Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1

Abstract

A thermoalkaline protease with a molecular weight of 22kDa was purified from the Bacillus cereus SIU1 strain using a combination of Q-Sepharose and Sephadex G-75 chromatography. The kinetic analyses revealed the Km, Vmax and kcat to be 1.09mgml−1, 0.909mgml−1min−1 and 3.11s−1, respectively, towards a casein substrate. The protease was most active and stable at pH 9.0 and between a temperature range of 45–55°C. It was fully stable at 0.0–2.0% and moderately stable at 2.5–10.0% (w/v) sodium chloride. Phenyl methyl sulfonyl fluoride, ethylene diamine tetra acetic acid and ascorbic acid were inhibitory with regard to enzyme activity, whereas cysteine, β-mercaptoethanol, calcium, magnesium, manganese and copper at concentration of 1.0mM increased enzyme activity. Sodium dodecyl sulfate, Triton X-100, Tween 80, hydrogen peroxide and sodium perborate significantly enhanced protease activity at 0.1 and 1.0% concentrations. In the presence of 0.1 and 1.0% (w/v) detergents, the protease was fairly stable and retained 50–76% activity. Therefore, it may have a possible application in laundry formulations. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the protease is predominantly a β-pleated structure and a detailed structural composition showed ∼50% β-sheets. The CD-based conformational evaluation of the protease after incubation with modulators, metal ions, detergents and at different pH values, revealed that the change in the β-content directly corresponded to the altered enzyme activity. The protease combined with detergent was able to destain blood stained cloth within 30min.