ИСТИНА

Formate dehydrogenase (EC 1.2.1.2., FDH) is an oxidoreductase which catalyzes the reaction of hydride anion transfer from substrate (formate anion) to C4-atom of nicotinamide moiety of NAD(P)+. In practice FDH is used in the reaction of chiral compounds synthesis with other oxidoreductases which use NAD(P)H as cofactor. FDH reduces oxidased form of coenzyme which is obtained in the main reaction. In our laboratory, systematic study of NAD+-depended FDHs from various sources is carried out for more than 25 years. Analysis of genome of the yeast Ogataea parapolymorpha DL-1 (GenBank assembly accession: GCA_000187245.3, former name Hansenula polymorpha) shows presence of the gene of NAD+-dependent formate dehydrogenase (GeneBank Accession NC_027860). A special feature of this source is ability to grow at 42 ºC. We have cloned and expressed in E.coli cells gene of FDH from the yeast O.parapolymorpha DL-1 (OpaFDH). Different conditions of OpaFDH expression were optimized and as result the yield of the enzyme achieved about 5000 U per L of cultivation medium (i.e. > 800 mg/L). Our recombinant OpaFDH did not have any additional tagged sequences to facilitate enzyme purification whereas in similar work (Yu S. et al, Appl. Microbiol. Biotechnol. 2014) His-tag was added to C-terminus of OpaFDH (ht-OpaFDH). In this work we determined kinetic parameters and studied the enzyme thermal stability at different temperatures, pH values and phosphate buffer concentrations. Comparison of our data with results of Yu S. and co-workers shows that the presence of His-tag at C terminus of OpaFDH results in increase of KM for NAD+ 7-fold, decrease of kcat 1.6-fold and decrease of KM for formate 3-fold. Study of enzyme thermal inactivation kinetics indicate that our OpaFDH is about 7-fold less stable in comparison with recombinant FDH from bacterium Pseudomonas sp.101. OpaFDH also demonstrated high stability in different ion liquids. This work was supported by grant of Russian Foundation for Basic Research (grant 15-54-78035-Ital-a). Authors also express special thanks to Prof. Olga Dontsova for kind supplement of O.parapolymorpha DL-1chromosomal DNA.