Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: Effect of N-terminus in thermal activity and stability
| Autor: | Olalla López-López, M. Isabel González-Siso, Lorenzo Pastrana, M. Esperanza Cerdán, M. Luisa Rúa, Estrella Atanes, Pablo Fuciños |
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| Rok vydání: | 2011 |
| Předmět: |
Glycosylation
Recombinant Fusion Proteins Molecular Sequence Data Esterase Nitrophenols Kluyveromyces Thermal stability Amino Acid Sequence Cloning Molecular Kluyveromyces lactis Analysis of Variance Chromatography biology Protein Stability Thermus thermophilus Esterases Temperature Wild type Hydrogen-Ion Concentration biology.organism_classification Yeast Biochemistry Protein Processing Post-Translational Biotechnology Mesophile |
| Zdroj: | Protein Expression and Purification. 78:120-130 |
| ISSN: | 1046-5928 |
| Popis: | Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(½)< 5 min) than the wild-type enzyme (t(½)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(½)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability. |
| Databáze: | OpenAIRE |
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