Separation And Purification Of Alkaline Protease And Its Application In Hydrolysis Of Soy Protein Isolate

Alkaline proteases are important industrial enzymes, and widely used in food, washing,leather, pharmaceutical, feed and other industries. The lab screeninged a strain of highproducing alkaline protease, it was identified that the strain was belonged to Bacillusaltitudinis, and the optimal fermentation and protease producing conditions of the strain wereResearched. On the basis of the previous studies, this paper was to purify the alkalineproteases from Bacillus altitudinis PY41and studied its properties. The optimal hydrolysisconditions were determined, soy protein isolate was hydrolyzed at the optimal conditions ofalkaline protease, Flavourzyme, Protamex. The capacity of antioxidant and the ACE inhibitionof the hydrolysate were analysed.The fermentation supernatant of the Bacillus altitudinis PY41was used as a crudeenzyme solution, from which alkaline proteases was separated by (NH4)2SO4segmentationprecipitation and purified by Gel filtration and hydrophobic column chromatography. TheSpecific activity was518U/mg, Purification fold was36.7, Yield was10.3%. AndSDS-PAGE studies demonstrated that the enzyme had a molecular weight of29.5kDa.The properties of alkaline protease from Bacillus altitudinis PY41was studied, theoptimal pH, temperature for enzyme activity were9.5and50℃.The Km value was15.5mg/mL, the Vmax was24.57μg/(min·mL). Ag+and Hg2+can significantly reduced theenzyme activity, while Mn2+could significantly enhance enzyme activity, SDS has no effecton protease activity.The optimal hydrolysis conditions of alkaline protease from Bacillus altitudinis PY41was researched.The conditions of hydrolyzing soy protein isolate were optimized by singlefactor experiment and orthogonal experiment. The optimal hydrolysis conditions weredetermined as follows:temperature50℃, pH9.5, substrate concentration4%, enzymeconcentration10000U/g, hydrolysis time2h, under those conditions,the degree ofhydrolysis was22.04%.soy protein isolate ware hydrolyzed respectively in the optimal conditions by alkalineprotease, Flavourzyme and Protamex. The capacity of antioxidant and the ACE inhibition of the hydrolysate were analysed and compared. the three kinds of hydrolysate had the capacity ofantioxidant and ACE inhibition. The hydrolysate of alkaline protease had the higherscavenging activity of OH·andO2·, the hydrolysate of Protamex had the greatest capacityof ACE inhibition than others.