The optimized medium composed by casitone (0.3%) and molasses (1%), previously described, was used for the production of extracellular proteases by Streptomyces sp. 594 in a bench scale fermenter at 200 rpm and I vvm. In these conditions maximum protease production, 99 U/ml, obtained after 120 It incubation at 30 degreesC, was not growth associated. Crude enzyme showed best activity at a temperature range from 55 to 70 degreesC, pH 6.0, and was stable from -20 to 40 degreesC. Proteolytic activity and stability were significantly enhanced by Ca2+ and Ba2+ at 55 degreesC after I h. Copper and EDTA caused a loss of 46 and 74% of the original activity, respectively. SDS-PAGE plus gelatin showed three active zones suggesting that Streptomyces sp. 594 synthesizes multiple thermophilic proteases with molecular masses different from those described in literature. This was confirmed by the inhibition with PMSF and phenantroline, which revealed the presence of distinct serine and metal requiring proteases. These indications and the wide specificity to various soluble and cheap insoluble proteinaceous substrates offer an interesting potential for enzymatic and/or microbiological hydrolysis at the industrial level. (C) 2004 Elsevier Inc. All rights reserved.