Characterization And Heterologus Expression Of Multicopper Oxidases For Degradation Of Biogenic Amines

Biogenic amines?BAs?are low molecular weight organic compounds that present in fermented foods.Excessive intake of BAs through food can cause allergic reactions such as vomiting,palpitations,and rising of blood pressure,which can be life-threatening in severe cases.Degradation of BAs by enzymes is the most promising method for reduction of BAs in fermented foods.However,enzymatic properties and application properties of biogenic amine degrading enzymes are still not clarified.Therefore,it is of great importance to find proper enzymes and work out their enzymatic and applied properties,for establishing the enzymatic method to degrade BAs in fermented foods.The aim of this study was to obtain multicopper oxidases?MCO?with the ability to degrade BAs,heterologous expression and purification of it,and to study the enzymatic properties and their ability in degrading BAs.The results may lay a foundation for establishment of enzymatic control of biogenic amines in fermented foods.The main results are listed below:?1?Screening of strains producing MCO that is capable of degrading BAs.Five strains that produce MCOs and are capable of degrading BAs were screened out of 8000 strains,through primary and secondary screening.Among them,W.cibaria 17,B.amyloliquefaciens XJ03 and L.fermentum Y29 showed strongest BAs degradation capability.Thus,they were selected for further study on characterization of their MCOs.?2?Heterologus expression of MCOs and characterization of their enzymatic properties.Genes encoding MCO from W.cibaria 17,B.amyloliquefaciens XJ03 and L.fermentum Y29 were successfully cloned in Escherichia coli BL21.Expression of corresponding MCOs were detected to be 149.8 U·L^-1,212.3 U·L^-1 and 484.2 U·L^-1.The enzymatic properties of MCOs were studied.The optimal reaction temperature for three MCOs was detected to be 55?,50?and 50?,respectively.The optimal reaction pH was found to be 3.5,3 and 3.5.The K_m values of the recombinant MCOs were determined to be 0.17mmol·L^-1,0.07 mmol·L^-1 and 1.30 mmol·L^-1.The V_max values of the recombinant MCOs were 3.67×10^-2 mmol·L^-1·min^-1,4.67×10^-2 mmol·L^-1·min^-1 and 7.67×10^-2 mmol·L^-1·min^-1.It was found that metal ions such as Cu²⁺,Ni²⁺,Ca²⁺could activate MCO,the enzymatic activity was increased by 2⁴ times in the presence of Cu²⁺.They are capable of degrading seven of the tested BAs,including tryptamine,phenylethylamine,putrescine,cadaverine,histamine,tyramine and spermidine.Recombinant MCOF exhibited the best degradation capability with reduction of histamine and tyramine by 51.6%and 40.9%.?3?Investigation of application properties of MCOs.Among the three recombinant MCOs,MCOW was the best tolerant to salt?18%NaCl?,followed by MCOF,and MCOB was not salt tolerant.The tolerance of MCO to 2.5%ethanol from tolerant to not tolerant was detected to be MCOB,MCOF and MCOW.Preliminary studies on degradation of BAs in soy sauce with recombinant MCOs showed that MCOW had the highest degradation rate of total BAs in soy sauce?13.5%?and the best degradation in tyramine?17.9%?.The degradation rate of total BAs by MCOF was 10.6%,and degradation of histamine was the highest?11.3%?.The degradation rate of total BAs in soy sauce by MCOB was found to be the lowest?4.4%?.?4?Enhancment of expression of MCOs.The expression of MCOW and MCOF were increased to 599.7 U·L^-1 and 1146.3 U·L^-1,respectively,by optimization of IPTG concentration and induction temperature.By optimizing the medium composition,the expression levels of MCOW and MCOF were further increased to 3071.6 U·L^-1 and 3545.7U·L^-1,which were 20.6 times and 7.3 times higher than those before optimization,respectively.