Purification and characterization of an extracellular cysteine proteinase produced by Micrococcus sp INIA 528

Micrococcus sp. INIA 528, a micro-organism isolated from raw ewe's milk Manchego cheese, produced an extracellular proteinase. This enzyme was purified to homogeneity from culture supernatant fluid in two chromatographic steps, with a 29-fold increase of specific activity and a 28% recovery of proteinase activity. The homogeneous protein was characterized biochemically. The molecular weight of the enzyme was determined to be 19.4 kDa by mass spectrometry. The purified enzyme was inhibited by E-64, PMSF and iodoacetamide and activated by cysteine, glutathione, dithiothreitol and beta-mercaptoethanol. These results suggest that the enzyme is a cysteine proteinase. Optimal conditions for activity on azocasein were 34 degrees C and a pH of 7.0. The proteinase preferentially degraded beta-casein, while after a longer incubation period alpha(s1)-casein was also extensively hydrolysed. The proteinase had a K-m value of 6.12 g l(-1) for casein and 2.20 g l(-1) for azocasein.