Sequence-based screening and characterization of cytosolic mandelate oxidase using oxygen as electron acceptor

• Sequence features for mandelate oxidase (MO) were summarized, and used in the screening of MO.
• By using sequence-based screening, the HmoSC and HmoAO were screened.
• The MO activities of HmoSC and HmoAO were identified, and characterized.
• The HmoSC showed a better characterization than HmoAO.

Sequence-based screening was carried out to find a type of cytosolic mandelate oxidase that converted l-mandelate to phenylglyoxylate using oxygen as the final electron acceptor. The sequence features of the cytosolic mandelate oxidase were summarized, and were used in the screening process. Mandelate oxidases from Streptomyces coelicolor (HmoSC) and Amycolatopsis orientalis (HmoAO) were screened and then they were heterologously expressed and characterized. At pH 7.3 40 °C, the HmoAO showed kcat and Km values of 140 min−1 and 10.2 mM, the HmoSC showed kcat and Km values of 105.1 min−1 and 2.06 mM. The HmoSC was thermal stable and retained its 90% activity at 60 °C for up to 5 h, while HmoAO lost most of its activity at this temperature. The HmoSC could effectively catalyze the conversion of l-mandelate to phenylglyoxylate at higher temperature using oxygen as the final electron acceptor.