Expression in Escherichia coli of an Unnamed Protein Gene from Aspergillus oryzae RIB40 and Cofactor Analyses of the Gene Product as Formate Oxidase

Abstract

An unnamed protein of Aspergillus oryzae RIB40 (accession no. XP_001727378), the amino acid sequence of which shows high similarity to those of formate oxidase isoforms produced by Debaryomyces vanjiriae MH201, was produced in Escherichia coli in C-His₆-tagged form. The gene product, purified by affinity column chromatography, catalyzed the oxidation of formate to yield hydrogen peroxide but showed no evidence of activity on the other substrates tested. The K_m and V_max values at 30 °C at pH 4.5 were 7.9 mM and 26.3 μmole/min mg respectively. The purified enzyme showed UV-visible spectra atypical of ordinary flavoproteins. The UV-visible spectra of the enzyme and the UV-visible spectra, fluorescence spectra, and mass spectrometry of the extract obtained by boiling the purified enzyme suggested that the enzyme has a non-covalently bound FAD analog, which is expected to be 8-formyl-FAD.