Studies On Extration Of Extracelluar Elastase Of Bacterial Strains And Characteristics Of Elastase

Pseudomonas aeruginosa SWU-M was studied in our lab which secrete extracelluar elastase. Its elastase activity could be 15u/ml in its fermentation medium.Bacterial cells were then removed by high-speed centrifugation and then elastase was purified in this medium.1. Salting-out and precipitation by organic solvents were used separately.Its specific activity was 47u/mg.pro in salting-out with fractionation of 30%-50% under the condition of 4℃ and pH4.91 and the yield of enzyme was high.While the specific activity in precipitation by acetone was only 37u/mg.pro which was complex and desired high refrigeration (-20℃) and during which acetone concentration was adjusted to 50%-75%(v/v) and the ionic strengthen was 0.2 mol/L under the condition of pH4.91.During this process the yield of enzyme was low and tended to lose its activity compared with the former. So the medium was centrifugated at 4000g for 30min at 4℃,and the crude elastase was precipitated from the culture supernatant fluid with solid ammonium sulfate.The resulting precipitate was dissolved and dialyzed aginst distilled water overnight at 4℃ with four changes of distilled water and ultrafiltrated at 4℃ and 0.5x 105 Pa.At last the elastase activity of the production was 75u/mL and the specific activity was 100u/mg. pro.2. Optimum pH of the bacterium elastase differed in different buffer.lt could be 7.8 in boric acid buffer and 8.48 in Tris buffer;it had maximal activity at temperature 50℃.Elastase was active and highly stable in solution from pH7 to 12 and at temperature under 40℃.3. Heavy metal could inhibit the elastolytic activity to some degree except calcium ;the presence of EDTA in the solution acted as an inhibitor to the enzyme .4. The addition of glycerol and 2-mercaptoethanol in the solution did some good to elastolytic activity and thermal stability;a trace of sulfate ammonium could activate the enzyme.5. The elastolytic activity was found to be enhanced by detergent,notwithstanding the degree of elastolytic activity was different.6. The kinetic parameters determined at 37℃ for orcein-elastin was 0.741% and the maximal velocity was 0.09512u/ml;experiment on the adsorption of enzyme onto elastin showed that the abilities of the adsorptin onto the elastin of nonelastolytic proteinases were considerably small in comparison with those of elastase and elastase showed a high priority on hydrolysis on elastin.7. In this experiment elastin> gluthu casein > albumuu egg albumin could be degraded by the elastase and their hydrolysate existed mainly in the form of proline and hydroxide proline.The bacterial elastase showed a high hydrolysis ability on meat sample rich in elastin such as cattle stomach,hogskin and pure elastin.For some meat such as beef and pork,the hydrolysis rate of elastase could be 59.9% and for some more tender meat such as fish, it could be 22.8%.8. In the experiment of meat tenderness,the effect of enzymes on meat tenderness of elastase-injected samples was superior to those of papain-injected samples.These findings suggested that this alkaline elastase is promising as a favorable meat tenderizer.