Work

Discovery and characterization of NADH oxidases for selective sustainable synthesis of 5-hydroxymethylfuran carboxylic acid

Year: 2024

Type: article

Abstract: Efficient regeneration of NAD + remains a significant challenge for oxidative biotransformations. In order to identify enzymes with higher activity and stability, a panel of NADH oxidases (Nox) was investigated in the regeneration of nicotinamide cofactors for the oxidation of hydroxymethyl furfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). We present novel Nox that exhibit remarkable catalytic activities, elevated thermal and pH stabilities, and higher intrinsic flavin loadings, thus eliminating the need for external flavin addition. The kinetic analysis of the NADH oxidases indicates that Ad Nox, Gd Nox, Cm Nox, and Lv Nox exhibit V max values of 86 U/mg, 50 U/mg, 4.3 U/mg, and 23 U/mg, respectively. When these NADH oxidases were applied in a HMF oxidation reaction, LvNox demonstrated the highest HMFCA yield of 97 % in the presence of 0.1 mM NAD and 10 mM HMF. In contrast to previously reported NADH oxidases from the same family, these NADH oxidases naturally accept NADPH as a substrate. Rapid kinetics experiments identified the oxidative reaction as the rate-limiting step of the reaction. NADH oxidases achieved high atom economy, a high reaction mass efficiency and a low process mass intensity. The findings contribute significantly to the field of biocatalysis and offer potential avenues for more environmentally friendly cofactor regeneration in chemical synthesis. • NADH oxidases from non-lactobacillus and lactobacillus organisms expand the catalyst toolbox for nicotinamide regeneration. • The NADPH-dependent actvity exceeds that of most previously reported NADH. • The high flavin loading of the NADH oxidases eliminates the cost for external or exogenous FAD addition. • LvNox achieved an impressive 97 % yield of HMFCA with a remarkable 100-fold decrease in cofactor requirement.

Source: Journal of Biotechnology

DOI: 10.1016/j.jbiotec.2024.11.009

Authors: Karishma Shah, Daniel Kracher, Peter Macheroux, Silvia Wallner, André Pick, Robert Kourist

Institutions: Graz University of Technology, Austrian Centre of Industrial Biotechnology (Austria)

Language: English

Topic: Enzyme Catalysis and Immobilization

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,830

Funders: Horizon 2020, H2020 Marie Skłodowska-Curie Actions