EXTRACELLULAR PROTEASE ACTIVITY IN ANTIBIOTIC-PRODUCING STREPTOMYCES-THERMOVIOLACEUS

Production of secondary metabolites was investigated in the thermophilic streptomycete Streptomyces thermoviolaceus grown at 45-degrees-C in a fermenter. Extracellular protein was secreted into the culture medium at the same time as an antibiotic granaticin; both were synthesized during the second slower phase of biphasic growth, which is most apparent at 45-degrees-C for this organism. Protease, assayed as azocaseinase, was identified as one component of, and marker for, the excreted protein. The effects of different growth temperatures revealed that the synthesis of extracellular protein, like that of the antibiotic, was maximal in cultures grown between 37-degrees-C and 45-degrees-C, whereas protease activity was greatest in 50-degrees-C grown cultures. A method was devised, based on acetone precipitation, for concentrating the protease activity from culture supernatants. Characterization of the concentrated activity using inhibitors suggested the presence of a serine and a metallo-type protease. A peptide substrate specific for the metallo-protease showed that it had a pH optimum for activity of 6.5-7.0. Approximately 50% of the activity was lost after 80 min of incubation at 70-degrees-C. Although calcium (5 mM) promoted increased thermotolerance such that around 65% of the activity remained after 100 min at 70-degrees-C, it seems that manganese and/or zinc may be more important for enzyme activity.