Abstract
NAD(P)+-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) is actively used in processes of chiral synthesis by oxidoreductases with systems of reduced cofactor regeneration. The efficient use of FDH in such systems requires simple and fast enzyme purification. Metal-chelate affinity chromatography is widely used for such purposes. The method requires the presence of at least six His residues at N- or C-terminus of protein. The addition of extra His residues can affect enzyme properties. The computer modeling of the structure of FDH from bacterium Pseudomonas sp. 101 with different positions of His6 sequence showed that the optimal case is His-tag at N-terminus. Three types of PseFDH with His6 were prepared: wild-type NAD+-dependent enzyme and two mutant NADP+-specific forms. New PseFDHs were obtained as homogeneous preparations through a one-step purification procedure. The comparison of PseFDHs with and without His-tag showed that they have similar kinetic properties.
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