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 -