Statistical optimization of production, purification and industrial application of a laundry detergent and organic solvent-stable subtilisin-like serine protease (Alzwiprase) from Bacillus subtilis DM-04

Abstract

Optimum protease production of 518 U by Bacillus subtilis DM-04 in submerged fermentation was attained by response surface method. An alkaline protease, exists as zwitterionic form at pH 7.0 was purified to 23.5-fold by a combination of cation and anion exchange chromatography, ethanol precipitation followed by reverse-phase HPLC. The purified protease (Alzwiprase) contributes 29.0% of overall extracellular proteases of B. subtilis DM-04, has a subunit molecular mass of 16.9 kDa and exists as a monomer. It shows optimum activity at 45 °C and pH 10.0, respectively. The Km and Vmax values of Alzwiprase towards casein were determined as 59 μM and 336 μg min−1, respectively. Irreversible inhibition of enzyme activity of Alzwiprase with serine protease inhibitors demonstrates that it belongs to serine protease family, more particularly endopeptidase K and/or subtilisin-like protease. The significant stability and compatibility towards organic solvents, urea, surfactants, commercial laundry detergents as well as excellent stain removal and dehairing properties of Alzwiprase hold a tremendous promise for its industrial application.