| Autor: |
Pometun AA; Bach Institute of Biochemistry, Federal Research Centre 'Fundamentals of Biotechnology', Russian Academy of Sciences, Moscow, 119071, Russia. aapometun@gmail.com.; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia., Shaposhnikov LA; Bach Institute of Biochemistry, Federal Research Centre 'Fundamentals of Biotechnology', Russian Academy of Sciences, Moscow, 119071, Russia.; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia., Zubanova SA; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia., Kovalevskii RP; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia., Atroshenko DL; Bach Institute of Biochemistry, Federal Research Centre 'Fundamentals of Biotechnology', Russian Academy of Sciences, Moscow, 119071, Russia.; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia., Pometun EV; Sechenov First Moscow State Medical University, Moscow, 119991, Russia., Savin SS; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia., Tishkov VI; Bach Institute of Biochemistry, Federal Research Centre 'Fundamentals of Biotechnology', Russian Academy of Sciences, Moscow, 119071, Russia. vitishkov@gmail.com.; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia. |
| Abstrakt: |
Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. Recombinant enzyme with additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in active and soluble form with a yield of more than 1 g per liter of the medium. In the present work, a detailed comparison of this enzyme with FDHs from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate effect of N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that addition of an Ala residue to the N-terminus reduces four-fold the rate constant of thermal inactivation compared with the addition of a Gly residue. Addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to acceleration of purification, practically does not affect kinetic parameters, but somewhat reduces thermal stability, which, however, can be restored to the level of OpaFDH_AK stability by adding 0.5 M NaCl. |
