Purification and characterization of a novel extracellular protease from a halo‐alkaliphilic Bacillus sp. 17N‐1, active in polar organic solvents

A novel enzyme of molecular mass about 29 kDa was purified from the strain halo‐alkaliphilic Bacillus sp. 17N‐1 and designated protease‐B‐17N‐1. This enzyme is likely to be a cysteine protease; it was found active in media containing EDTAK2 and dithiothreitol, it maintained considerable activity at temperatures 14°C to 33°C and pH 6.50 to 8.50 with optimum k cat / Km and/or k cat values at pH 7.00 and 25°C. The activity of protease‐B‐17N‐1 was strongly affected by the specific irreversible inhibitor of cysteine proteases E‐64, while it remained unaffected by the 3,4‐dichloro‐isocoumarine, an irreversible inhibitor specific for serine proteases. Protease‐B‐17N‐1 retained full activity at 25°C after 30 min incubation at 8°C or at 33°C; moreover, it was found to be stable and active in the polar organic solvents DMSO and acetonitrile. The enzyme hydrolyzed the substrate Cbz‐FR‐pNA via Michaelis–Menten kinetics, while it showed insignificant activity for the substrate Suc‐AAA‐pNA. Valuable pKa s, rate constan...