期刊
ACTA NATURAE
卷 14, 期 1, 页码 82-91出版社
RUSSIAN FEDERATION AGENCY SCIENCE & INNOVATION
DOI: 10.32607/actanaturae.11665
关键词
formate dehydrogenase; Pseudomonas sp. 101; catalytic properties; thermal stability; site-directed mutagenesis
资金
- Russian Federation [MD-349.2021.1.4]
A mutant formate dehydrogenase (PseFDH) from Pseudomonas sp. 101 bacterium with improved thermal stability has been generated through single-point amino acid replacements. The mutant with E170D substitution exhibits increased thermal stability compared to the starting mutant and the wild-type enzyme.
Formate dehydrogenase from Pseudomonas sp. 101 bacterium (PseFDH, EC 1.2.1.2) is a research model for the elucidation of the catalytic mechanism of 2-oxyacid D-specific dehydrogenases enzyme superfamily. The enzyme is actively used for regeneration of the reduced form of NAD(P)H in chiral synthesis with oxidoreductases. A multi-point mutant PseFDH SM4S with an improved thermal and chemical stability has been prepared earlier in this laboratory. To further improve the properties of the mutant, additional single-point replacements have been introduced to generate five new PseFDH mutants. All new enzymes have been highly purified, and their kinetic properties and thermal stability studied using analysis of thermal inactivation kinetics and differential scanning calorimetry. The E170D amino acid change in PseFDH SM4S shows an increase in thermal stability 1.76- and 10-fold compared to the starting mutant and the wild-type enzyme, respectively.
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