ИСТИНА

Native enzymes usually do not have properties which are fully suitable for application in biotechnological processes. That is why that practically all enzymes are subjected to directed modi-fication of properties before practical application. In present report we will show examples of ra-tional design of some practically important enzymes. Formate dehydrogenase (FDH, EC 1.2.1.2.). We cloned genes of new FDHs from bacte-rium Staphylococcus aureus, thermotolerant yeast Ogataea parapolymorpha, bakery yeast Sac-charomyces cerevisiae (SauFDH, OpaFDH, and SceFDH, respectively). It was found that SauFDH has the highest value of specific activity compared to other FDHs (25 and 10 U/mg of protein, re-spectively). OpaFDH shows the same low KM values with formate and NAD+ as plant FDHs but it has much higher thermal stability. Chemical stability of FDH from Pseudomonas sp.101 was im-proved at least by four orders. Wild-type SauFDH and mutant PseFDH show the best chemical sta-bility compared to all described FDHs. Wild-type and mutant FDHs from bacteria, yeasts and plants were tested for activity and stability in ionic liquids. It was shown that different amino acid changes on the surface of protein globule had various effects on stability and activity FDHs in water and ionic liquids. New highly efficient mutant PseFDHs with changed coenzyme specificity from NAD+ to NADP+ were obtained. Two variants of fusion proteins of NADP-PseFDH with cytochrome P450 BM3 (CYP102A1) from the soil bacterium Bacillus megaterium were prepared and it was found that all variants show both types of activity. D-amino acid oxidase (DAAO, EC 1.4.3.3). Different multi-points mutants (mpmDAAO) of yeast DAAO have been prepared. mpmDAAO showed better chemical stability and higher activ-ity with cephalosporin C compared to wild-type enzyme. α-Amino acid esters hydrolase (AEH, EC 3.1.1.43). Gene of AEH from Xanthomonas ru-brilineans (XrAEH) has been cloned and overexpressed in E. coli. It was shown that recombinant XrAEH is more efficient compared to native XrAEH. This work was supported by Russian Science Foundation (grant 16-14-00043), Russian Foundation for Basic Research (grants 15-54-78035 Ital_a and 17-04-01469а) and Alexander von Humboldt Foundation (fellowships for VIT and AAP).