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°C to 35°C, and the maximum activity was achieved at 30°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 Km value and the Vmax value were 1.95 g/L and 26.23mmol/min, respectively. These properties demonstrated PA-1 was an ideal choice in low temperature food processing.
Pseudomonas cold-adapted, Protease isolation, Purification serine
© The Author(s) 2013. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.