title: [Characterization of recombinant L-isoleucine-4-hydroxylase from Bacillus thuringiensis and its application in 4hydroxyisoleucine biosynthesis].
abstract: OBJECTIVE
  L-isoleucine-4-hydroxylase (IDO) encoding gene ido from Bacillus thuringiensis TCCC 11826 was cloned and expressed, followed by enzyme characterization. In addition, recombinant strain was tested for its 4-Hydroxyisoleucine (4-HIL) biotransformation.
  
  
  METHODS
  Ido gene was amplified from B. thuringiensis TCCC 11826 genomic DNA and expressed in BL-IDO. Recombinant IDO was extracted, purified and characterized. Recombinant strain used for biotransformation of 4-HIL was constructed.
  
  
  RESULTS
  Composed of 723 nucleotides encoding 240 amino acids (sharing 97.47% and 97.91% identities with that of B. thuringiensis 2-e-2), ido gene was cloned from B. thuringiensis TCCC 11826. The recombinant IDO contained a His1-X-Asp/Glu-Xn-His2 motif that is specific for Fe(II)/alpha-ketoglutarate-dependent hydroxylases and catalyzed L-isoleucine to 4-HIL. Normal hyperbolic kinetics was observed with L-Ile in the reaction by recombinant IDO. Lineweaver-Burk treatment of the data yielded apparent Km and the Vmax was 0.18 mmol/L and 2.10 micromol/min/mg, respectively. The optimum temperature and pH for the recombinant IDO was 35 degrees C and 7.0 respectively; moreover, the relative activity of the enzyme remain 85.10% after 5 h incubation at 35 degrees C. In all, recombinant strain harboring ido transformed 89.28% of L-isoleucine to 4-HIL.
  
  
  CONCLUSIONS
  In this study, an ido (Accession No. KC884243) with novel sequence was isolated and enzymatic characteristics of recombinant IDO was systematically analyzed. In addition, we successfully achieved the biotransformation of 4-HIL from L-isoleucine. This work will lay theoretical foundation and practical basis on the microbial manufacture technology of 4-HIL and other amino acid derivatives. This work will lay theoretical foundation and practical basis on the microbial manufacture technology of 4-HIL and other amino acid derivatives.
authors.forename: Chenglin
authors.initials: C
authors.lastname: Zhang
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Ning
authors.initials: N
authors.lastname: Xue
authors.forename: Xinxin
authors.initials: X
authors.lastname: Wang
authors.forename: Xixian
authors.initials: X
authors.lastname: Xie
authors.forename: Qingyang
authors.initials: Q
authors.lastname: Xu
authors.forename: Ning
authors.initials: N
authors.lastname: Chen
mesh_terms: D001413:Bacillus thuringiensis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007532:Isoleucine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: C104672:4-hydroxyisoleucine; D001426:Bacterial Proteins; D007532:Isoleucine; C104673:isoleucine hydroxylase; D006867:Hydrolases