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

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Synthesis

Background:

• Fe(II)- and α-ketoglutarate-dependent dioxygenases are key catalysts for synthesizing non-natural amino acids.
• The cost of the cosubstrate α-ketoglutarate (αKG) is a significant economic barrier in these processes.

Purpose of the Study:

• To develop a more economical and efficient method for producing non-natural amino acids.
• To couple in situ αKG production with αKG-dependent dioxygenase activity in a cascade reaction.
• To evaluate the performance of immobilized enzymes under process-near conditions.

Main Methods:

• A two-step cascade reaction was designed, combining N-succinyl amino acid hydroxylase (SadA) with L-glutamate oxidase (LGOX).
• Both enzymes were immobilized and tested in a preparative scale setup.
• Key parameters like oxygen supply, enzyme loading, and substrate concentration were optimized before scaling up to a 1 L reactor.

Main Results:

• The L-glutamate oxidase achieved a space-time yield of 14.2 g/L/h with a substrate concentration of 73.6 g/L and was recycled up to 3 times.
• The hydroxylase SadA achieved a space-time yield of 1.2 g/L/h at a product concentration of 9.3 g/L.
• Oxygen supply was identified as a critical factor for both cascade reactions.

Conclusions:

• The study highlights the robustness and suitability of α-ketoglutarate-dependent dioxygenases for cell-free applications.
• Immobilized enzyme systems offer a viable strategy for the cost-effective production of non-natural amino acids.
• This cascade approach enhances the efficiency of αKG regeneration and utilization in biocatalytic processes.