Isolation of a halophilic V<i>irgibacillus</i> sp. EMB13: Characterization of its protease for detergent application

Abstract

Isolation and characterization of a moderately halophilic bacterium, <i style="mso-bidi-font-style:normal">Virgibacillus </i>sp. EMB13, from the West coast of India has been described in the present study. It produced five different extracellular proteases. Conditions were optimized for maximum protease production. Under optimized conditions, <i style="mso-bidi-font-style: normal">viz</i>., a medium containing (%, w/v) yeast extract, 1.0; peptone, 0.5; mannitol, 0.5; NaCl, 0.5; KCl, 1.0; CaCl₂, 0.03 and MgSO₄, 0.04 with <i style="mso-bidi-font-style:normal">p</i>H 8, it produced 270 U protease mL^-1. The protease was partially purified by DEAE ion exchange and Sephadex G-75 gel filtration chromatography. The purified preparation contained two unseparated proteases with apparent molecular masses of 49 and 54 kDa, respectively. The preparation was stable at different concentrations of NaCl (0-15%, w/v) and <i style="mso-bidi-font-style:normal">p</i>H range 6.0-12.0. It exhibited maximal activity at <i style="mso-bidi-font-style: normal">p</i>H 7.5, temperature 50°C and 1% (w/v) NaCl. The K_m and V_max values towards casein were 7.5 mg mL^-1 and 156.25 µg min^-1 mL^-1, respectively. Inhibition of their proteolytic activity by EDTA and 1,10-phenanthroline indicated them to be metalloprotease in nature. The proteases exhibited remarkable stability in the presence of salt and organic solvents. These properties enable them for novel applications in non-aqueous enzymology and as an additive in detergent formulations.