| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 34369 | Process Biochemistry | 2014 | 9 Pages |
•A thermophilic membrane-associated esterase from Thermus thermophilus HB27 was expressed in Escherichia coli.•E34Tt-His6 was purified from the E. coli membrane as a dimer in a single step by using metal affinity chromatography.•Detergent was essential for activity and stability. The esterase had preference for short chain esters.•The Rosso's model was used to describe the enzyme activity as a function of pH and temperature.•Activity was maximal at 58 °C and pH 6.3. The enzyme was active in a wide temperature (19.7–79.4 °C) and pH range (4.0–9.3).
The 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.
