Characterization Of Recombinant L-isoleucine Dioxygenase And The Synthesis Of 4-hydroxyisoleucine In The Reorganization System

4-hydroxyisoleucine（4-HIL） which is a kind of nonproteinogenic amino acid, could promote the secretion of insulin and was used to lower cholesterol, â…¡ diabetes treatment, etc. L-isoleucine dioxygenase（IDO） can catalyse the hydroxylation of L-isoleucine（L-Ile） to generate 4-HIL, and the reaction accompany by the oxidative decarboxylation of α-Ketoglutaric acid（α-KG） into succinate. Through expression and purification the recombinant of IDO from Bacillus subtilis, analyzed its catalytic conditions, so as to build the crude enzyme and whole-cell catalytic synthesis of 4-HIL reaction system.（1） The recombinant IDO was purified by Ni-NTA affinity chromatography from the available recombinant E. coli BL21/p ET28a-ido. The enzymatic conditions of the recombinant IDO catalyzing hydroxylation were investigated using L-Ile as the substrate. Based on activity assay of the recombinant IDO catalyzing hydroxylation of L-Ile, the kinetic parameters of the enzyme were obtained as Km 0.247 mmol·L-1, kcat 1.260 s-1 and kcat/Km 5.101 s-1·mmol-1. Compared with other homologous enzyme from B. thuringiensis 2-e-2, the recombinant IDO performed higher substrate affinity and catalytic efficiency. The recombinant IDO was more active at 20 ℃ and p H 7.0, and was more stable at the temperatures below 35℃. The optimal concentration of Fe2 + was 1 mmol·L-1 in the catalytic system. The optimum reaction time for the system was 10 h; when the concentration of L-Ile and α-KG were 10²0 mmol·L-1, the molar yield of 4-HIL relatively high. The recombinant IDO was active to a variety of L-amino acids, of which L-isoleucine, L-norleucine, and L-methionine were more suitable for the IDO catalyzing hydroxylation.（2） To keep other components in the crude enzyme reaction system were changeless, by single factor experiment, related factors of L-Ile hydroxylation reaction optimized, in order to determine their impacts on hydroxylation reaction and the optimum adding amount. Fe SO 4·7H2O 0.001%, Vc 5 mmol·L-1, glucose 40⁵0 mmol·L-1, crude enzyme concentration 15%, α-KG 30 mmol·L-1, the substrate L-Ile 40⁵0 mmol·L-1.Within the scope of 0⁸ mmol·L-1,Isocitrate lyase inhibitors itaconic acid and oxalic acid increased the production of 4-HIL, the tendency for the optimum adding amount were 2 mmol·L-1; Isocitrate dehydrogenase activation agent Mn²⁺ could promote the formation of 4-HIL, the optimum adding amount of Mn SO4?H2O was 2 mmol·L-1; Zn SO4?H2O was not conducive to generate 4-HIL; α-Ketoglutaric acid dehydrogenase inhibitors sodium hypochlorite and hydroxyl ammonium both had a certain role in promoting the production of 4-HIL, the optimum adding amount were 2 mmol·L-1 and 4 mmol·L-1.（3） The whole cell reaction system of other components unchanged, by single factor experiment, L-Ile hydroxylation reaction system related factors optimized, in order to determine their impacts on the hydroxylation reaction and the optimum adding amount. Among them, Fe SO4 ·7H2O 0.01%, Ca Cl2 1 mmol·L-1, Vc 5 mmol·L-1, bacteria concentration 10%, glucose 30 mmol·L-1, α-KG 30 mmol·L-1, the substrate L-Ile 40 mmol·L-1, culture conditions for oxygen. Within the scope of 0⁸ mmol·L-1, Isocitrate lyase inhibitors itaconic acid and oxalic acid increased the production of 4-HIL, the optimum adding quantity were 6 mmol·L-1; Isocitrate dehydrogenase activation agent Mn²⁺ could promote the formation of 4-HIL, the optimum adding amount of Mn SO4?H2O was 2 mmol·L-1, Zn SO4?H2O was not conducive to generate 4-HIL; α-Ketoglutaric acid dehydrogenase inhibitors sodium hypochlorite and hydroxyl ammonium sulphate both had a certain role in promoting the production of 4-HIL, the optimum adding amount were 4 mmol·L-1 and 6 mmol·L-1.