Journal
PROCESS BIOCHEMISTRY
Volume 49, Issue 6, Pages 927-935Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2014.03.006
Keywords
Thermus thermophilus HB27; Thermostable lipolytic enzyme; Cloning; Escherichia coli expression; Chromatography; Response surface methodology
Categories
Funding
- Spanish Ministry of Science and Technology [CTQ2005-05609/PPQ]
- Conselleria de Innovacion e Industria from the Xunta de Galicia, Spain [PGIDITO6REM38302PR]
- European FEDER [PPQ20013361]
- Consolidacion program from CEOU (Xunta de Galicia, Spain)
Ask authors/readers for more resources
The esterase E34Tt (YP_004875.1) from Therms 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 beta-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-His(6) was active in a wide temperature (19.7-79.4 degrees C) and pH range (4.0-9.3), and maximal activity was determined at pH 6.3 and 58.2 degrees C, which is 10-18 degrees C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His(6) preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9 min at 85 degrees C), suggesting that E34Tt-His(6) has potential for industrial applications. (C) 2014 Elsevier Ltd. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available