Abstract:[Objective] In order to construct a biocatalytic system to synthesize 4-hydroxyl isoleucine (4-HIL), L-isoleucine (L-Ile) dioxygenase (IDO) from Bacillus subtilis expressed in recombinant Escherichia coli was purified and characterized. [Methods] The recombinant IDO was purified by Ni-NTA affinity chromatography from recombinant Escherichia coli BL21/pET28a-ido. The enzyme was the characterized by using L-Ile as the substrate. As the necessary cofactor of IDO, the concentration of α-ketoglutaric acid (α-KG) in the enzymatic system was further optimized to improve the catalytic efficiency. [Results] Kinetic parameters of the enzyme were obtained as Km 0.247 mmol/L, kcat 1.260 s?1, and kcat/Km 5.101 L/(mmol·s). Compared with other homologous enzymes, the recombinant IDO had higher substrate affinity and catalytic efficiency. The recombinant IDO was more active at 20 °C and pH 7.0, and more stable at the temperatures below 35 °C. The optimal concentration of Fe2+ was 1 mmol/L in the catalytic system. 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. By optimization of α-KG concentration in enzymatic catalysis, 4-HIL with the yield of 66.20% was achieved from 70 mmol/L L-Ile by adding 30 mmol/L α-KG in the reaction system. Thus, the addition of α-KG as the reaction-coupled cofactor had a significant impact on the reaction efficiency of recombinant IDO-mediated L-Ile hydroxylation. [Conclusion] This study provides the basis for enzymatic conversion of 4-HIL and other hydroxylated amino acids.
