BIOPHYSICAL CHARACTERIZATION OF A THERMOALKALOPHILIC ESTERASE FROM Geobacillus sp.

摘要

Esterases are a class of enzyme with broad industrial applications in stereo-specific synthetic approachesto metabolic processing of drugs and antimicrobial agents. As such, preparation of an efficient esterase that isrobust to denaturation under a wide range of temperatures and solvent conditions would be of great practicalutility. To this end, an esterase cloned from the thermophilic bacteria Geobacillus sp. was subjected to biophysicalcharacterization. In the presence of reducing agents (e.g., dithiothreitol), equilibrium studies of heat- and chemicalinduceddenaturation were apparently two-state and reversible. Derived parameters from unfolding studiesusing fluorescence and circular dichroism were similar, indicating that unfolding is largely cooperative. Aspredicted, the esterase is highly stable, with Tm app = 75.8 °C and ..Gunf app = –69.8 kJ/mol at 25°C. Refoldingstudies carried out at 25°C reveal the presence of one or more folding intermediates. Taken together, these resultssuggest that this esterase is an excellent candidate for use in industrial applications, and indicate that the systematicremoval of Cys to recover reversible folding in the absence of reducing agents will be a feasible approach tofurther improving the utility of this enzyme.