Improving The Application Characteristics Of Multicopper Oxidase Through Molecular Modification

Biogenic amines?BAs?are a group of organic compounds that present in fermented foods.The excessive intake of biogenic amines is possible harmful to human health.Some enzymes belong to the multicopper oxidase?MCO?family can degrade a variety of biogenic amines.This confers a good application prospect of MCO in reducing ammonia?amine?hazards in fermented foods.However,problems including low catalytic efficiency,poor tolerance to low pH or hyperosmolar and secretory expression,need to be solved for the application of MCO.The foundation for industrial production and application of enzymatic reduction of BAs in fermented foods will be laid by doing this.In this study,the multicopper oxidase MCOB from Bacillus amyloliquefaciens was modified by combining site-directed single mutation and complex mutation to improve its catalytic efficiency,salt tolerance and degradation of histamine.MCOB was successfully extracellularly expressed in Escherichia coli by fusing the signal peptide to the N-terminal of the enzyme,and the expression of MCOB was optimized.The main results are as follows:?1?Improvement of the catalytic efficiency of multicopper oxidase by site-directed single mutation.Two single mutants of MCOB,T317N and L386Y,were successfully constructed,and their expression levels in Escherichia coli were 1126.5 and 1093.7 U?L^-1,which were 1.65and 1.60 times as much as the wild type,respectively.By analyzing the enzymatic properties of the mutants,it was found that the catalytic efficiency?Kcat/Km?of T317N and L386Y were10.52 and 14.19 L??mol^-1?s^-1,which were 43.0%and 94.0%higher than the wild type,respectively;the specific activities of T317N and L386Y were 4.63 and 5.72 U?mg^-1,which were 1.39 and 1.72 times as much as the wild type,respectively.The optimal reaction pH of mutants T317N,L386Y and the wild type MCOB were all 3.0,and the optimal reaction temperature were all 55°C.The stability of the single mutants under different pH conditions were improved,but the stability of the single mutants under different temperature conditions were reduced compared to the wild type.?2?Enhancement of the salt tolerance of multicopper oxidase by site-directed complex mutation.Based on the single mutants,MCOB double mutant T317N/L386Y,triple mutants T317N/L386Y/S427E and T317N/L386Y/A110E,and quadruple mutant T317N/L386Y/S427E/A110E were successfully constructed.The catalytic efficiency of the double mutant T317N/L386Y and the triple mutant T317N/L386Y/S427E were significantly higher than the wild type MCOB,which were 2.16 and 1.95 times as much as the wild type MCOB,respectively;the catalytic efficiency of other complex mutants were all reduced.In addition,the salt tolerance of the triple mutant T317N/L386Y/S427E was significantly improved compared to the wild type,and its tolerance to 15%NaCl was increased by 61.3%.The optimal reaction pH and optimal reaction temperature of the complex mutants were unchanged,and the stability of the complex mutants under different pH conditions were improved,but the stability of the complex mutants under different temperature conditions were reduced compared to the wild type.Degradation of histamine by the MCOB mutant T317N/L386Y/S427E?with the best salt tolerance?was much better than the wild type in th presence of 15%NaCl.When using 15000 U?L^-1 enzyme,the degradation rate of the triple mutant T317N/L386Y/S427E was 29.4%,which was 40.0%higher than that of the wild type MCOB.?3?Secretory expression of multicopper oxidase.The Secretory expression of MCOB in Escherichia coli was successfully achieved through fusion of the signal peptide PhoA to the N-terminal of MCOB,and the extracellular enzyme activity reached 69.8 U?L^-1.Extracellular MCOB enzyme activity increased to 105.2 U?L^-1 after optimization of induction conditions,which was 50.7%higher than that before optimization.The optimal induction conditions for MCOB expression were determined to be:the induction temperature was 25°C,the IPTG concentration was 0.05 mmol/L,induced when the cell density(OD₆₀₀)reached 1.0.The Secretory expression of MCOB was enhanced to 238.1 U?L^-1 after 40 h of fermentation by adding 150 mmol?L^-1 glycine 6 h after induction.The extracellular enzyme activity was increased by 2.4 times.