ИСТИНА

Nowadays enzymes are widely used in different processes of production of fine organic chemicals. The main advantages of enzymes are high substrate specificity, mild conditions of the reaction, high velocity of the process and absence of side chain reactions. NADPH is widely used in different synthetic processes using enzymes, so that the problem of its regeneration is still actually nowadays. One of the most appropriate enzymes for cofactor regeneration is formate dehydrogenase (FDH, EC 1.2.1.2.) which catalyzes the reaction of formate oxidation coupled with reduction of NAD(P)+ to NAD(P)H. FDH is a well studied enzyme, found in different bacteria, yeasts, fungi and plants. The majority of FDHs are more specific to NAD+. Consequently the experiments of changing substrate specificity of FDH from NAD+ to NADP+ are carried out. Formate dehydrogenases from different sources are studied in our laboratory. One of the most interesting FDHs is formate dehydrogenase from bacterium Pseudomonas sp 101 (PseFDH). It has high thermal stability and catalytic constant compared to FDHs from yeast, fungi and plants. Analysis of PseFDH structure showed, that one of the residues, which is responsible for coenzyme binding is Asp221. The substitution of this negatively charged residue to neutral amino acids might eliminate repulsion between carboxyl group of Asp221 and 2'-phosphate group of NADP+. In this work we present several new mutant forms with substitutions in position 221. The genes, encoding mutant enzymes were expressed in E.coli cells and new FDHs were obtained as active and soluble enzymes. Kinetic parameters in reaction with NAD+ and NADP+ were determined. It was shown that mutant enzymes had a high specificity to NADP+. Also the thermal stability of new mutant forms was studied by kinetic of inactivation and differential scanning calorimetry (DSC).