Purification and characterization of a halotolerant intracellular protease from Bacillus subtilis strain FP-133.

A halotolerant strain FP-133, able to grow at concentrations of 0-12.5% (w/v) NaCl, was isolated from a fish paste and identified as Bacillus subtilis . B. subtilis strain FP-133 produced an intracellular protease which showed catalytic activity under saline conditions. The enzyme was purified to homogeneity 143-fold with a yield of 0.9%. The purified enzyme showed an optimum activity at a concentration of 5% (w/v) NaCl. After storage in 7.5% (w/v) NaCl at 4 degrees C for 24 h, the enzyme kept 100% of its activity. The molecular mass of the protease was determined to be 59 kDa by gel filtration; the protein consisted of four subunits each with a molecular mass of 14 kDa. The enzyme showed aminopeptidase activity. It acted on L-leucyl-p-nitroanilide, L-leucyl-beta-naphthylamide, and oligopeptides containing glycine, L-histidine, or L-leucine. The K(m ) and V (max) values for L-leucyl-p-nitroanilide were 18 microm and 2.2 mm/h mg, respectively. The enzyme was activated by Fe(2+), Fe(3+), and Ni(2+) in synergism with Mg(2+).