Biochemical characterization of a low salt-adapted extracellular protease from the extremely halophilic archaeon Halococcus salifodinae.

Extracellular proteases from haloarchaea can expand the application fields of proteases. Exploring novel robust proteases is of great importance. An extracellular protease HlyA from Halococcus salifodinae was obtained by heterologous expression, affinity chromatography, in vitro refolding and gel filtration chromatography. Its activity was optimal at 45 °C, pH 9.0 and 1.5-2 M NaCl. Interestingly, although HlyA was from an extremely halophilic archaeon, it retained >75% of maximal activity in a broad NaCl concentration of 0.5-4 M. It displayed relatively stable activities over a wide range of temperature, pH and salinity. Thus, HlyA exhibited good temperature, pH and especially, salinity tolerance. Ca2+, Mg2+ and Sr2+ significantly enhanced the protease activity. HlyA activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting it is a serine protease. HlyA showed good tolerance to some surfactants and organic solvents. The Km and Vmax values of HlyA for azocasein were calculated to be 0.72 mM and 21.98 U/µg, respectively. HlyA was able to effectively degrade several protein substrates, including bovine hemoglobin, casein and azocasein. Generally, HlyA from the extremely halophilic archaeon Hcc. salifodinae is an alkaliphilic and low salt-adapted halolysin with high activity, thus representing an attractive candidate for various industrial uses.

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 °C, 0-5 M NaCl, and pH 3-13. Optimum activity, however, was at 70 °C, 3 M NaCl, and pH 7. The enzyme was thermo stable at 70 °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 ß-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.

Thermophilic and halophilic ß-agarase from a halophilic archaeon Halococcus sp. 197A.

An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a ß-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl2 or MgCl2. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65-80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl2, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.