Screening And Expression Of Microbial Isoleucine Dioxygenase And Its Application In Biosynthesis Of Hydroxyisoleucine

4-Hydroxy-L-isoleucine (4-HIL) extracted from plants has been used in pharmaceuticalsto treat type Ⅱ diabetes and lower cholesterol, because it could increase glucose-inducedinsulin release. Recently, the synthesis of4-HIL by microbial enzymatic has been highlydesired by researchers increasingly, but the number of related functional enzymes still remaininsufficient and the yield of transformation is not high enough. Here, we show that theisoleucine dioxygenase (IDO) screened from the nature could specifically hydroxylateisoleucine (L-Ile) to4-HIL. And the ido gene was cloned and expressed in Escherichia coli.Using the whole recombinant cell, we obtained4-HIL by hydroxylating L-Ile.(1) Bacillus was screened from a number of soil samples via heat treatment, consideringthe higher hydroxylation efficiency of dioxygenases from this kind of strains. And then thestrains with oxygenase activity were screened from Bacillus by the reaction coloration withGibbs assayed by spectrophotometer. At last, the strain with dioxygenase activity wasscreened by thin-layer chromatography (TLC). The original strain identified as Bacillussubtilis was named XB6.(2) Functional genes ido from XB6were obtained by PCR according to the sequencehomology of IDO-encoded genes from Bacillus. The purified PCR products and pET28a wereconnected to construct recombinant plasmid pET28a-ido, which was transformed to E. coliBL21(DE3). The induced expression conditions were optimized as0.5mmol L-1IPTG and30oC, under which the foreign protein was expressed actively and the correspondingrecombinant strain was verified to have the function of hydroxylation of L-Ile.(3) The whole cell reaction system and conditions were optimized. With low substrateconcentration like10mmol L-1, the endogenous-KG and ascorbic acid could meet thetransformation needs, so there was no need to add them. According to the single factorexperimental results, the optimal concentration of cells、L-Ile and FeSO4·7H2O was5%、10mmol L-1and0.5mmol L-1. The optimal reaction condition was pH7.5and28oC.It facilitated the absorption of substrate and the formation of product when feeding at10h after reaction started with20mmol L-1L-Ile. And the feeding strategy could improve theproduction to1.88times of the control with no feeding. The addition of glucose wasconducive to-KG accumulation, so as to promote the coupling reaction and increase theproduction. However, cells after permeability treated went against the reaction because of theimpaired cell metabolism. Compared with Tris-HCl buffer, the sodium carbonate buffercontributed to the dissolution and absorption of the substrate, and it could increase the yield tomore than85%even with the20mmol L-1L-Ile. Similarly, the yield of transformation withthe whole recombinant cells was increased to more than85%by incubating the cellsovernight at30oC. According to the above optimized conditions,40mmol L-1L-Ile could beused to generate23.74mmol L-1HIL by reaction for24h.