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Formate dehydrogenase (EC 1.2.1.2, FDH) is widely used for coenzyme regeneration of NADH or NADPH in different biocatalytic processes. Company Degussa (now Evonik) developed and introduced biocatalytic process of L-tert-leucine preparation with FDH reduced coenzyme regeneration. Till now this process if the is the largest scale process of chiral synthesis with purified enzymes till now [1]. The research in our laboratory is focused on the systematic study of formate dehydrogenases from different sources [2]. Recently the gene of a novel NAD+-dependent formate dehydrogenase from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL-1 was cloned (GenBank assembly accession: GCA_000187245.3). In this work wild-type OpaFDH was overexpressed, purified and characterized. Kinetic parameters were determined. Thermal stability of enzyme was studied 1) through thermal inactivation kinetics at different temperatures, pH values, phosphate buffer concentrations and 2) with differential scanning calorimetry. OpaFDH showed the lowest values of Michaelis constants (KM) both for NAD+ and formate among all described FDHs from yeast and bacteria. The enzyme also demonstrated high thermal stability, which was lower only in comparison with the FDHs from bacteria Pseudomonas sp.101 and Staphylococcus aureus. A synthetic gene of OpaFDH was also cloned in work [3]. Distinctive feature of this enzyme was a presence of additional six histidines at C-terminus of enzyme. Comparison of our data with results of Yu S. and co-workers shows that the presence of additional histidines results in increase of KM for NAD+ 6-fold, decrease of kcat 2-fold and decrease of KM for formate 2-fold. This work was supported by grant of Russian Foundation for Basic Research (grants 17-04-01469-а). Authors also express special thanks to Academician O.A. Dontsova for kind supplement of O.parapolymorpha DL-1chromosomal DNA.
№ | Имя | Описание | Имя файла | Размер | Добавлен |
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1. | Biotech_Cong_Mosc_2017_Zarubina.pdf | Biotech_Cong_Mosc_2017_Zarubina.pdf | 106,7 КБ | 20 мая 2017 [tishkov] |