结核分枝杆菌H37Rv组氨醇脱氢酶基因的克隆、表达及酶学性质分析

Cloning and Expression of Histidinol Dehydrogenase Gene from Mycobacterium tuberculosis H37Rv and Properties of the Recombinant Histidinol Dehydrogenase

以结核分枝杆菌H37Rv基因组为模板,扩增hisD基因,构建pET-28a-HDH重组质粒;转化重组质粒到E.coli BL21(DE3)并诱导表达,纯化可溶性的结核分枝杆菌L-组氨醇脱氢酶(HDH),并对其性质进行研究,结果表明:重组结核分枝杆菌L-组氨醇脱氢酶能以L-组氨醇和NAD+为底物催化L-组氨醇生成L-组氨酸;该酶的最适pH值为8.3,最适温度为45℃,比活力为1.788 U/mg;Mn2+,Ca2+,Zn2+,Co2+等对酶促反应有激活作用;底物NAD+和L-组氨醇的米氏常数分别为0.9765 mmol/L和2.755μmol/L;25℃时重组蛋白的二级结构中有20.5%的α-螺旋,40.9%β-折叠,4.2%β-转角,34.3%无规卷曲.

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 Mn^2＋ ,Ca^2＋ ,Zn^2＋ and CO^2＋ . The kinetic constants was determined：Km for NAD^＋ was found to be 0. 9765 mmol/L and for histidinol 2. 755 μmlol/L. Circular dichroism studies on the MtHDH indicated that the secondary structure of the recombinant protein had about 20.5 % a-helix, 40.9 % β-sheet, 4.2 % β-turn,34.3 % random coil at 25 ℃.