Abstract:4-Hydroxyisoleucine(4-HIL) is a promising drug that promotes insulin secretion, improves peripheral tissue resistance to insulin, and regulates dyslipidemia. L-isoleucine hydroxylase (IDO) is commonly used in the production of 4-HIL. First, the L-isoleucine hydroxylase derived from Bacillus thuringiensis was cloned, and successfully expressed in E. coli BL21(DE3). Secondly, the site-directed mutation of I156 locus was achieved through homology modeling and protein structure analysis based on the principle of changing the amino acid residue bound to the supstrate side chain from the hydrophilic or long-chain hydrophobic structure to Ala. The mutation aimed at increasing the supstrate binding pocket and widening the supstrate channel to increase the yield of 4-HIL. Finally, the biochemical properties of wild-type and mutated enzymes were studied. The hydroxylation reaction system of mutated enzyme was studied. Under the optimal catalytic conditions, the supstrate was fed in batches to produce 4-HIL. The results of enzymatic properties showed that the optimum temperature and pH of wild-type enzyme and mutated enzyme I156A were 25 ℃ and pH 7.0, and the I156A mutated enzyme activity was 1.9 times higher than that of the wild-type enzyme with 28% increased L-ILe conversion rate. The optimal conversion conditions for the hydroxylation reaction system were: 20 mmol/L L-ILe, 20 mmol/L α-ketoglutarate, 8 mmol/L Fe2+, 30 mmol/L ascorbic acid, and HEPES (50 mmol/L, pH 7.0) buffer. Under the optimal transformation conditions, the recombinant E. coli BL21/pET28a-idoI156A was fed in batches for a time interval of 4 h. After 32 h, 77.3 mmol/L 4-HIL was obtained, and the highest conversion rate of supstrate was 98.35%.
