Discovery of Two Novel Oxidases Using a High‐Throughput Activity Screen

Discovery of novel enzymes is a challenging task, yet a crucial one, due to their increasing relevance as chemical catalysts and biotechnological tools. In our work we present a high‐throughput screening approach to discovering novel activities. A screen of 96 putative oxidases with 23 substrates led to the discovery of two new enzymes. The first enzyme, N‐acetyl‐D‐hexosamine oxidase (EC 1.1.3.29) from Ralstonia solanacearum, is a vanillyl alcohol oxidase‐like flavoprotein displaying the highest activity with N‐acetylglucosamine and N‐acetylgalactosamine. Before our discovery of the enzyme, its activity was an orphan one ‐ experimentally characterized but lacking the link to amino acid sequence. The second enzyme, from an uncultured marine euryarchaeota, is a long‐chain alcohol oxidase (LCAO, EC 1.1.3.20) active with a range of fatty alcohols, with 1‐dodecanol being the preferred substrate. The enzyme displays no sequence similarity to previously characterised LCAOs, and thus is a completely novel representative of a protein with such activity.