ИСТИНА

Enzymes are highly efficient biocatalysts but different processes require enzymes with different properties and native sources can not provide them. Moreover, high cost of enzymes preparation (especially for chiral synthesis and pharmaucetical industry) very often prevent biocatalysts introduction into practice. Protein engineering methods permit to get biocatalysts with prescribed properties but initial source plays important role because volume (and cost) of work depend on properties of initial enzymes. In our our laboratory we carry out systematic works to find new sources of enzymes with the most suitable ofr target process. Bioinformatic search and selection of genes of interest were followed by cloning, overexpression. Gene structure is modified to improve expression level, stability and activity of enzyme. Crystallization and determination of 3D structure provide background for rational design of enzymes. Using this approach we found and cloned genes of new formate dehydrogenases and D-amino acid oxidases with new properties. Five crystal structures were determined. The second direction of experiments is creation of new hybrid biocatalysts which are not observed in nature. We prepared hybrid chiral biocatalysts consisting on oxidoreductase (two types of monooxigenases) with two types of mutant NADP+-dependent formate dehydrogenases. Genetic construction had different order of genes. Hybrid enzymes were expressed and purified as active soluble proteins. In one case the length of polypeptide chain was about 1200 amino acid residues. New hybrid biocatalysts showed higher catalytic efficiency compared to mixture of individual enzymes. This work was supported by Russian Foundation for Basic Research (grants 17-04-01469а, 17-04-01487а, 17-04-01662а, 18-34-00594 and 18-34-20098_mol_ved), RFBR and Moscow city Government (grant 19-34-70036), Russian Science Foundation (grant 18-74-00146) and DAAD (fellowships for PDP).