title: N-terminal purification tag alters thermal stability of the carboxylesterase EstGtA2 from G. thermodenitrificans by impairing reversibility of thermal unfolding.
abstract: The novel thermostable carboxylesterase EstGtA2 from G. thermodenitrificans (accession no. AEN92268) was functionally expressed and purified using an N-terminal fusion tag peptide. We recently reported general properties of the recombinant enzyme. Here we report preliminary data on thermal stability of EstGtA2 and of its tagged form. Conformational stability was investigated using circular dichroism and correlated with residual activity measurements using a colorimetric assay. The tag peptide had no considerable impact on the apparent melting temperature: T(m) value = 64.8°C (tagged) and 65.7°C (cleaved) at pH 8. After thermal unfolding, the tag-free enzyme rapidly recovered initial activity at 25°C (1.2 Umg(-1)), which was corroborated by substantial refolding (83%) as determined by far-UV CD transitions. However, after thermal unfolding, the purification tag drastically decreased specific activity at 25°C (0.07 Umg(-1)). This was corroborated by the absence of refolding transition. Although the purification tag has no undesirable impact on activity before thermal unfolding as well as on Tm, it drastically hinders EstGtA2 refolding resulting in a major loss of thermal stability.
authors.affiliation: Departement de chimiebiologie, Universite du Quebec a Trois-Rivieres, Trois-Rivieres, Quebec, Canada.
authors.forename: David M
authors.initials: DM
authors.lastname: Charbonneau
authors.forename: Fatma
authors.initials: F
authors.lastname: Meddeb-Mouelhi
authors.forename: Marc
authors.initials: M
authors.lastname: Beauregard
mesh_terms: D016477:Artifacts; D043182:Carboxylesterase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology*; D011487:Protein Conformation; D058849:Protein Refolding; D058767:Protein Unfolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
chemical_list: D011993:Recombinant Fusion Proteins; D043182:Carboxylesterase