières (Québec) G9A 5H7, Centre de Recherche sur les Matériaux Lignocellulosiques, Université du Québec à Trois-Rivières, Trois-Rivières (Québec) G9A 5H7, PROTEO (Quebec Network for Research on Protein Function, Engineering and Applications), Université Laval, Québec (Québec) G1V 0A6, CRML, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières (Québec)