Cofactor Generation Cascade for ?-Ketoglutarate and Fe(II)-Dependent Dioxygenases.

ABSTRACT: Fe(II)- and ?-ketoglutarate dependent dioxygenases have emerged as important catalysts for the preparation of non-natural amino acids. The stoichiometric supply of the cosubstrate ?-ketoglutarate (?KG) is an important cost factor. A combination of the N-succinyl amino acid hydroxylase SadA with an l-glutamate oxidase (LGOX) allowed for coupling in situ production of ?KG to stereoselective ?KG-dependent dioxygenases in a one-pot/two-step cascade reaction. Both enzymes were used as immobilized enzymes and tested in a preparative scale setup under process-near conditions. Oxygen supply, enzyme, and substrate loading of the oxidation of glutamate were investigated under controlled reaction conditions on a small scale before upscaling to a 1 L stirred tank reactor. LGOX was applied with a substrate concentration of 73.6 g/L (339 mM) and reached a space-time yield of 14.2 g/L/h. Additionally, the enzyme was recycled up to 3 times. The hydroxylase SadA reached a space-time yield of 1.2 g/L/h at a product concentration of 9.3 g/L (40 mM). For both cascade reactions, the supply with oxygen was identified as a critical parameter. The results underline the robustness and suitability of ?-ketoglutarate dependent dioxygenases for application outside of living cells.

SUBMITTER: Busch F 

PROVIDER: S-EPMC7493210 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature