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.
Conclusion: 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.
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