Study On The Improvement Of Stability Of Aminopeptidase From Bacillus Subtilis

Aminopeptidases are exopeptidases which have wide applications in food, industry,chemical engineering, medicine and agriculture. The stability, especially the thermostabilityof aminopeptidase is very important in its extraction and application. In this paper, differentstabilizers and modification methods were used to improve the stability of a specificaminopeptidase from Bacillus subtilis Zj016, and the application of the enzyme in thepreparation of yeast extract was discussed.The effects of14different stabilizers on the thermostability and activity ofaminopeptidase were studied. Among them, glycerol, sodium chloride and Tween80werechosen for further testing. The optimal combination of stabilizers was confirmed byorthogonal experiment: glycerol10%, NaCl20mmol/L and Tween800.001%. After theaddition of combined stabilizers, the thermal stability, pH stability and storage stability of theenzyme are significantly improved. The enzyme activity remains94%after70d storage in thepresence of composite stabilizers, while it remains only78.6%without adding any stabilizer.Under the optimal conditions, succinic anhydride was chosen for the chemicalmodification. After chemical modification, the enzymatic activity and the affinity for substratewere increased. Other characteristics of the enzyme are also changes with the change of theconformation. The optimal temperature of the modified aminopeptidase increases from60℃to65℃, and the optimal pH shifts from8.5to10.0. Most importantly, the thermostability issignificantly improved after modification. After incubation at70℃for5h,70%of activitywas remained, whereas only35%of activity was remained for unmodified enzyme.Finally, aminopeptidase was combined with neutral protease to prepare yeast extract. Theoptimal hydrolysis conditions: substrate concentration8.5%, autolysis promoter NaCl2%,aminopeptidase powder770U/g, neutral protease28U/g. After hydrolyze at55℃for24hwith150rpm rotation, the total amino acids in hydrolyzate is determined to be11.73mg/mL,glutamic acid to be1.72mg/mL. The peptides with molecular weight less than500Da areaccounted to be82.78%. Compared with autolysis and sole use of aminopeptidase, thecombination use of two enzymes achieves better hydrolysis results in yeast extractionpreparation.