Improvement Of Multicopper Oxidase Applied Properties In Degradation Of Biogenic Amines By Employment Of Optimized Expression Strategies

Biogenic amines(BAs)are potential amine hazards that detected in fermented foods and alcoholic beverages.Excessive intake of biogenic amines may bring allergic symptoms such as vomiting and fever.Degradation of biogenic amines by enzyme catalysis is currently the most promising method,for its unnecessary changing of the food fermentation process and leaving no effect on food nutrition and flavor.Some enzymes belonging to the multicopper oxidase(MCO)family can degrade BAs by oxidation of them into harmless aldehydes and water.However,problems such as low activity under acidic conditions,poor catalytic stability,low expression level and no food-grade expression system available procrastinate the application of MCOs in degradation BAs in fermented foods.Therefore,improvement of the catalytic stability and activity of MCOs under application conditions,and enhancement of enzyem expression are of great importance for commercialization of MCOs.In this work,the catalytic stability and efficiency of MCO in degrading BAs were improved by fusional expression of MCO with catalase(CAT).Then recombinant Lactococcus lactis strains for food-grade expression of MCO were constructed and the expression level of MCO was highly improved.The results provide a good reference for reducing amine hazards in fermented foods through enzymatic degradation.The main results are as follows:(1)Improvement of catalytic stability of MCO by fusion expression of MCOF with CAT.It was found that H₂O₂decreased the activity of MCOF(MCO from Lactobacillus fermentum)significantly.Fused MCOFs with Bacillus subtilis catalase(CAT)were successfully expressed in Escherichia coli BL21.The tolerance to H₂O₂of eight fused MCOFs was increased by 51%–68%.Among them,the catalytic stability of CAT&MCOF in degrading histamine was increased by 17.3%.(2)Evaluation of biochemical and applied properties of fused MCO.The optimal reaction p H and reaction temperature of CAT&MCOF were determined to be 3.5 and 45?,respectively.Using ABTS as the substrate,the substrate affinity(K_m),the catalytic efficacy(k_cat/K_m)and the molar specific activity of CAT&MCOF were found being 1.00,1.71 and1.23 folds higher than those of MCOF,respectively.The stability of CAT&MCOF under acidic conditions(p H 2.5–4.5)and at moderate temperatures(35–55?)was also improved.Moreover,the degradation rates of putrescine,cadaverine and histamine by CAT&MCOF were 31.7%,36.0%and 57.8%,respectively.They were increased by 132.5%,45.7%and38.9%,compared to that of MCOF.(3)Recombinant Lactococcus lactis strains for expression of food-grade MCOB_L0(MCO from Bacillus amyloliquefaciens)and its mutants(MCOB_L1?MCOB_L2?MCOB_L3)were successfully constructed.The expression of MCOB_L0and its mutants was improved by optimizing the induction conditions and addition of Cu²⁺.The expression level of MCOB_L0reached to 2,235.84 U·L^-1,increased by 9.06 folds than that of the initial level(231.11 U·L^-1).Expression of mutants MCOB_L1,MCOB_L2and MCOB_L3reached to 4488.06 U·L^-1,4289.78U·L^-1and 2254.45 U·L^-1,increased by 12.35 folds,12.25 folds and 6.97 folds,respectively.(4)Evaluation of biochemical and applied properties of food-grade MCO.By studying the biochemical properties of MCO_Ls(MCOB_L0,MCOB_L1,MCOB_L2,MCOB_L3),it illuminated that the optimal reaction p H and temperature of MCOB_Ls were p H 4.5 and 65°C.These are different from MCOB_Es expressed in Escherichia coli(p H 3.0,55?).Moreover,in the p H range of 2.5–5.5,MCOB_Ls showed higher stability and catalytic efficiency than that of MCOB_Es.Among them,the catalytic efficiency of MCOB_L0using ABTS as the substrate is59.75%higher than that of MCOB_E0.Using histamine as the substrate,the degradation rates of MCOB_L0was 63.41%higher than of MCOB_E0.