Production, purification and characterization of protease produced from Cronobacter sakazakii

      In this study, five isolates of Cronobacter sakazakii  were obtained from biotechnology department, college of science, of AL-Nahrain university, primarily identified according to their cultural , morphological characteristics and biochemical tests. The ability of these isolates in protease production was examined. Results showed that all five isolates of C.sakazakii were protease producers with variable degrees. Among them, the isolate symbold  C.sakazakii R4 was the most efficient in protease production. The specific activity of protease producer in crude filtrate was 35.8 U/mg protein. Upon that, this isolate was selected to determine  optimum conditions for protease production. Results showed that maximum protease production was achieved after supplementation of the production medium (pH7) with 0.5% glucose, 1.5% tryptone and 0.1% KH2PO4 and inoculation with a 10⁴ CFU of fresh bacterial culture and incubated at 30°C in shaker incubator (150 rpm) for 24hrs. Under these conditions, the specific activity of protease produced in culture medium was sharply increased to 80.4U/mg protein. Protease produced under the optimum conditions was purified in four purification steps, first by precipitation with 60% saturation of ammonium sulfate,  dialysis, ion exchange chromatography using DEAE-Cellulose column, and the gel filtration chromatography throughout Sephacryl S-200 column .Specific activity of the purified enzyme was 285.7 U/mg with 3.5 folds of purification and 26.7% enzyme recovery. Purified protease was characterized by studing some  biochemical characteristics of the enzyme. Results showed that the molecular weight of protease produced by C.sakazakii R4  was about 68000 Dalton, the optimum pH for enzyme activity was 7 while that for stability was 6. Optimum temperature for enzyme activity was 35°C, hence the enzyme was stable with full activity at a range of temperatures between 20-40°C. The effect of chelating and reducing agents and heavy-metal ions on purified protease activity was investigated. Results showed that protease produced by the C. sakazakii R4  was activated by  Mg⁺², Ca⁺² and Cu⁺²,but Zn⁺² did not affect the activity of the protease. While EDTA was able to inhibit the protease activity and remaining activity reach to 36% which indicated that the enzyme is a metalloenzyme, but Iodoacetic acid showed no effect.