| Summary: | D-amino acids are valuable building blocks for the synthesis of biologically active compounds and pharmaceuticals. The asymmetric synthesis of chiral amino acids from prochiral ketones using stereoselective enzymes is a well-known but far from exhausted approach for large-scale production. Herein, we investigated a pyridoxal-5′-phosphate-dependent D-amino acid transaminase from <i>Haliscomenobacter hydrossis</i> as a potential biocatalyst for the enzymatic asymmetric synthesis of optically pure aliphatic and aromatic D-amino acids. We studied the catalytic efficiency and stereoselectivity of transaminase from <i>H. hydrossis</i> in the amination of aliphatic and aromatic α-keto acids, using D-glutamate as a source of the amino group. We constructed a one-pot three-enzyme system, which included transaminase and two auxiliary enzymes, hydroxyglutarate dehydrogenase, and glucose dehydrogenase, to produce D-amino acids with a product yield of 95–99% and an enantiomeric excess of more than 99%. We estimated the stability of the transaminase and the cofactor leakage under reaction conditions. It was found that a high concentration of α-keto acids as well as a low reaction temperature (30 °C) can reduce the cofactor leakage under reaction conditions. The obtained results demonstrated the efficiency of transaminase from <i>H. hydrossis</i> in the asymmetric synthesis of enantiomerically pure D-amino acids.
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