Purification and characterization of subtilisin inhibitors ‘Marinostatin’ produced by marine Alteromonas sp.

The four novel peptide subtilisin inhibitors, which have been named Marinostatin B‐1, B‐2, C‐1 and C‐2, were purified from the culture supernatant fluid of a marine bacterium, Alteromonas sp. A combination of Diaion HP‐20 and CM‐cellulofine ion exchange column chromatographies, Sephadex G‐25 gel filtration and preparative high performance liquid chromatography (HPLC) were employed. Final preparations gave a single peak on HPLC. The molecular weights by gel filtration of Marinostatin B‐1 and C‐1 were estimated to be 1500, and those of B‐2 and C‐2 were 1700. All the inhibitors were stable in acidic (pH range 4–6) but less stable in alkaline solutions (pH 10). The metal ions Co 2+ and Fe 2+ repressed the inhibitory activity by 30 and 20%, respectively. The four inhibitors had inhibitory effects on serine proteases like α‐chymotrypsin.