Identification Of Aminopeptidase-producing Strain,Purification And Characteristics Of The Enzyme

Aminopeptidase is one kind of hydrolytic enzymes，hydrolyzing the amino acids fromthe N-terminal sequence of the peptides or proteins.Aminopeptidase exists widely. It is usedfor proteolytic debittering, depth protein hydrolyzate, preparation of bioactive peptides,medical research, etc. Therefore, it is of great research significance to separate and purifyaminopeptidase of high-purity, and to explore its enzymatic properties.In this paper, we preserve an aminopeptidase-producing strain and research themorphological, physiological and biochemical characteristics.then, by sequencing the16SrDNA and the method of specific PCR, we identified the strain as Bacillus Cereus.Therefore, the strain was named as Bacillus Cereus CZ.The purification of the enzyme typically involves three basic steps: extraction,purifycation, crystallization or preparation. Firstly, we obtain a crude enzyme solution bycentrifugation, secondly increasing the concentration by salting-out, and then apply thepurification technology of gel filtration and ion exchange chromatography. Finally,SDS-PAGE is used to detect the purity and the molecular mass of the enzyme. The molecularweight of the enzyme was110kDa.After the study of the enzymatic properties of aminopeptidase, we have the followingconclusions: the optimum pH is9.0and the optimum reaction temperature is58℃. Theenzyme exhibits excellent thermostability，and the pH stability range is8.0~9.0. As for themental ions, Zn2+、Mg2+show obvious promotions to enzyme activity while Ni2+、Cu2+showobvious inhibitions. As for the inhibitions, serine protease inhibitors PMSF and Pepstain haveno effect on aminopeptidase activity while protein-denaturant SDS has a strong inhibitoryeffect. Especially, metal-chelating agent EDTA strongly inhibits the enzyme activity of aminopeptidase. The Michaelis-constant (Km) is for7.73mmol L-1, and the maximum reactionvelo-city (Vmax) is for22.22μg mL-1min-1. In this study, we sucessfully identified the strain of the species relationship, obtaining thepurified aminopeptidase, and study the enzymatic properties. Through the study, we desire toprovide basis for industrial production of the aminopeptidase.