N-terminal Purification Tag Alters Thermal Stability of the Carboxylesterase EstGtA2 from G. thermodenitrificans by Impairing Reversibility of Thermal Unfolding

作者：Charbonneau David M; Meddeb Mouelhi Fatma; Beauregard Marc^*

来源：Protein and Peptide Letters, 2012, 19(3): 264-269.

• Carboxylesterase
• thermal stability
• thermal unfolding
• tag peptide
• circular dichroism
• Geobacillus thermodenitrificans

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 degrees C (tagged) and 65.7 degrees C (cleaved) at pH 8. After thermal unfolding, the tag-free enzyme rapidly recovered initial activity at 25 degrees 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 degrees 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.

• 出版日期2012-3