Purification and properties of the extracellular Metallo-proteinases of Chromobacterium lividum (NCIB 10926)

Abstract

Four extracellular proteolytic enzymes (I–IV) (EC 3.4.22.—) were identified in static cultures of Chromobacterium lividum (NCIB 10926) by agar gel electrophoresis and isoelectric focusing. Proteinases I–III were freed of nonenzymic protein by chromatography on TEAE-cellulose and CM-cellulose. The enzyme mixture was then fractionated in a pH gradient by isoelectric focusing. All three enzymes were shown to be heat-labile metallo-enzymes. Optimal activity occurred at pH 5.6 for enzyme I and at pH 6.2 for enzymes II and III. Remazolbrilliant Blue-hide powder was a sensitive substrate for these enzymes. Proteinase I was also shown to degrade haemoglobin and casein effectively, but not myoglobin, ovalbumin or bovine serum albumin. Proteinases I–III exhibited molecular weight values of 75 000, 72 000 and 67 000 by exclusion chromatography and 71 000 and 66 000 by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis for enzyme I and II, respectively. The amino acid compositions of enzymes I and II were somewhat similar. Proteinase I was inhibited by EDTA, 1,2-di(2-aminoethoxy)ethane- N, N, N′, N′-tetraacetic acid or 1,10-phenanthroline and both Ca 2+ and Co 2+ were required for maximal activity. Mg 2+ could substitute for Ca 2+ or Mn 2+ for Co 2+. The interrelationship of proteinases I–III is discussed.