Cold Resistant Bacteria Pseudomonas Sp., W7 The Separation And Purification Of Low Temperature Protease Producing And Enzymology Properties

The cold-adapted protease has been widely studied for its high activity in lowertemperature as well as potential high values for application on food, detergence,cosmetics, medicine and environment biotechnology governance. Therefore, thecold-adapted protease has aroused interested by scientists around the world. In thispaper, the cold-adapted protease which is produced by the psychrotrophs Pseudomonassp.W7preserved in the laboratory has been extracted and purified, and then studied thecharacterizations of purified enzyme systematically. It will be a foundation for the studyof its gene clone and further industrial application.The strain of psychrotrophs Pseudomonas sp.W7was cultured at20℃for48h, andthen the protease was extracted by ultrafiltration, ammonium sulfate fractionation anddialysis, Sephadex G100gel filtration was next carried out for protease purification.SDS-PAGE analysis revealed a protein band of around50.0kDa. The specific activityreached7386.19U/mg and which was48.86folds higher than the crude fermentingliquor and the yield was up to11.40%.The characterizations of purified cold-adapted protease was consequently studied.The optimal temperature of the cold-adapted protease was37℃, The enzyme activityremained at least80%, when placed at the temperature from20℃to35℃for1h, theenzyme was thermal lability, the activity lost after incubated60min at55℃. Theoptimal pH of the protease was7.2, it was stable at range from pH4to10. The proteaseactivity was stimulated by Mn²⁺and inhibited by Ag⁺, Hg²⁺and Fe³⁺., Combined withthe inhibitor EDTA, it has almost lost the protease activity. It may be belongs tometalloproteinase. Organic solvents have a little influence on protease activity. Proteaseactivity was stimulated by glycerol, When the final concentration came to10%, theprorease was inhibited by isopropanol and isobutanol. The inhibitory rate reached to80.71%and63.12%. Among the protective agent, sodium alginate was suitable for thelong-term protection, Ca²⁺did so for short-term.The protease catalyzed the hydrolysis of casein, bovine haemoglobin and bovineserum albumin, With a Km of0.2369g/L for casein,0.4184g/L for bovine haemoglobin, as for bovine serum albumin was not observed. The result of dynamicsshowed that the casein was the optimal substrate. With the temperature increased, Kcatraised gradually. K_cat/K_mremained unchanged at10℃,20℃,30℃,40℃, and that suitsthe characterization of the cold-adapted protease. The protease was stable from10℃to40℃. Compared to mesophilic papain protease, more free amino acids from milkprotein hydrolyzed by the cold-adapted protease was deteacted, which was4.33timesthan mesophilic papain hydrolysis,34.05times than non-joined protease hydrolysis.