Purification And Properties Of The Extracellular Elastase Produced By Bacillus Subtilis

Elastase is a kind ofprotease that can specially degrade elastin.Elastase can be drawn from animal pancreas and microbe culture.With the broad-spectrum hydrolyzing activity,elastase has extensive applications in medical therapy,food processing and commodity chemicals industry.On the base of optimized culture conditions and fermentation technics,we had purified the Bacillus subtilis elastase with the aid of Sephadex G-100 column chromatograph and SDS-PAGE,and studied its enzymology property as well as its kinetics of enzyme catalyzing reactions.Then elastase hydrolyzation and tenderizing effect on meat was studied,contrasted with pancreatic elastase and Papain. The results were as follows:1.By the way of ammonium sulfate subsection salting-out,dialysis and gel chromatography on Sephadex G-100,we got two apices and collected the two products respectively.By activity test we found the latter had higher elastase activity.SDS-PAGE showed that its molecular weight was about 30KDa.Moreover,the lmmol/L Ca²⁺ kept elastase activity well during purification process.Until now,there was no interrelated report about adding Ca²⁺ in elastase purification procedure at home and abroad.2.The Bacillus subtilis elastase's optimal temperature for activity was 50℃and it could endure high temperature.The elastase was kept at 60℃for 4 hours,its activity was just decreased by 35%.It was active and highly stable in solution from pH 8 to 11,but there was a inflexion in pH 5.The study showed that its optimal pH for activity was associated with the different buffers we selected.It was about pH7.4 in Borax-boric acid buffer,and about pH8.6 in Tris-HC1 buffer.The elastase activity could be improved slightly by 0.3 mmol/L Na⁺.It also could be stimulated obviously by Ca²⁺ and Zn²⁺ when their concentration was 3mmol/L.The effect of Ca²⁺ was better than Zn²⁺.Most metal ions including Li⁺,K⁺, Mg²⁺,Ba²⁺ and Mn²⁺ could inhibit the enzyme activity.The stronger inhibition with higher concentration.By adding certain quantity of EDTA to the enzyme reaction system,we found that EDTA apparently had inhibited the enzyme activity and when the concentration of EDTA was increased to 50mmol/L,the activity was just 17%as high as that of enzyme reaction system without EDTA.3.The results clearly showed that different Bacillus subtilis elastase concentrations had different effect on elastolytic process.The elastin-degrading rate was greatly increased under the higher elastase concentration.The elastolytic processes under different substrate concentrations were not the same,too much or too low concentration had no increasing effect on elastolysis.The kinetic parameters for elastin-orcein was 0.0057g/ml and the maximal velocity was 0.0308U/ml,the catalyzing kinetic equation was V=0.0308[S]/0.0057+[S].4.The elastin,casein,glutin and bovine serum albumin(BSA) could be degraded by the Bacillus subtilis elastase,pancreatic elastase.Papain could hydrolyze above proteins except elastin.It was important that the Bacillus subtilis elastase showed the strongest hydrolysis ability on edible proteins which were rich in elastin such as beef,cattle stomach,cattle ligamentum,pork and hogskin.The hydrolysis ability of pancreatic elastase on such edible proteins was weaker than Bacillus subtilis elastase.Papain's hydrolysis ability was the weakest,moreover,the papain hardly hydrolyzed on cattle ligamentum.The shearing force test and sensory evaluation also demonstrated the conclusion.