L-lysine-ε-oxidase, Lod A, Purify, Enzyme Activity Analysis, Active Reconstruction

L-amino acid oxidases (LAO EC1.4.3.2) catalyze the reaction of oxidativedeamination of L-amino acids followed by formation of hydrogen peroxide andcorresponding α-keto acid and ammonium ion along with the consumption of oxygen.Most of amino acid oxidase has low affinity with the essential amino acid. L-Lysineplays an important role in our daily life. It not only can regulate metabolic balanceand enhance the body’s absorption of protein for cereals, also improve human dietaryand promote growth and development.L-Lysine oxidase containing L-lysine-α-lysine oxidase and L-lysine-ε-oxidase(Lod A). L-lysine-ε-oxidase is a novel type of lysine oxidase. Unlike other L-aminoacid oxidases, Lod A is not a flavoprotein but contains a quinone cofactor. As far aswe know, the only L amino acid oxidase described that is not an FAD protein is theM. mediterranea Lod A, which contains a non-dissociable quinone cofactor. Lod A isencoded by an operon with two genes, Lod A and Lod B. In the native system, Lod Bis required for the synthesis of a functional Lod A. How does Lod B work is unclear.L-lysine-ε-oxidase showing higher affinity for substrate than L-lysine-α-lysineoxidase. LodA is an extracellular enzyme very specific for L-Lys that catalyzes theoxidative deamination of L-lysine into6-semialdehyde2-aminoadipic acid, ammonia,andhydrogen peroxide.We have obtained crystals of the recombinant Lod A proteinthat belonged to the monoclinic P2space group. The monomer is made up by726amino acids of which only the first686are visible in the crystallographic structure,the missing amino acids corresponded to the C-terminal region of the sequence. TheLodA monomer has a unique structure with two arms of antiparallel β-strandprotruding from the main body composed mostlyof a β-barrel made by three β-sheetsand several of α-helices and antiparallel β-sheets. The covalent-linkage betweenTrq581and Cys516is most likely assigned to a thioether bond by analogy to cysteinetryptophylquinone (CTQ) which was previously identified in quinohemoproteinamine dehydrogenase (QHNDH).Lod A and lod B constitute an operon. Lod B appears to start immediatelydownstream of lod A.LOD activity is detected regardless of the presence of lod A orlod B in the same or different transcriptional units. This study is successful expressed and the Lod A and Lod B protein in E. coli,and the co-expression of Lod A/B success. Lod A and Lod B protein was purified.Based on SDS-PAGE and gel filtration chromatography of Lod A, Lod B and Lod A/B,the results show, Lod A and Lod B, are polymer form when it single expression.Lod A majority exists as a dimer of in solution when Lod A and Lod B co-expression.After purification of the Lod A, Lod B and Lod A/B testing of enzyme activity,it suggest that single expression protein does not have L-lysine oxidase activity. Theactivity increased significantly after Co-expression of Lod A/B protein purify byanion exchange chromatography. Speculate it may be related to protein folding rightand anionic gel column adsorption. We also study preliminary of reconstruction theL-lysine oxidase activity.but Lod B can not fold or improve the Lod A activity afterLod A extracellular.This reach is not only important for filter high expression lysine strain but alsoimportant on L-lysine oxidase biosensor,to detecting the concentration of lysine infood or human.