Cloning and expression of the histidinol dehydrogenase gene from mycobacterium tuberculosis H37Rv and properties of the recombinant histidinol dehydrogenase

Abstract

The Mycobacterium tuberculosis hisD gene encoded histidinol dehydrogenase(MtHDH) was amplified by PCR from Mycobacterium tuberculosis H37Rv strain genomic DNA and cloned into a prokaryotic expression vector pET-28a(+) to construct the recombinant expression plasmid pET-28a-HDH.Then this recombinant plasmid was transformed into the strain E.coli BL 21(DE3) and highly expressed after induction with IPTG.Purified MtHDH can catalyse L-histidinol to the corresponding amino acid L-histidine.The optimal pH and temperature of the MtHDH were 8.3 and 45 ℃,respectively.The specific activity of MtHDH was 1.788 U/mg and the relative activity was promoted in the presence of Mn2+,Ca2+,Zn2+ and Co2+.The kinetic constants was determined: Km for NAD+ was found to be 0.9765 mmol/L and for histidinol 2.755 μmol/L.Circular dichroism studies on the MtHDH indicated that the secondary structure of the recombinant protein had about 20.5% α-helix,40.9%β-sheet,4.2%β-turn,34.3% random coil at 25 ℃.