PARTIAL-PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR METALLOPROTEASES WITH CASEINOLYTIC, AMINOPEPTIDOLYTIC AND COLLAGENOLYTIC ACTIVITIES FROM VIBRIO-ANGUILLARUM

Proteases produced by Vibrio anguillarum were isolated from culture supernatant by ultrafiltration, gel chromatography and ion-exchange chromatography. The enzyme(s) were shown to be collagenolytic when assayed with native collagen substrates. In addition, the enzyme(s) hydrolysed azocasein, azocollagen, the collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg and the aminopeptidase substrate L-Leu-pNA effectively. Separation of the proteases by Mono Q ion exchange chromatography and native polyacrylamide gel electrophoresis revealed four distinct protein bands containing caseinase activity. However, only two of the bands showed aminopeptidase activity. The aminopeptidase activities could be separated from the caseinase activities by isoelectric focusing. Secreted proteases of different serotypes of V. anguillarum showed a heterogeneous caseinolytic pattern. The molecular mass of the major enzyme was estimated at 35 kDa as determined by its mobility on SDS-polyacrylamide gels. Serine protease inhibitors like PMSF, TPCK, TLCK and benzamidine had no inhibitory effects on the proteolytic activity when tested with azocasein as substrate. However. the enzyme was strongly inhibited by metal chelators like EDTA and 1,10-phenanthroline. Also, normal salmon serum and purified alpha(2)-macroglobulin From salmon serum strongly inhibited the caseinolytic activity of the enzyme.