Provider: Ingenta Connect
Database: Ingenta Connect
Content: application/x-research-info-systems
TY - ABST
AU - M. Charbonneau, David
AU - Meddeb-Mouelhi, Fatma
AU - Beauregard, Marc
TI - N-terminal Purification Tag Alters Thermal Stability of the Carboxylesterase EstGtA2 from G. thermodenitrificans by Impairing Reversibility of Thermal Unfolding
JO - Protein and Peptide Letters
PY - 2012-03-01T00:00:00///
VL - 19
IS - 3
SP - 264
EP - 269
KW - Geobacillus thermodenitrificans
KW - thermal unfolding
KW - tag peptide
KW - enantioselectivity
KW - polyhistidine
KW - circular dichroism
KW - Conformational stability
KW - EstGtA2
KW - thermal stability
KW - Carboxylesterase
N2 - 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: Tm 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.
UR - https://www.ingentaconnect.com/content/ben/ppl/2012/00000019/00000003/art00002
M3 - doi:10.2174/092986612799363181
UR - https://doi.org/10.2174/092986612799363181
ER -