| Esterases (EC 3.1.1.1) catalyze the hydrolysis or synthesis of ester bonds and have evoked significant industrial interest because of their potential use in applications such as the resolution of racemic mixtures. In spite of these opportunities, esterase biocatalysis has yet to support large-scale industrial biotechnology, because of problems with catalyst instability in process mandated reaction environments such as organic solvents and nonoptimal pH and temperature. Although techniques such as protein and solvent engineering can help to alleviate these problems, strategic catalyst selection from other enzyme sources may be used in concert with these techniques to further improve enzyme stability. One such source of enzymes is hyperthermophiles (microorganisms that grow at 80°C or higher), as their biocatalysts can function in the extreme environments characteristic of industrial processes due, in part, to their inherent structural rigidity. As such, the effect of reaction environment, as it affects the catalytic, structural, and functional properties of hyperthermophilic esterases, needs to be examined in greater detail.; The focus of this work was a carboxylesterase from the extreme thermoacidophile Sulfolobus solfataricus P1 (Sso EST1) and the influence of reaction environment on its biocatalytic function. Sso EST1 was cloned and expressed in Escherichia coli and purified in a one step chromatographic process. Co-solvent experiments showed that while most additives were deleterious to the enzyme, DMSO had an activating effect on Sso EST1 at low concentrations (3.5% v/v) between 50–85°C. In fact, in certain instances, DMSO could be used in place of thermal activation to achieve comparable values of catalytic turnover. Sso EST1 was also shown to have excellent resolving power towards enantiomers of (R, S) Naproxen methyl ester (%ee(S)-Acid ≥ 90%). The addition of co-solvents and changes in pH resulted in only slight improvements in the enzyme's resolving power. However, enantioselectivity (%ee(S)-Acid) was optimal at temperatures significantly below the enzyme's melting temperature of 103°C. This result indicated that the resolution of the enantiomers by Sso EST1 was governed by enthalpic interactions between 4–70°C, in contrast to results obtained for this same resolution with mesophilic esterases from Candida rugosa and Rhizomucor miehei, which were under entropic control. |
