Poster
Presentation 25:
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.
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