Preparation Of Genetic Recombinant Lysozyme And Study On Its Enzymatic Properties

In recent years,the use of genetic engineering methods to construct new genetically engineered strains and the production of recombinant lysozyme through large-scale fermentation of microorganisms has important implications for its industrialization.In this project,a methanol-induced chicken lysozyme-recombinant Pichia pastoris strain NCY-2wort fermentation broth was used as the research object.The centrifugation,ultrafiltration and ion exchange treatment were performed to optimize the separation of egg white lysozyme Purification process parameters,the obtained concentrated solution,vacuum drying to prepare lysozyme powder,the enzymatic properties of recombinant lysozyme dry enzyme powder were studied,the results are as follows:?1?Optimization of ultrafiltration conditions:30 kDa polyethersulfone?PES?membrane was used to optimize the optimal conditions:transmembrane pressure was 0.20MPa,pH 6.5,the yield of recombinant lysozyme was 96.6%,and the enzyme activity2612.1 U·mL^-1,1.78 times the original enzyme activity.?2?Optimization of ion exchange conditions:Adsorption and elution of D152 resin are used to optimize the optimal process conditions:the amount of resin accounts for 15%of the volume of the material liquid,the adsorption time is 4 h,and the concentration of eluent is 1.0 mol·L^-1.The recovery rate of enzyme activity of recombinant lysozyme was95.7%,and the enzyme activity was 3879.6 U·mL^-1,which was increased by 0.5 times and3.1 times of the original enzyme activity.?3?Research on enzymatic properties:the recombinant lysozyme dry enzyme powder has an inhibitory effect on Micrococcus lysogenes,the enzyme activity is 12573.6 U·mg^-1;the optimal temperature is 50?,and the thermal stability is good within the range of20-60?;The optimum pH is 6.5;the enzyme activity of recombinant lysozyme will be inhibited by Fe²⁺,Fe³⁺,Zn²⁺and Cu²⁺,while a Na⁺and Mg²⁺will activate the enzyme activity,and has no obvious effect on Mn²⁺,K⁺,Ca²⁺;it can also be inhibited by Span 80,activated by Tween 20,Tween 80 and glycerin.