ISOLATION AND CHARACTERIZATION OF METALLOPROTEINASE FROM BACILLUS-MEGATERIUM

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).