Purification and kinetic characterization of an esterase from Pyrococcus furiosus overexpressed in Escherichia coli
S. M. C.Alquéres1*; R. V. Almeida1,2; T. M. Alves2; O. B. Martins1
1Laboratório de Biologia Molecular – IBqM – UFRJ; 2Laboratório de Bioprocessos – COPPE – UFRJ; *e-mail: alqueres@bioqmed.ufrj.br
Carboxylesterases (E.C. 3.1.1.1) are enzymes that catalyse the hydrolysis of esters bonds. Their broad synthetic potential make them ideal biocatalysts for oleochemistry and organic synthesis. Despite that, the greatest limitation found in using enzymes in industrial scale resides in their short time of half-life time when compared to inorganic catalysts. The enzymes from extremophilic archaea posses the ability to remain catalytically active under extremes of temperature, salinity, pH and solvents, what circunvent the biotechnological conflict between industrial conditions and the fragility of biological components. However, extremophilic microorganisms cultivation is difficult and expensive. On the other hand, these enzymes can be cloned and expressed in microorganisms easier to cultivate. In previous work, we have cloned and expressed an esterase gene from Pyrococcus furiosus fused to a thiorredoxin in Escherichia coli(DE3). The present study reports the purification and the characterization of this enzyme.
The recombinant esterase was purified in a one-step affinity chromatography procedure. The enzyme was treated with enterokinase to cleavage the thiorredoxin tag and both, fused and cleaved enzyme, were characterized. The optimum temperature and pH were similar to both enzymes, 80oC and 7.0, respectively. However, the activity of cleaved enzyme was higher than fused one, suggesting different temperature dependence. Kinetic studies indicated that the enzyme with the thirredoxin tag had values of apparent K(m) and V(max) corresponding to 20mM and 0,38mmoles x mg prot-1 x min-1 for 4-methylumbelliferone-Acetate and 20mM and 1,25mmoles x mg prot-1 x min-1 for 4-methylumbelliferone-Heptanoate. The enzyme without the tag had values of apparent K(m) and V(max) corresponding to 20mM and 0,43mmoles x mg prot-1 x min-1 for 4-methylumbelliferone-Acetate and 20mM and 1,80mmoles x mg prot-1 x min-1 for 4-methylumbelliferone-Heptanoate. These data strongly suggest that this biocatalyst can be used in biotechnological applications in a near future.
Supported by CNPq and FAPERJ.
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