Purification and characterization of an extracellular metalloprotease from Pseudomonas fluorescens.

An extracellular metalloprotease was purified from the culture supernatant of Pseudomonas fluorescens strain KT1 to apparent homogeneity and shown to consist of a single polypeptide chain (M(r) 46,000-47,000). The enzyme was strongly inhibited by chelating agents such as EDTA and o-phenanthroline, and activated by certain detergents. Among the peptidyl 4-methylcoumaryl-7-amide (MCA) substrates examined, t-butyloxycarbonyl-Arg-Val-Arg-Arg-MCA was the best one. With this substrate, the enzyme exhibited a pH optimum of around pH 5.5 in the absence of Co2+ ions, whereas it showed two different pH optima (at pHs around 5.5 and 8-9) in the presence of Co2+ ions due to remarkable activation by Co2+ ions in the alkaline pH range. On the other hand, a single broad pH optimum of around 6 to 8 was obtained with some peptides in both the presence and absence of Co2+ ions, and no activation by Co2+ was observed. The enzyme showed trypsin-like specificity, preferentially cleaving certain arginyl peptide bonds, and hydrolyzed the basic protein, histone, most rapidly among various proteins examined. Partial amino acid sequence analysis revealed that the enzyme is highly homologous with proteases of the serralysin family, a group of zinc metalloproteases.