Three proteolytic enzymes-the metalloproteinase, SFMP, and two serine proteinases, SFSP and SFTP-have been isolated and purified from the culture fluid of Streptomyces fradiae using chromatography on bacitracin-silochrome, bacitracin-Sepharose, DEAE-cellulose and fractionation by ammonium sulfate. Study of physico-chemical and functional properties of the enzymes and structural analysis revealed that SFMP is a cysteine-containing metalloendopeptidase with M(r) of 36 kDa, has a peak activity for synthetic substrates at pH 7.0-7.5 and at 60-65 degrees C and is stable at pH 7.0-9.0. The serine proteinase SFSP is related to subtilisin-like enzymes, has a M(r) of 29 kDa and a pH optimum at 7.5-8.5 at temperature up to 50 degrees C. The proteinase is stable at pH 4.0-9.0 and retains 30% of its activity at 70 degrees C. The other serine proteinase, SFTP, has a M(r) of 26 kDa and is related to trypsin-like enzymes. Its activity for synthetic substrates of trypsin is maximal at pH 6.8-8.8 at 50 degrees C. The enzyme is stable at pH 4.5-8.5 and at temperature below 50 degrees C. It has been shown that Streptomyces fradiae, like Streptomyces griseus and other Streptomycetes, possesses an ability to secrete serine proteinases (SFSP and SFTP) related to two evolutionally distinct families of serine proteinases, i.e., subtilisin and chymotrypsin families. SFMP and SFSP have been isolated and characterized for the first time.

Stepwise application of affinity chromatography on bacitracin-silochrome, gel filtration on Acrylex P-10, rechromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-15, a homogeneous metalloproteinase (M(r) = 35,000 Da) has been isolated from the cultural filtrate of B. megaterium strain 599. The amino acid composition and N-terminal sequence (20 amino acids) of the enzyme have been determined. The proteinase is not inhibited by diisopropyl-fluorophosphate, is inhibited by o-phenanthroline, EDTA, and Zn2+, and is activated by Co2+. The enzyme has a peak activity at 60-65 degrees C. The maximum of the enzymatic activity after hydrolysis of synthetic substrates is at pH 6.5-7.0. The enzyme is stable at pH 7.0-9.0 and retains its stability at 45-60 C for several hours. In acid media the enzyme undergoes irreversible inactivation. The dependence of kcat/Km on pH points to the involvement of an ionogenic group with pKa 7.5 in the catalytic act, most probably of the imidazole group of histidine. The metalloproteinase hydrolyzes synthetic peptide substrates at the bonds formed by the amino groups of hydrophobic amino acids-Phe, Leu, Ile and Val.

A homogenous metalloproteinase with molecular weight 35 kD was isolated from the culture medium of Bacillus megaterium strain 599 by stepwise application of affinity chromatography on bacitracin-silochrom, gel filtration on Acrylex P-10, rechromatography on bacitracin-Sepharose 4B, and gel filtration on Sephadex G-25 The amino acid composition and N-temminal sequence (20 amino acids) were determined The proteinase is not inhibited by diisopropyl fluorophosphate. It is suppressed by o-phenanthroline, EDTA, and Zn2+ but activated by Co2+. The enzyme shows peak activity at 60-65 degrees C. The activity for synthetic substrates is maximal at pH 6.5-7.0. The enzyme is stable at pH 7.0-9.0 and retains its stability at 45-60 degrees C for several hours. In acid media the enzyme is irreversibly inactivated The pH dependence of k(cat)/K-m suggests the involvement of an anionic group with pK(a) 7.5 (probably the imidazole group of histidine) in catalysis. The metalloproteinase hydrolyses synthetic peptide substrates at the bonds formed by amino groups of hydrophobic amino acids (Phe, Leu, Ile, Val).