ИСТИНА

Formate dehydrogenase (FDH, EC 1.2.1.2.) can be found in bacteria, yeasts, fungi and plants. Plant FDHs are considered to be very attractive due to its high affinity to the coenzyme. The gene, encoding FDH from soybean Glycine max (SoyFDH) was cloned and expressed in E.coli cells. SoyFDH shows the lowest values for KMNAD+ and KMHCOO- compared to FDHs from other sources. That is why SoyFDH is a very promising enzyme for cofactor regeneration. The disadvantage of SoyFDH is low thermal stability. Rational design approach was applied for enhancing thermal stability of the enzyme. Several single-point mutations were selected. Here we present the combination of the substitutions with highest stabilization effect. Several new mutant enzymes with multiply amino acid changes were prepared and fully characterized. Thermal stability was studied by inactivation kinetics and differential scanning calorimetry. It was found that combination of mutations resulted in synergetic stabilization effect. All multiply mutants showed higher stability and catalytic efficiency compared to FDH from Candida boidinii.