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

Florian Busch, Florian Busch, Jan Brummund, Elia Calderini, Martin Schürmann, Robert Kourist*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)8604-8612
Number of pages9
JournalACS Sustainable Chemistry and Engineering
Volume8
Issue number23
DOIs
Publication statusPublished - 15 Jun 2020

Keywords

  • Biocatalysis
  • Cascade reactions
  • Cell-free system
  • Immobilized enzymes
  • In situ production
  • l -Glutamate oxidase
  • N-Succinyl amino acid hydroxylase
  • Non-natural amino acids
  • Recycling
  • Sustainability

ASJC Scopus subject areas

  • Chemistry(all)
  • Environmental Chemistry
  • Chemical Engineering(all)
  • Renewable Energy, Sustainability and the Environment

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