Molecular Modification Of L-isoleucine Dioxygenase From Bacillus

4-hydroxyisoleucine?4-HIL?can reduce insulin resistance and promote insulin secretion.It is a promising potential drug for the treatment of type II diabetes.Isoleucine dioxygenase?IDO?can catalyze the C-4 hydoxylation of L-isoleucine?Ile?to generate 4-HIL.Our previous study showed that the stability and activity of IDO is not high;meanwhile,its activity is inhibited by Ile to a certain extent when the concentration of Ile is too high.In this paper,the IDO derived from Bacillus weihenstephanensis was modified by molecular engineering,and the transformation of 4-HIL by whole cells of Escherichia coli BL21?DE3?expressing mutant enzyme was achieved.The main results are as follows.1.The IDO gene?ido?derived from B.weihenstephanensis was synthesized and expressed in E.coli BL21?DE3?.The Ido protein was induced,purified by Ni-NTA,and its enzymatic properties were determined.Ido existed as a soluble protein and its molecular weight was approximately 29 kDa.Its optimum p H was 5.7 and its optimum temperature was30°C.It was stable at pHs between 4.5-6.0 and temperatures between 20-40°C.When Ile was used as the substrate,its K_m and V_max were 2.37 mmol/L and 0.98?mol·mg^-1·min^-1,respectively.2.For the directed evolution of ido,error-prone PCR was used at first to create a library of mutants.Five mutants were screened out from approximately 2000 mutants by paper chromatography,and finally two positive mutants(Ido^N126H and Ido^S47G/I191V)were obtained by assaying the specific activity of the crude enzyme solution.The specific activity of Ido^N126H was 2.8 times that of the wild type Ido.3.For the site-specific mutagenesis of ido,4 mutation sites were designed based on the primary structure alignment of Idos of several Bacillus strains and four mutants were eventually obtained,with Ido^T130K the best one.The specific activity of Ido^T130K increased by60%compared to the wild type Ido.4.To combine the beneficial sites,site-specific mutagenesis of ido^N126H was then performed and the mutant Ido^N126H/T130K showed the best properties.Its K_m and V_max were1.51 mmol/L and 1.50?mol·mg^-1·min^-1,respectively,both improved as compared to the wild type Ido.Therefore,Ido^N126H/T130K is a more excellent enzyme.5.To analyze the effect of mutant Ido on 4-HIL production,the whole cell transformation of recombinant E.coli BL21?DE3?strains expressing wild-type ido and mutant ido^N126H/T130K was performed.After 24 h,the 4-HIL yield of the recombinant strain expressing mutant ido reached 66.50±0.99 mmol/L and the conversion ratio of Ile to 4-HIL reached 66.5%,while the yield of 4-HIL of strain expressing wild-type ido was only26.10±1.80 mmol/L and the conversion ratio was 26.1%.After optimization of the whole cell transformation conditions,the 4-HIL yields of the mutant and wild-type ido expression strains increased to 79.15±0.50 mmol/L and 46.49±6.19 mmol/L,respectively.Therefore,the mutant Ido^N126H/T130K was more beneficial for the whole cell transformation of 4-HIL.