Journal
MOSCOW UNIVERSITY CHEMISTRY BULLETIN
Volume 75, Issue 4, Pages 250-257Publisher
PLEIADES PUBLISHING INC
DOI: 10.3103/S0027131420040057
Keywords
formate dehydrogenase; Pseudomonassp; 101; affinity chromatography; catalytic properties; thermal stability; His-tag
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Funding
- Russian Science Foundation [18-74-00146]
- Russian Science Foundation [18-74-00146] Funding Source: Russian Science Foundation
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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 bacteriumPseudomonassp. 101 with different positions of His(6)sequence showed that the optimal case is His-tag at N-terminus. Three types of PseFDH with His(6)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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