Characterization of recombinant L-isoleucine-4-hydroxylase from Bacillus thuringiensis and its application in 4-hydroxyisoleucine biosynthesis

Abstract:

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)/α-ketoglutaratedependent 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 μmol/min/mg，respectively．The optimum temperature and pH for the recombinant IDO was 35℃ and 7.0 respectively; moreover，the relative activity of the enzyme remain 85.10% after 5 h incubation at 35℃．In all，ecombinant 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．

Key words:Keywords: 4-Hydroxyisoleucine，L-isoleucine-4-hydroxylase，enzymatic characteristics，Bacillus thuringiensis，microbial transformation