Production and characterization of haloalkaline protease from ascidian-associated Virgibacillus halodenitrificans RSK CAS1 using marine wastes

Abstract

A marine ascidian-associated bacterium, Virgibacillus halodenitrificans RSK CAS1, was optimized for protease production by response surface methodology using marine waste as substrate. The central composite design was employed, and the optimal medium constituents for maximum protease production (1461.11 U/ml) were determined to be shrimp shell powder (15.32 g/l), casein (5.37 g/l), MgSO4 (3.0 g/l) and NaCl (55.31 g/l). The protease was purified from the culture supernatant to homogeneity in a three-step procedure consisting of ammonium sulfate precipitation, ion exchange chromatography (DEAE-cellulose column) and gel-filtration chromatography (Sephadex G-75 column), resulting in a 8.7-fold-change in purified protein. This protein had a specific activity of 1,086.78 U/mg and a molecular weight of 21 kDa. It exhibited optimal activity at 50 °C, pH 9 and 25 % NaCl. The significant stability of this protein at higher levels of salt, metal ions, organic solvents and commercial detergents and at higher, temperature, as well as its application as a cleaning additive in blood stain removal, suggests its possible use the laundry detergent industry.