Multiple proteases from Streptomyces moderatus. I. Isolation and purification of five extracellular proteases.

The presence of multiple proteases in the culture filtrate of Streptomyces moderatus was detected. After preliminary purification by ammonium sulfate precipitation and decolorization using DEAE-cellulose, the fractionation of various proteases was carried out using CM-trisacryl cation-exchange chromatography. By this procedure, four different protease fractions (Fr.) were separated (Fr. I, II, III, and IV). The first fraction was further separated into two different proteolytically active fractions (Fr. IA and Fr. Ib) by DEAE-trisacryl anion-exchange chromatography. Fraction Ia was purified further by affinity chromatography on N-carbobenzoxy-D-phenylalanyl triethylenetetramine-Sepharose 4B. The second fraction (Fr. Ib) was purified by gel filtration on Ultrogel AcA 44. For the purification of the other protease fractions (Fr. II, III, and IV) single-step affinity chromatography methods were employed. Protease fractions II and III were purified by epsilon-aminocaproyl-4-(4-aminophenylazo)phenylarsonic acid Sepharose 4B and protease fraction IV was purified on epsilon-aminocaproyl trialanine-Sepharose 4B. All five proteases purified were found to be apparently homogeneous by gel electrophoretic methods.