Studies On Purification, Modification And Cytotoxicity Of The Extracellular Metalloprotease Of The Pathogenic Vibrio Anguillarum

The marine bacterium Vibrio anguillarum is the causative agent of vibrosis, one of the major bacterial disease affecting marine animals. The extracellular metalloprotease has been considered as a putative virulence factor involved in the pathogenic mechanism of Vibrio anguillarum. Two pathogenic Vibrio anguillarum strains CW-1 and CW-4 originally isolated from diseased fish in Qingdao of Shandong province, China. This paper described the studies on purification, modification and cytotoxicity of the extracellular metalloprotease.The two strains CW-1 and CW-4 were studied on their growth and the expression of their extracellular metalloproteases under various growth conditions. The optimal temperature for bacterium growth is 28℃. When grown at 37℃, both the growth of the bacteria and the secretion of the extracellular metalloproteases were inhibited with different levels. 1mmol/L Zn2+ and Cu2+ can obviously prevent bacterium growth and reduce the enzymatic activity of Vibrio anguillarum CW-1 to respectively 83.25% and 81.4% of the control one, CW-4 to 71.71% and 78.76%, while Fe3+, Ca2+ and Mg2+ did not. Glucose, sucrose, maltose or some other carbons added to the medium can greatly increase bacterium growth, especially for glucose, while they greatly decreased the secretion of the enzymes. Both ampicillin and EDTA can strongly inhibit bacterium growth and the secretion of the enzymes, while EGTA had little effect.The extracellular protease was partially purified from the culture solution of Vibrio anguillarum strain CW-1 by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow and Sepharose 4B chromatography. The biochemical and physicochemical properties of the enzyme were studied. Molecular weight of the purified enzyme was 65.3kDa by...