Recombinant Expression And Activity Analysis Of Aconitase And Isocitrate Dehydrogenase In Escherichia Coli

α-ketoglutaric acid is a kind of important short chain carboxylic acid,and it is widely used in food,medicine and cosmetics industries.Under the action of aconitase（aconitase,ACO,EC 4.2.1.3）,citric acid can be produced by dehydration and water reaction of isocitrate,isocitric acid can be catalyzed by isocitrate dehydrogenase（isocitrate dehydrogenase,IDH,EC 1.1.1.42）,theα-ketoglutaric acid and CO₂ are produced by oxidative decarboxylation,and the coenzyme NADP⁺into the form of NADPH.C itric acid is rich in source and inexpensive,co-immobilization of the composition of ACO and IDH compound,explores a new approach to the production ofα-ketoglutaric acid.In this study,ACO and IDH were used as the research object,and the gene of them is expressed and purified in Escherichia coli,which come from Escherichia coli and Bacillus subtilis respectively,and the activity and kinetic parameters of IDH were determinated,this laid the foundation for directional covalent immobilization of ACO and IDH.In this study,ACO and IDH were used as the target protein for immobilization,and the ACO gene acnA,acn B and the IDH gene icd1 from Escherichia coli and the ACO gene cit B and the IDH gene icd2 from Bacillus subtilis were combined with the His-tag and the4’-phosphate pantothenoxyethylamine transferase（Sfp）modified short peptide SRP tag,successfully constructed five recombinant expression vector pET30a-His-SRP-acnA,pET28b-His-SRP-acnB,p ET28b-His-SRP-cit B,p ET28b-His-SRP-icd1,pET28b-His-SRP-icd2,and expressed and purified the target protein,and named them ACO1,ACO2,IDH1,ACO3,IDH2.In addition,the activity and kinetic parameters of Escherichia coli isocitrate dehydrogenase（IDH1）and Bacillus subtilis isocitrate dehydrogenase（IDH2）were determinated.and after repeated experiments to find the average,the results showed that the activity of IDH1 and IDH2 was 36.40 U,30.23 U respectively,the K_m was 0.14 mM and 0.05 mM respectively,and the K_cat was 152.84 s^-1 and 111.06 s^-1 respectively,therefore,IDH1 is more efficient than IDH2 when the substrate is sufficient.On this basis,In this study,we selected a zinc finger domain gene zif with a specific DNA binding ability of 243 bp,and mixed it with the icd1,constructed two recombinant expression vector p ET28b-His-SRP-icd1-TEV（RS）-zif,p ET30a-zif-TEV（RS）-icd1-SRP-His,and they were expressed and purified,the two enzymes were named IDH3 and IDH4,respectively.In addition,The results of the determination of the enzymatic parameters of IDH3 and IDH4 were shown,the K_m of IDH3 and IDH4 was 0.1547 mM and 0.1474 mM respectively,and the difference was less than the front.The enzyme activity of IDH3 and IDH4 was 1.29 U and 3.70 U respectively,The K_cat was 14.44 s^-1 and 39.67 s^-1 respectively,it was clear that the catalytic efficiency of IDH3 is much greater than IDH4 when the substrate is sufficient.