Characterization of Extracellular Protease from the Haloarcheon Halococcus sp. Strain GUGFAWS-3 (MF425611)

Halococcus agarilyticus GUGFAWS-3 (MF425611) was isolated from a marine white sponge of Haliclona sp., inhabiting the rocks in the intertidal region of Anjuna, Goa, India. Uniquely, the microbe simultaneously produces two halo-extremozymes in 25% NaCl, namely protease and lipase at 49.5 +/- 0.4 and 3.67 +/- 0.02 (U mL(-1)), respectively. The protease is constitutively produced in starch mineral salts medium with consistent 4 +/- 1.0 mm zone of enzyme production, regardless of the non-availability of protein as substrate. The ethanol precipitated enzyme on dialysis and Sephadex G-200 gel filtration chromatography was partially purified to 12.26-fold and was active between 20 and 80 degrees C, 0-5 M NaCl, and pH 3-13. Optimum activity, however, was at 70 degrees C, 3 M NaCl, and pH 7. The enzyme was thermo stable at 70 degrees C with 50.26 +/- 2.40% of relative enzyme activity at 75 min. Furthermore, it was stable in the presence of polar and non-polar organic solvents, detergents, and hydrocarbons. Several metal cations enhanced its activity in the order of Ca2+ > Ni2+ > Fe3+ > Co2+ > Mg2+ > Cu2+ > Mn2+. Dependence of enzyme on cysteine; serine, and metal ions was confirmed by beta-mercaptoethanol; PMSF and EDTA, respectively which induced its partial inhibition. Additionally, protease inhibited in vitro biofilm formation in Staphylococcus aureus. Conclusively, the production of a neutral halo-thermophilic protease is reported for the first time in the genus Halococcus.