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 (Hmo(sc)) and Amycolatopsis orientalis (Hmo(Ao)) were screened and then they were heterologously expressed and characterized. At pH 7.3 40 degrees C, the Hmo(Ao) showed k(at) and K-m values of 140 min(-1) and 10.2 mM, the Hmosc showed kat and K-m values of 105.1 min(-1) and 2.06 mM. The Hmo(sc) was thermal stable and retained its 90% activity at 60 degrees C for up to 5 h, while Hmo(Ao) lost most of its activity at this temperature. The Hmo(sc) could effectively catalyze the conversion of L-mandelate to phenylglyoxylate at higher temperature using oxygen as the final electron acceptor. (C) 2014 Elsevier Inc. All rights reserved.