Food‑grade expression of multicopper oxidase with improved capability in degrading biogenic amines

Biogenic amines(BAs)are potential amine hazards that are detected in fermented foods and alcoholic beverages.Excessive intake of BAs may lead to allergic symptoms such as difculty in breathing,nausea,and vomiting.Degradation of BAs by multicopper oxidase(MCO)is a promising method as it has little efect on the fermentation process,food nutrition,and favor.However,the application of MCO in food industry was restricted due to its poor catalytic properties and low productivity.In this work,food-grade expression of the Bacillus amyloliquefaciens MCO(MCOB)and its three mutants were successfully constructed in Lactococcus lactis NZ3900.The expression level of MCOB in L.lactis NZ3900 was dramatically enhanced by optimizing the cultivation conditions,and the highest expression level reached 4488.1 U/L.This was the highest expression level of food-graded MCO reported so far,to our knowledge.Interestingly,the optimal reaction pH of MCOB expressed in L.lactis NZ3900 switched to 4.5,it would be more suitable for degrading BAs in food as the pH value of most fermented foods was found to be 4.5.Moreover,MCOB expressed in L.lactis NZ3900 was quite stable(with more than 80%residual activity)in the pH range of 4.0–5.5,the catalytic rate constant(kcat)and specifc activity of MCOBLS were all dramatically increased compared with that of MCOB expressed in Escherichia coli.Using histamine as the substrate,the degradation of BAs within 24 h by MCOB expressed in L.lactis NZ3900 was 69.7%higher than that expressed in E.coli.The results demonstrated the potential applications of MCOB in food industry for reduction of biogenic amines.