An esterase from Thermus thermophilus HB27 with hyper-thermoalkalophilic properties: Purification, characterisation and structural modelling

• Published in: Journal of molecular catalysis. B, Enzymatic Enzymatic, 2011-07, Vol.70 (3), p.127-137
• Main Authors: Fuciños, P., Pastrana, L., Sanromán, A., Longo, M.A., Hermoso, J.A., Rúa, M.L.
• Format: Article
• Language: English
• Subjects: amino acids; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; carboxylic ester hydrolases; catalytic activity; cell membranes; Cell-bound esterase; consensus sequence; esters; Fundamental and applied biological sciences. Psychology; half life; heat inactivation; Methods. Procedures. Technologies; molecular weight; peptides; Purification; sequence analysis; Structure; Sus scrofa; temperature; thermal stability; Thermostability; Thermus thermophilus
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2011.02.017

[Display omitted] ► A membrane-associated esterase (E34Tt) was detected in Thermus thermophilus HB27. ► E34Tt was purified, and identified by peptide mass fingerprinting. ► CHAPS was essential for activity and stability. E34Tt has preference for C10 esters. ► High thermophilicity; optimal temperature was >80 °C and t 1/2 at 85 °C > 135 min. ► A theoretical structural model was proposed using a prolyl oligopeptidase as template. A membrane-associated esterase (E34Tt) was detected in Thermus thermophilus HB27. The enzyme was purified to homogeneity in a three-step protocol. Detergent (CHAPS) above the CMC was found to be essential to solubilise the enzyme from cell membranes as well as for maintaining activity and stability. By using mass fingerprinting, peptides were found to share identity with the YP_004875 protein, which was annotated as putative esterase in the genome analysis of T. thermophilus HB27, although experimental evidence was lacking. No homology was detected with any known lipase or esterase. However, a comparison with the high-scored sequences from a BLASTp search identified the consensus sequence for lipases/esterases between amino acids 157 and 161 (Gly-Cys-Ser 159-Ala-Gly). Further inhibition assays with E600 confirmed that Ser 159 was involved in the catalytic mechanism. The monomeric enzyme had a molecular mass of 34 kDa and exhibited esterase activity with preference for medium chain-length esters (C10). E34Tt was noticeable for its high thermal stability; the optimal reaction temperature was higher than 80 °C and the half-life of thermal inactivation at 85 °C was 135 min, which makes it even more thermostable than some hyperthermophilic esterases. These properties convert E34Tt into a very attractive enzyme for biotechnological purposes. A theoretical structural model was constructed using as template a prolyl oligopeptidase from Sus scrofa, and a putative catalytic triad (Ser 159, Glu 255 and His 293) with high similarity to the template was identified.