Purification and properties of an extracellular cold-active protease from the psychrophilic bacterium Pseudoalteromonas sp. NJ276

The extracellular cold-active protease from the psychrophilic bacterium Pseudoalteromonas sp. NJ276 was purified by 22.5-fold using precipitate of saturation (NH₄)₂SO₄, DEAE-Sephadex A50 and Sephadex G-75. It was shown that purified enzyme was homogeneous in terms of SDS-PAGE with molecular mass estimate of 28 kDa. The protease depicted an optimal pH of 8.0 and was stable at pH 7.0-9.0, and its optimal temperature was at 30 °C. The protease was completely inhibited by PSFM. It was partially inhibited by metal salts, especially, had high tolerance to a wide range of NaCl concentrations (0-3 M NaCl). The highest k_cat and k_cat/K_m values were observed at 35 °C, and 35 and 54% of their highest values retained at 0 °C, respectively. Milk protein treated by this protease released more free amino acids than those treated by mesophilic papain at 4 °C. These results suggested that Pseudoalteromonas sp. NJ276 protease had broad substrate specificities and potential application in low-temperature food processing.