Discovery and characterization of high-activity formate oxidase with low Km value under non-acidic conditions

Formate oxidase (FOX) has applications in enzymatic assays, pollutant biodegradation, and bioorganic chemistry. However, its sources are few and none of them have significant activity above pH 7. We performed bioinformatic mining of FOX and identified 579 potential sequences. Two FOXs from Aspergillus nomiae (AnFOX) and Trichophyton rubrum (TrFOX) were expressed in E. coli. AnFOX had an alkalinity-tolerant pH activity range. It not only had a Km value for formate one order of magnitude lower, but also had far higher catalytic activity under non-acidic conditions. Spectroscopic and mass spectrometry analyses found that AnFOX contains an 8-formyl FAD cofactor. AnFOX enabled the H2O2-dependent sulfoxidation of thioanisole over pH 6-8. Structural analysis and kinetic studies revealed that the acidic residue E142, positioned on the surface adjacent to the isoalloxazine ring of flavin, could contribute to the unique pH preference. This novel FOX could greatly expand the applicability under non-acidic conditions.