Immobilization Of Alkaline Protease And Its Application

Alkaline protease is a proteolytic enzyme which belongs to endonuclease serine protease.Its outstanding feature is that it is suitable for higher pH environment and is widely used in food and washing industry.The immobilized enzyme has the advantages of high operating stability and easy recovery compared with the free enzyme.In this paper,the enzyme method was used to catalyze the hydrolysis of valsartan methyl ester to screen the suitable immobilized alkaline protease material.The influence of immobilized enzyme material on the catalytic performance of alkaline protease was investigated from the amount of protease binding and the enzyme activity recovery.A better immobilized alkaline protease carrier was obtained.The main work is as follows:First,starting from the immobilization method and carrier,the protease binding amount and the activity of immobilized enzyme were used as indicators.It was preliminarily screened that the recovery of immobilized alkaline protease by 1000EPN amino resin covalent binding method was 79%,the binding amount of protease reached 49.5 mg/g carrier,and the activity recovery of immobilized alkaline protease by agarose embedding method reached 76%.The immobilized process of 1000EPN amino resin was optimized.The results obtained by using 8%glutaraldehyde to activate 1 hour 1000EPN amino resin,immobilized condition under 20?,adding 0.1 M pH 7.5 phosphoric acid buffer solution immobilized 50mg protease,immobilized for 4 hours,protease binding rate up to 99.5%,and protease recovery up to 86%.Under optimal immobilization conditions,1000EPN amino resin covalently binding protease was stored in a refrigerator at 2-8?,storing for 180 days yet remaining 24.19%of enzyme activity.Second,the immobilized process of the selected agarose was studied.The results showed that the 0.1 M pH 9.5 carbonic acid buffer solution was selected and the mass fraction of 2.5%agarose was covered with 100 mg protease,and the enzyme activity recovery was as high as 80%.Under optimal immobilization conditions,the enzyme was stored in a refrigerator at 2-8 ? for 70 days,and the enzyme activity remained 30.37%.Finally,the hydrolysis of valsartan methyl ester was catalyzed by free enzyme,and the optimum catalytic hydrolysis conditions were determined.The results showed that under 40 ?,20 g/L protease catalyzed hydrolysis of 1 g/L valsartan methyl ester.After 44 hours reaction,the catalytic rate reached 86%.Based on the free enzyme catalysis process,the catalytic processes and operating stability of the two immobilized enzymes were investigated.The results showed that the catalytic properties of the two immobilized alkaline proteases were different from those of the free enzymes.The 1000EPN amino tree fat immobilized protease was used for 5 times after the hydrolysis of 50%valsartan methyl ester as the target.Enzyme activity remained 60.71%,agarose immobilized alkaline protease was reused for 5 times,and enzyme activity remained 35.52%.