A family AA5_2 carbohydrate oxidase from Penicillium rubens displays functional overlap across the AA5 family.

Copper radical alcohol oxidases belonging to auxiliary activity family 5， subfamily 2 (AA5_2) catalyze the oxidation of galactose and galactosides， as well as aliphatic alcohols. Despite their broad applied potential， so far very few AA5_2 members have been biochemically characterized. We report the recombinant production and biochemical characterization of an AA5_2 oxidase from Penicillium rubens Wisconsin 54-1255 (PruAA5_2A)， which groups within an unmapped clade phylogenetically distant from those comprising AA5_2 members characterized to date. PruAA5_2 preferentially oxidized raffinose over galactose; however， its catalytic efficiency was 6.5 times higher on glycolaldehyde dimer compared to raffinose. Deep sequence analysis of characterized AA5_2 members highlighted amino acid pairs correlated to substrate range and conserved within the family. Moreover， PruAA5_2 activity spans substrate preferences previously reported for AA5 subfamily 1 and 2 members， identifying possible functional overlap across the AA5 family.