Stability and Folding of Endoglucanase I (CEL7B) from Humicoia Insolens

Abstract

Cellulases are of economic significance, particularly in the detergent and textile industries, where they are subjected to a wide range of operating conditions affecting their stability. To increase our insight into the properties of this class of enzymes, we have carried out a study of the stability and folding behavior of the 413-residue endoglucanase I (Ce17B) from Humicola insolens. Data from chemical denaturation in guanidinium chloride agree satisfactorily with calorimetric measurements, revealing an optimum stability of ca. 20 kcal mol⁻¹ around p^H 7 and a peak half-width of 3 -4 p^H units. Stability and activity show very similar p^H-profiles, but this is probably fortuitous. Judging from equilibrium m-values (the dependence of the log of the equilibrium unfolding constant on the denaturant concentration), the denatured state becomes significantly more compact outside p^H 6–9.

Folding and unfolding proceed very slowly with relaxation half times up to 6h. Single- and double-jump kinetic data at p^H 7 suggest a folding scheme involving two intermediates with native-like secondary structure but varying degrees of tertiary structure.

Key Words:

• Endoglucanase
• Cel7B
• Stability
• Folding
• Intermediate
• Kinetics Abbreviations
• D, denatured state
• GdmCl, guanidinium chloride
• N, native state