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

• Published in: ACS sustainable chemistry & engineering 2020-06, Vol.8 (23), p.8604-8612
• Main Authors: Busch, Florian, Brummund, Jan, Calderini, Elia, Schürmann, Martin, Kourist, Robert
• Format: Article
• Language: English
• ISSN: 2168-0485; 2168-0485
• DOI: 10.1021/acssuschemeng.0c01122

Fe(II)- and α-ketoglutarate dependent dioxygenases have emerged as important catalysts for the preparation of non-natural amino acids. The stoichiometric supply of the cosubstrate α-ketoglutarate (αKG) 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 αKG to stereoselective αKG-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 α-ketoglutarate dependent dioxygenases for application outside of living cells. An efficient cell-free system for the production of a redox cosubstrate using a recyclable enzyme with a product formation rate of up to 14.16 g/L/h facilitates industrial usage of αKG-dependent dioxygenases.