Abstract:

4-Hydroxyisoleucine(4-HIL) was synthesised using L-isoleucine(L-Ile) as substrate, the recombinant Escherichia coli BL21/pET28a-ido heterologously expressing Fe(Ⅱ)/2-ketoglutarate-dependent dioxygenase, and the whole cell as a catalyst. Based on the catalytic properties and conditions of L-Ile dioxygenase catalyzing isoleucine hydroxylation, the optimization of single factors included ferrous sulfate(FeSO₄·7H₂O), α-ketoglutaric acid(α-KG) and substrate concentration. As a result, the optimized yield of 190 mmol/L of 4-HIL was obtained in the 50 mmol/L trimthylolamine hydrochloride(Tris-HCl) buffer system comprising FeSO₄·7H₂O(2 g/L), with the molar ratio of substrate to α-KG molarity as 1∶1. Associated with the optimized conditions in shake flask level, the stirring speed and the cell concentration were further optimized at the reactor level, to achieve continuous regulation of isoleucine hydroxylation at high substrate concentration. Consequently, a whole-cell transformation system and process was established to produce 400 mmol/L 4-HIL, with the conditions of FeSO₄·7H₂O(2 g/L), molar concentration ratio of substrate and α-KG 1∶1, wet bacteria 50 g/L, and rotation speed 400 r/min.

Key words: L-Isoleucine, Whole cell catalysis, 4-Hydroxyisoleucine, Dioxygenase, Process regulation