Thermostable Alkaline Protease from a Novel Marine Haloalkalophilic Bacillus Clausii Isolate

Abstract

An oxidative and SDS-stable alkaline protease secreted by a marine haloalkalophilic Bacillus clausii isolated from the tidal mud flats of the Korean Yellow Sea near Inchon City was investigated in batch fermentation in shake flasks and in a bioreactor under a range of conditions. The isolate produced maximum protease yields (15,000 U ml−1) under submerged fermentation conditions at 42 °C for 40 h with an aeration of 1.5 v/v/min and agitation of 400 rev/min in a formulated soybean—casein medium (pH 9.6) containing (w/v): soybean meal (2%), casein (1%), corn starch (0.5%), NH4Cl (0.05%), NaCl (0.05%), KH2PO4(0.04%), K2HPO4(0.03%), MgSO4(0.02%), yeast extract (0.01%) and Na2CO3(0.6%). The optimal pH and temperature of activity of the partially purified enzyme were 11.5 and 80 °C, respectively. The alkaline protease showed extreme stability towards SDS and oxidizing agents, retaining its activity above 96 and 75% on treatment for 72 h with 5% SDS and 5% H2O2, respectively. The inhibition profile exhibited by phenylmethanesulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases.