Abstract:
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 percent NaCl, namely protease and lipase at 49.5 plus or minus 0.4 and 3.67?plus or minus?0.02 (U mL sup(?1)), respectively. The protease is constitutively produced in starch mineral salts medium with consistent 4 plus or minus 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 plus or minus 2.40 percent 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 Ca sup(2+) greater than Ni sup(2+) greater than Fe sup(3+) greater than Co sup(2+) greater than Mg sup(2+) greater than Cu sup(2+) greater than Mn sup(2+). 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.
