里氏木霉酸性蛋白酶的纯化、酶学性质及稳定剂筛选OA

[Objective]To purify and study the enzymatic properties of acid protease from the Trichoderma reesei strain,and to screen the stabilizer formula.[Methods]The acid protease is purified by ammonium sulfate precipitation,dialysis,and anion exchange column chromatography.Then,its molecular weight is determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and its enzymatic properties are studied.Finally,the stabilizer formula of acid protease is optimized,with an investigation into its effect on acid protease storage stability.[Results]For the purified acid protease,its specific enzyme activity is 4 849 U/mg,purification fold is 3.28 times,and relative molecular weight is 40.The optimal reaction condition of the enzyme is a pH of 3.0 and a temperature of 55℃,with satisfactory stability in the pH range of 3.0 to 6.0 and below 60℃.The enzyme activity retention rate is 69%at 60℃,indicating high temperature resistance.In metal ions,Na+,Zn2+,and Mn2+protect acid protease stability.Stored at a concentration of 20%sodium chloride for 7 days,acid protease still retains certain activity,exhibiting high salt tolerance.The optimal formula of acid protease stabilizer is:maltose 26%,mannitol 12.5%,sodium chloride 12%,and zinc chloride 0.1 mol/L.Added with stabilizer,the enzyme activity of acid protease is 92.8%after storage at 45℃for 48 days.[Conclusion]Acid protease is high in heat and salt resistance,while the stabilizer formula can significantly improve acid protease stability.