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Cloning, expression, purification and characterization of an oligomeric His-tagged thermophilic esterase from Thermus thermophilus HB27

AutorFuciños, P. ; Atanes, Estrella; López-López, Olalla; Solaroli, Michele; Cerdán, M. Esperanza; Siso, M. I. G. ; Pastrana, Lorenzo ; Rúa, M. Luisa
Palabras claveThermus thermophilus HB27
Thermostable lipolytic enzyme
Escherichia coli expression
Response surface methodology
Fecha de publicación2014
CitaciónProcess Biochemistry 49(6): 927-935 (2014)
ResumenThe esterase E34Tt (YP_004875.1) from Thermus thermophilus HB27 was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The gene sequence was subcloned into a T-vector, released with the restriction enzymes BamHI and HindIII, ligated to a pET-21d(+) vector, and transferred to E. coli BL21 (DE3) cells. Inducer concentration (isopropyl β-d-1-thiogalactopyranoside, IPTG) and cultivation time before and after induction were optimized. Best results were obtained by adding 0.25 mM IPTG after 8 h of cultivation and maintaining the induction during 4 extra hours. Most of the enzyme (94%) remained membrane-associated and had to be extracted with a detergent. From the membrane crude extract, the His-tagged E34Tt was purified as a dimer (71.8 kDa) in a single purification step by using metal affinity chromatography. The Rosso's model was used to optimize the reaction conditions. E34Tt-His6 was active in a wide temperature (19.7–79.4 °C) and pH range (4.0–9.3), and maximal activity was determined at pH 6.3 and 58.2 °C, which is 10–18 °C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His6 preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9 min at 85°C), suggesting that E34Tt-His6 has potential for industrial applications.
Descripción9 páginas, 7 figuras, 3 tablas
Versión del editorhttp://dx.doi.org/10.1016/j.procbio.2014.03.006
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