Improving The Properties Of Glucose Oxidase By Rational Design

Glucose oxidase exists in plants,animals and microorganisms,although the production of natural enzyme is low,GOD can be large-scale produced in industry by engineering technology now.Recently,GOD is very important in food industry,medicine,feed additives,test,biological sensors.Methionine is one of essential amino acid in protein,but the methionine residue is easily oxidized to methionine sulfoxide,then methionine sulfoxide is oxidized to methionine sulfone,this process is irreversible.When methionine are oxidized in the protein,the structure and function of the protein will change,which may cause the protein lose its activation.Because GOD can oxidize glucose into gluconic acid and hydrogen peroxide,hydrogen peroxide is a strong oxidizer,which can effect the methionine in glucose oxidase.In this study,we improve the antioxidation of GOD meanwhile keep the activation of GOD by site-directed mutation.The modification of enzyme include rational design strategy and irrational design strategy.Irrational design strategy mainly refers to directed evolution of protein,its method are error-prone PCR,stagger extension process PCR and DNA shuffling.Rational design strategy requires to understand the tertiary structure and catalytic mechanism of protein,based on these to control the structure of enzyme.The rational design of enzyme include design based on experimental results and Computer-aided design.Design based on experimental results is site-directed mutation,modification of loop,replacement of catalytic metal ion.Directed evolution of protein is the most effective,widely method to get new enzyme currently.Directed evolution of protein has achieved great success in improving the catalytic activity and stability of the enzyme,changing substrate specificity of enzyme and changing the enantiomer-specific of enzyme.But,because of advantages of having definite objective,more convenient and faster than irrational design strategy,also,as in recent years,more and more tertiary structures and catalytic mechanism have been established,the application of rational design growing fast.The future development of modification of enzyme is combination of both rational design strategy and irrational design strategy.Directed evolution can undertake preliminary screening for the modification of enzyme,and rational design can make site-directed mutation based on directed evolution to achieve the desired aims.This study's method is rational design,we design three mutants by computational design,analyze them by homology modeling,then let mutants dock glucose by FlexibleDocking.The mutated gene was cloned into the expression vector pPIC9K,then transform the recombinant plasmid into P.pastoris GS115 by electroporation.After fermentation in shake flask 120h,purified by ion exchange chromatograph,test the activity and antioxidation.Finally,we got three mutation,the first mutation which is 8-sites mutations,we tested it had no activity.The specific activity of GOD is 3.24U/?g.The second mutation is GOD-M523L-M524L,its specific activity is 0.0457U/?g.The third mutation is GOD-M524L-M528L,its specific activity is 3.59U/?g.GOD and GOD-M524L-M528L were incubated by hydrogen peroxide for 3.5h,the residual enzyme activity of GOD is 20%,while the residual enzyme activity of GOD-M524L-M528L is 30%.