Biochemical Characterization of a Carboxylesterase from the extreme thermoacidophile Sulfolobus solfataricus P1

Amitabh C. Sehgal “David”1, W. Callen2, E.J. Mathur2, J. Short2 and Robert M. Kelly1
1Department of Chemical Engineering, North Carolina State University, Raleigh NC;
 2Diversa Corporation, San Diego, CA
acsehgal@eos.ncsu.edu

 Esterases are enzymes which traditionally have been used in the hydrolysis of ester bonds.  Although hydrolysis is the principal reaction observed in aqueous systems, esterases readily perform the reverse reactions in systems with low water content (i.e., organic solvents).  For example, esterases have been used in: the resolution of racemic mixtures, synthetic reactions, blocking or unblocking of catalytic groups in peptide chemistry, and the modification of sugars.  This inherent flexibility has made esterases promising candidates for potential industrial applications.  Despite these advantages, few examples of industrial processes using esterases exist.  A major drawback in bringing this technology to an industrial level is enzyme lability, especially in organic solvent systems.  Thermophilic and hyperthermophilic esterases offer a promising alternative because of their inherent thermostability and thus increased resistance to denaturation in organic solvents. 
 
Sulfolobus solfataricus is an aerobic extreme thermoacidophile which is capable of growth at very low pH (<2.0) and relatively high temperatures (Topt of 75°C). Given that protocols have been developed for its growth to high cell densities (> 10⁹ cells/ml) and that its genome is currently being sequenced, S. solfataricus is an excellent source of thermostable biocatalysts. In addition, extracellular and membrane-associated enzymes produced by this organism, function at low pH as well as high temperatures.  Here, we describe the biochemical characteristics of a novel esterase produced by S. solfataricus. The gene encoding this 33 kDa single subunit enzyme has been cloned and expressed in Escherichia coli.  In addition, the enzyme has been purified to homogeneity by heat treatment and weak anionic exchange chromatography, and evaluated from the perspective of biochemical function.  The physiological role of this enzyme appears to be intracellular given its pH optimum of 7.7.  The esterase was most active towards p-nitrophenyl caproate. Further efforts to evaluate this enzyme in nonaqueous media will be discussed.