ИСТИНА

D-amino acid transaminases (DAATs) are pyridoxal-5'-phosphate-(PLP)-dependent enzymes that catalyze the reversible and stereoselective transfer of an amino group from D-amino acid to a prochiral α-keto acid, forming a new D-amino acid and α-keto acid. D-amino acids play an important role in physiological processes of living organisms as neurotransmitters, immunity regulators, and as part of antibacterial and anticancer compounds [1]. DAATs are promising biocatalysts for the synthesis of optically pure D-amino acids because chiral purity of chemical products is one of the important requirements in fine organic synthesis. We discovered a sequence encoding transaminase in a genome of the mesophilic bacteria Aminobacterium colombiense (Amico). The transaminase was expressed in E. coli cells, purified and investigated. We found that Amico was active with various D-amino acids with D-glutamate being the best substrate. Amico was found to catalyze transamination in a temperature range 30-60 °C, at pH 8-9. We investigated properties of Amico as a potential biocatalyst for amination of α-keto acids. We studied specificity of Amico to various keto acids, stabilization of holo form and apo form of Amico as well as the effect of pH and temperature on the Amico activity and stability. We studied the PLP leakage and approaches to stabilize the holo form. A one-pot-three-enzyme system to produce D-amino acids was developed. Product yields and enantiomeric excess (e.e.) in the reactions were determined. Optimization of one-pot-three-enzyme system conditions resulted in the production of 215 mM of norvaline and 300 mM of 2-aminobutyrate with e.e. more than 99%. We found that substitution K237A led to a shift of the pH-optimum of Amico activity to acidic pH 5-6 while maintaining the level of activity.