Fermentation Optimization Of Multicopper Oxidase Strain And Immobilization Of Enzyme For Degradation Of Histamine

Biogenic amines(BAs)are a group of organic compounds with low molecular weight containing nitrogen,which are mostly found in foods with high protein content.Excessive intake of biogenic amines will interfere with the body’s detoxification function,its toxic effects can cause adverse reactions such as headache,diarrhea,vomiting,palpitations,and can be life-threatening in severe cases.Therefore,it is necessary to control the content of biogenic amines in fermented food,enzymatic degradation is one of the important methods to reduce the content of biogenic amines.This study intends to screen strains which have the ability to degrade bioamines from fermented bean curd and fish sauce,and used it as the initial strain.Firstly,the fermentation conditions are optimized to improve its ability to produce multicopper oxidases(MCOs).Then separate and purify MCOs and study its related enzymatic properties.Finally,the immobilized MCOs were studied.The research contents and results are as follows:(1)Optimization of medium component and fermentation parameters for producing MCOs by strain.Screening several strains which can grow normally on histamine-containing plates from fermented bean curd and fish sauce,among them,the strain with the highest enzyme activity was identified as Bacillus tropicus,and identified as the object of subsequent experimental research.The medium component and fermentation parameters were determined by single factor experiment.Plackett-Burman experiment was used to determine the pH,loading amount,inoculation amount and temperature as four significant factors affecting enzyme production.The central combination of response surface design was determined by the steepest ascent design.Through response surface design,the optimal medium component(g·L-1)and fermentation parameters were obtained as follows:yeast extract 4,beef extract 5,peptone 10,glucose 20,MgSO4 0.2,MnSO4 0.05,K2HPO4 2.0,triammonium citrate 2.0,sodium acetate 5,tangu80 1,fermentation temperature 28℃,initial pH 6,inoculation amount 0.9%,loading amount 53 mL.Under these conditions,the enzyme activity was 167 U,which was 67%higher than that before optimization.(2)Isolation,purification and enzymatic properties of MCOs.MCOs was preliminarily purified by ammonium sulfate fractional precipitation,dialysis desalting,DEAE anion exchange chromatography and Sephadex G-100 gel chromatography,and obtained the crude enzyme of MCOs with less miscellaneous protein.Studies on the enzymatic properties of MCOs showed that:the optimum reaction temperature of MCOs was 50℃,the optimum reaction pH was 4.0,Cu2+,Ni2+,Mn2+ could activate the enzyme activity,while Fe2+and Ca2+inhibited it.MCOs had a high salt tolerance,MCOs had good stability at 40℃,the remaining enzyme activity was 52%at 50℃ for 40 min,while less than 1/3 at 60℃.(3)Study on the immobilization of MCOs.The immobilized enzyme was prepared by embedding,cross-linking and adsorption respectively.Comparing the enzyme activities of the three methods,it was found that chitosan cross-linking method has the best immobilization effect on MCOs.Through the analysis of several influencing factors in the preparation process,the optimal conditions for immobilization are:chitosan 3%,glutaraldehyde 1%,enzyme dosage 5 ml(3 g Immobilized pellet),enzyme solution pH 6.0,crosslinking time 4 h.The study on the enzymatic properties of immobilized enzyme showed that the optimum pH and temperature did not change,the thermal stability was improved.After reuse for 5 times,the immobilized enzyme could retain 60%activity.