Identification of redox activators for continuous reactivation of glyoxal oxidase from Trametes versicolor in a two-enzyme reaction cascade

Abstract Glyoxal oxidases, belonging to the group of copper radical oxidases (CROs), oxidize aldehydes to carboxylic acids, while reducing O 2 to H 2 O 2 . Their activity on furan derivatives like 5-hydroxymethylfurfural (HMF) makes these enzymes promising biocatalysts for the environmentally friendly synthesis of the bioplastics precursor 2,5-furandicarboxylic acid (FDCA). However, glyoxal oxidases suffer from inactivation, which requires the identification of suitable redox activators for efficient substrate conversion. Furthermore, only a few glyoxal oxidases have been expressed and characterized so far. Here, we report on a new glyoxal oxidase from Trametes versicolor ( Tv GLOX) that was expressed at high levels in Pichia pastoris (reclassified as Komagataella phaffii ). Tv GLOX was found to catalyze the oxidation of aldehyde groups in glyoxylic acid, methyl glyoxal, HMF, 2,5-diformylfuran (DFF) and 5-formyl-2-furancarboxylic acid (FFCA), but barely accepted alcohol groups as in 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), preventing formation of FDCA from HMF. Various redox activators were tested for Tv GLOX reactivation during catalyzed reactions. Among them, a combination of horseradish peroxidase and its substrate 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) most efficiently reactivated Tv GLOX. Through continuous reactivation of Tv GLOX in a two-enzyme system employing a recombinant Moesziomyces antarcticus aryl-alcohol oxidase ( Ma AAO) almost complete conversion of 8 mM HMF to FDCA was achieved within 24 h.