Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi
| Autor: | Effrosini Boutou, Garabed Antranikian, Constantinos E. Vorgias, Harald Trauthwein, Ksenia Egorova, Patrick Mueller, Stefan Verseck |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Protein Denaturation
Protein Folding Pyrococcus abyssi Hot Temperature Archaeal Proteins medicine.disease_cause Nitrilase Pyrococcus horikoshii Aminohydrolases medicine Escherichia coli Aeropyrum pernix Cloning Molecular Thermostability biology Circular Dichroism Aeropyrum Hydrogen-Ion Concentration biology.organism_classification Chromatography Ion Exchange Isoelectric point Biochemistry Pyrococcus furiosus Biotechnology |
| Zdroj: | Protein expression and purification. 47(2) |
| ISSN: | 1046-5928 |
| Popis: | Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py. abyssi. This recombinant nitrilase was purified by heat treatment of E. coli crude extract followed by anion-exchange chromatography with a yield of 88% and a specific activity of 0.14 U/mg. The recombinant enzyme, which represents the first archaeal nitrilase, is a dimer (29.8 kDa/subunit) with an isoelectric point of pI 5.3. The nitrilase is active at a broad temperature (60-90 degrees C) and neutral pH range (pH 6.0-8.0). The recombinant enzyme is highly thermostable with a half-life of 25 h at 70 degrees C, 9 h at 80 degrees C, and 6 h at 90 degrees C. Thermostability measurements by employing circular dichroism spectroscopy and differential scanning microcalorimetry, at neutral pH, have shown that the enzyme unfolds up to 90 degrees C reversibly and has a T(m) of 112.7 degrees C. An inhibition of the enzymatic activity was observed in the presence of acetone and metal ions such as Ag(2+) and Hg(2+). The nitrilase hydrolyzes preferentially aliphatic substrates and the best substrate is malononitrile with a K(m) value of 3.47 mM. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect