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

Abstract

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 constants, activation energies and other important features were estimated from the profiles of parameters k cat / Km , k cat and Km , versus pH, temperature, and [NaCl]. In addition, interesting results were obtained from the effect of different metallic ions and polar organic solvents on the Michaelis–Menten parameters of protease‐B‐17N‐1, showing that it performs catalysis via a (Cys)‐S−/(His)‐Im+H ion‐pair, as well as its industrial and biotechnological potential, respectively.