Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4.

An extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43,000 and contained 2 g atom Ca/mol. It was active over a pH range 4.8-9.5 and had optimum activity on casein at pH 7.0 with Km = 0.17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 degrees C; above 50 degrees C activity declined rapidly, but significant activity persisted at 4 degrees C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.