Purification, Properties of a Cold-adapted Protease PA-1 Isolated from Marine Pseudomonas sp.

A cold-adapted protease named PA-1 was produced by Pseudomonas sp. isolated from the yellow-sea sediments. The protease was purified orderly by ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Superdex 200 molecular sieve. SDS-PAGE displayed that the protein molecular weight was 47kDa. PA-1 was stable at temperature range of 20 degrees C to 35 degrees C, and the maximum activity was achieved at 30 degrees C. The cold-adapted protease was stable in the pH range of 6.0-8.0, and the optimum pH was 7.0, indicating it belongs to the neutral protease. The influence of metal ions was evaluated. The addition of Mg2+, K+ enhanced the proteolytic activity of PA-1, while Li+, Na+ slightly inhibited the protease activity, Cu2+, Zn2+, Ag+ made the protease completely inactivated. Metalloprotease inhibitor EDTA and EGTA had no effect on the protease activity. The serine protease inhibitor PMSF and DFP significantly inhibited the protease activity which indicated that the cold-adapted protease belongs to the serine protease. Casein was chosed as substrate for determination of Michaelis-Menten kinetics. The K-m value and the V-max were 1.95 g/L and 26.23mmol/min, respectively. These properties demonstrated PA-1 was an ideal choice in low temperature food processing.