Catalytic and thermodynamic characterization of protease from Halobacterium sp. SP1(1)

Osmolytes KCl, glycerol, mannitol, trehalose, sucrose, betaine, proline and Na-glutamate at different concentrations (5-30%) were investigated as effective solutes for retaining the activity of Halobacterium sp. SP1(1) protease in the absence of NaCl. Maximum activity was observed in the presence of 30% Na-glutamate. Kinetic and thermodynamic parameters for casein hydrolysis revealed that the protease was equally efficient in the presence of Na-glutamate as in NaCl. The enzyme was active over a broader range of temperature (20-80 °C) and was highly stable even at 80 °C with Na-glutamate. Thermodynamic parameters (ΔH*, ΔS*, ∆G*) for irreversible inactivation of protease at different temperatures (20-80 °C) were determined in the presence of Na-glutamate and NaCl. The efficiency of these osmolytes for thermal stability of protease was 30% (1.6 M) Na-glutamate >4 M (∼25%) NaCl >2 M (∼10%), suggesting that the effect exerted by the osmolyte depends not only on its chemical nature but also on its concentration. Na-glutamate was thus found to play an important role in thermal stabilization of enzyme substituting for NaCl. Moreover, substitution of NaCl by Na-glutamate may increase the applicability of halophilic enzymes in biotechnology and industry, which is otherwise limited to high NaCl concentrations.