RESUMO
The kinetics of the thermal inactivation of recombinant wild-type formate dehydrogenase from Candida boidinii yeast was studied in the temperature range of 53-61(o)C and pH 6.0, 7.0, and 8.0. It was shown that the loss of the enzyme's activity proceeds via a monomolecular mechanism. Activation parameters ∆Ð(-) and ∆S(-) were calculated based on the temperature relations dependence of inactivation rate constants according to the transition state theory. Both parameters are in a range that corresponds to globular protein denaturation processes. Optimal conditions for the stability of the enzyme were high concentrations of the phosphate buffer or of the enzyme substrate sodium formate at pH = 7.0.
RESUMO
A comparative study of the thermostability of NAD+-dependent formate dehydrogenases (FDHs; EC 1.2.1.2) from both methylotrophic bacteria Pseudomonas sp. 101 and Moraxella sp. Cl, the methane-utilizing yeast Candida boidinii, and plants Arabidopsis thaliana and Glycine max (soybean) was performed. All the enzymes studied were produced by expression in E. coli cells. The enzymes were irreversibly inactivated in one stage according to first-order reaction kinetics. The FDH from Pseudomonas sp. 101 appeared as the most thermostable enzyme; its counterpart from G. max exhibited the lowest stability. The enzymes from Moraxella sp. Cl, C. boidinii, and A. thaliana showed similar thermostability profiles. The temperature dependence of the inactivation rate constant of A. thaliana FDH was studied. The data of differential scanning calorimetry was complied with the experimental results on the inactivation kinetics of these enzymes. Values of the melting heat were determined for all the enzymes studied.
