The Preparation And Immobilization Of L-Glutamate Oxidase

L-glutamate oxidase(Gox)[EC 1.4.3.11]catalyzes the oxidative deamination of an L-glutamate to a a-ketoglutarate along with the production of ammonia and hydrogen peroxide via an imino acid intermediate.Gox is classified as an flavoenzyme that has high substrate specificity and also is a kind of flavoenzyme using FAD as coenzyme.Cellulose binding domain(CBD)was capable of specifically binding the cellulose carrier,but it has no catalysis to cellulose.Cellulose is the most abundant material,which has potential applications of renewable energy.The fusion protein was bound to cellulose by recombinant proteins expression,and which can play the role of function,respectively.In this paper,the existing glutamic acid oxidase gene were cloning,heterologous expression,purification and determination of related enzymology properties.The immobilized glutamate oxidase was preliminary researched.In this paper,the expression vectors pET-24a(+)-Gox and pETM-10-Gox-CBD were constructed,and which we could obtain desired recombinant proteins L-glutamic oxidase(Gox)and L-glutamic oxidase with cellulose binding domain(Gox-CBD)by optimization of expression conditions in E.coli.Gox is heterologous expression in E.coli,which forms a sufficient amount of cofactor FAD,and the cofactor in the separation process is not easy to fall off,and there is no additional add.The final amount of fusion protein immobilized on MCC was 9.0 mg per gram of MCC at 4? for 4 hours.The enzymatic properties of the recombinant type,the free and immobilized enzyme(Gox-CBD)were studied.When compared immobilized Gox-CBD with free Gox,the specific activity of the recombinant protein Gox-CBD slightly lower,but the thermal stability of the immobilized enzyme than the free enzyme has improved a lot,and also retained 75%of the activity after 30 min at 60?,however,the free enzyme activity is completely lost.At the same time,the fusion protein of Gox-CBD can be immobilized on the MCC in a single step.Only the protein of Gox-CBD was detected by SDS-PAGE.Gox as one of flavoenzyme,which was widely used in many fields.In this paper,the activity Gox was obtained by E.coli expression system.By a single step purification operation,fusion with Cellulose-binding domain of Gox has bound to MCC,and its easy to operation,strong specificity,and economical.There is no significant difference compared to recombination Gox on the enzyme activity.In this paper,the bound to MCC of Gox characteristics were studied,which was obtained by fusion expression.This preliminary study is provided a theoretical basis for the subsequent application of biosensor and the preparation of a-keto acid.