ИСТИНА

NAD(P)+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is used for enzyme regeneration of cofactor reduced forms NAD(P)H in processes of synthesis of chiral compounds catalysed by different oxidoreductases. For example, FDH highly efficiently works for NADPH regeneration in reaction of Baeyer-Villiger oxidation catalysed by various monooxigenases1. Drawbacks of currently used FDHs are rather low specific activity and low operating (chemical) stability. After thorough bioinformatic search we cloned, expressed in E.coli and characterized new FDHs. It was found that FDH from bacterium Staphylococcus aureus shows the highest (at leas 2.5-fold) specific activity compared to all known FDHs. Gene of chimera FDH consisting on parts of FDH genes from Pseudomonas sp.101 and Moraxella sp.C1 was constructed. Using protein engineering new mutant FDHs with increased chemical and thermal stability we obtained. The other practically important enzyme is D-amino acid oxidase from yeast Trygonopsis variabilis (TvDAAO) which is also studied in our laboratory. We studied the enzyme activity and stability at different concentrations and temperatures. It was shown that oligomeric state of TvDAAO strictly depends on enzyme concentration and thermal inactivation proceeds through dissociative denaturation mechanism. Regions of concentration for existence of different oligomeric forms were determined. Kinetic parameters for two stages of inactivation were determined at different temperatures. References 1. Schwarz-Linek, U., Krödel, A., Ludwig, F.-A., Schulze, A., Rissom, S., Kragl, U., Tishkov, V.I., Vogel, M. Synthesis, 2001, 33(6), 947. This work was supported by Russian Science Foundation, project 16-14-00043.