Purification,Structural Analysis And Enzymatic Characterization Of Amine Degrading Enzyme From Enterococcus Faecium Ef2

In recent years,people pay more and more attention to the harm of biogenic amines in foods,so find an effective way to reduce the content of biogenic amines has become an urgent problem in the field of food safety.It is an effective way to reduce the content of biogenic amines by adding biogenic amine degrading enzyme into food during food fermentation.In this research,the Enterococcus faecalis Ef2 with biogenic amine degrading enzyme activity was used as the object of research,by evaluating its safety,optimizing the induction conditions for enzyme production,isolating and purifying the biogenic amine degrading enzyme,to explore the degradation characteristics of biogenic amine degrading enzyme,and provide theoretical support for better utilization of biogenic amine degrading enzyme.The main results and conclusions were as follows:1.The hemolysis test,antibiotics susceptibility test,and PCR amplification of virulence genes and drug resistance genes were used to assess the safety of Enterococcus faecium Ef2.The result of hemolysis test was negative,not sensitive to gentamicin and streptomycin,sensitive to vancomycin and most other antibiotics,and no virulence genes were detected,so it could be preliminarily determined that the strain is safety and could be used for following test and has potential application prospects.2.The single factor and orthogonal experiments were adopted to optimize the medium and culture conditions for biogenic amine degrading enzyme production from Enterococcus faecium Ef2.The optimal components of the medium were lactose 50 g/L,putrescine 6 g/L,yeast extract 8 g/mL,MgSO₄ 3 g/L,MnSO₄ 0.03 g/L,and sodium acetate 1.5 g/L,the optimum culture conditions were initial pH 7.0,inoculum amount 0.5%,culture time 36 h,and culture temperature 32?.The activity of enzyme increased from 10.90 U/mL to 26.12U/mL,which increased 139.63%.3.The ammonium sulfate fractional precipitation test,ion exchange column and gel filtration column were used to isolate and purify of biogenic amine degrading enzyme from Enterococcus faecium Ef2.It was showed that the optimal ammonium sulfate salt-out concentration was 70%;the Native-PAGE and SDS-PAGE had less other proteins,which indicated that the obtained enzyme had higher purity;the specific activity of the enzyme was increased from 0.49 U/mg to 3.53 U/mg,with a purification fold was 7.21 and recovery rate was 35.07%,indicated that the purification was well.4.The purified enzyme was identified and characterized by LC-MS/MS and FT-IR.The results showed that the purified biogenic amine degrading enzyme was aminotransferase with a molecular weight was 49.045 kDa and amino acid number was 391;the contents of?-helix?b-sheet?b-ture and random coil were about 23.39%,36.29%,12.1%and 28.23%,respectively.5.By researching the enzymatic characterization of purified enzyme,obteined the optimum reaction temperature was 30?,and had good thermal stability in temperature range of 25?⁴0?,the enzyme activity fell sharply when more than 40?;The optimal pH was 7.0,with acid-base stability in the pH range of 6.6⁷.8;1 mmol/L Cu²⁺could promote the enzyme activity;common inhibitors like SDS,EDTA,b-mercapto ethanol,DTT and PMSF could inhibit the enzyme activity,and EDTA had the strongest inhibitory ability;the optimum substrate was putrescine.