An extracellular aspartyl proteinase from Mucor mucedo DSM 809 submerged cultures was purified by a two-steps chromatographic procedure. The enzyme had a molecular weight (MW) of 32.7 kDa, and an isoelectric point (pI) value of 4.29; no evidence of N-linked glycosylation was found. As judged by mass spectrometry, the primary structure of the M. mucedo enzyme presented homology with Rhizopus spp. proteinases. The secondary structure showed 4% alpha-helix, 48% beta-sheet and 48% random coil structure in 20 mM phosphate buffer (pH 5.8), as evidenced by circular dichroism spectroscopy. When acting on milk to provoke curd formation, the proteinase showed maximum potency at pH 5.0 and at 40 degrees C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55 degrees C for 10 min. These results indicate that the milk-clotting enzyme from M. mucedo can be considered as a potential substitute for bovine chymosin in cheese manufacturing. (C) 2012 Elsevier Ltd. All rights reserved.