Extraction And Characterization Of Producing Antihypertensive Peptide Protease From Lactobacillus Delbrueckii QS306

In this study,Lactobacillus delbrueckii QS306 with angiotensin converting enzyme(ACE)inhibitory activity was selected as the research object.The ACE inhibition rate of fermented milk was 54.50±1.06%after 48 h fermentation.On the basis of ACE inhibitory activity of its fermented milk,the fermentation conditions and enzyme extraction conditions of producing antihypertensive peptide protease produced by strain QS306 were optimized,and the extracted enzyme was isolated,purified and identified.The ACE inhibitory activity of the enzyme hydrolyzed milk protein and the enzymatic properties were studied.The conclusions are as follows:(1)Fermentation time,initial p H and temperature were used as variables.Through single factor and orthogonal experiments,the optimal fermentation conditions for producing antihypertensive peptidase by strain QS306 were determined as follows:When the initial p H of the medium was 7.0 and cultured at 37?for 16 h,the enzyme activity was 5.77±0.05 U/m L.Taking lysozyme dosage,pyrolysis temperature and pyrolysis time as variables,single factor and orthogonal experiments were carried out to determine the optimal conditions for protease extraction:When lysozyme was added 2mg/m L and incubated at 53?for 4.5 h,the enzyme activity was 8.40±0.05U/m L.The supernatant obtained by centrifugation was the crude enzyme solution.(2)The crude enzyme was purified by ultrafiltration and Sephadex G-75gel chromatography.The results showed that the specific activity of the crude enzyme could reach 109.02±5.46 U/mg and the purification ratio was 3.74.The purified protease was used to hydrolyze milk protein and the ACE inhibition rate was 31.50±3.38%.4 protein bands were obtained by SDS-PAGE gel electrophoresis,and their molecular weights were about 90k Da,70 k Da,40 k Da and 30 k Da respectively.The results of LC-MS/MS proteomics analysis were analyzed by in gel digestion.A total of 791 proteins,5132 polypeptides and 20 proteases were identified.(3)The Michaelis constant(Km)of the purified proteases from Lactobacillus delbrueckii QS306 was 2.78 mm,and the maximum reaction rate(Vmax)was 0.636.Using Me Osuc-Arg-Pro-Tyr-p NA(MS-Arg)as a specific substrate,the highest activity of this protease was obtained at 42?and p H 6.0.The protease activity can be activated by K⁺,Fe³⁺,Ba²⁺and Cu²⁺,and inhibited by Mg²⁺,Ca²⁺.The activity of the protease can be inhibited by EDTA,which indicates that the conformation of the active center of the protease is related to metal ions.