作　　者： ; ; ; ; (罗兆飞）; (陈发忠）; (侯守海）; (廖东庆）;

机构地区： 广西大学生命科学与技术学院广西亚热带生物资源保护利用重点实验室

出　　处： 《中国生物工程杂志》 2006年第7期42-47,共6页

摘　　要： 1,3-丙二醇(1,3-propanediol,1,3-PD)是一种重要的化工原料,越来越受到广泛的关注。以弗氏柠檬酸菌(Citrobacter freundii)基因组DNA为模板,通过PCR得到1,3-丙二醇氧化还原酶(1,3-propanediol dehydrogenase,PDOR)的基因dhaT,序列显示与来源于C．freundii DSM 30040 (Genbank U09771)相应基因的相似性为78％。将此基因构建于表达载体pSE380,得到重组质粒pSE-dhaT。重组质粒转化到宿主菌E．coli JM109中进行了表达,重组酶通过镍柱及Sephacral S- 300进行纯化,重组酶SDS-PAGE结果显示有非常明显的单一的42kDa特异性蛋白条带出现。以丙醛为底物测定重组酶还原反应的最适温度为37℃、最适pH为8．0,对丙醛的Km值为10．05mmol／L,最大反应速度Vmax为37．27μmoL／min／mg;以1,3-PD为底物测定重组酶氧化反应的最适温度为25℃、最适pH为10．5,对1,3-PD的Km值为1．28mmob／L,最大反应速度Vmax为25．55μmol／min／mg。重组酶的还原反应比活为49．50U／mg,氧化反应比活为79．72U／mg。该酶同样具有假定的结合Fe2+的G-X-X-H-X-X-A-H-X-X-G-X-X-X-X-X-P-H-G模体保守结构。 1,3-propanediol （1,3-PD） is an important material for chemical industry, therefore, there is much interest in the production of 1,3-PD. The gene dhaT encoding 1,3-propanediol dehydrogenase （PDOR） of Citrobacter freundii was amplified by PCR. Sequence analysis of the similarity at the nucleotide and amino acid level between the gene encoding C. freundii PDOR and that of C. freundii （ U09771 ） were 78% and 90%, respectively. The recombinant plasmid pSE-dhaT was constructed by inserting dhaT gene into expression vector pSE380 and then transformed E. coli JM109. The recombinant strain was induced by IPTG to express dhaT. Further more the recombinant enzyme was purifed from recombinant E. coli by Ni-nitrilotriacetate affinity chromatography followed by Sephacral S-300 gel filtration. A single obvious protein about 42kDa could be obtained by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of recombinant enzyme. The purified enzyme was used to determined enzyme property on the substrate of propionaldehyde and 1, 3-PD. The optimal temperature and optimal pH of the purified enzyme were 37℃, 8.0 for reduction and 25℃, 10.5 for oxidation, respectively; and the kinetic property of PDOR about Km and V max were 10.05mmol/L, 37.27μmol/min/mg for propionaldehyde and 1.28mmol/L, 25.55μmol/min/mg for 1,3-PD, respectively; The deduced dhaT gene product （388 amino acids） showed a specific reduction activity of 49.50U/mg and oxidation activity of 79.92U/mg. There also have a putative iron-binding motif （G-XX-H-X-X-A-H-X-X-G-X-X-X-X-X-P-H-G） as a fingerprint pattern in the recombinant enzyme, the motif is fully conserved among these 1,3-propanediol dehydrogenase. It is beneficial to the researches of high producing 1, 3-propanediol by gene engineering strain.

关 键 词： 弗氏柠檬酸菌 丙二醇氧化还原酶 克隆 表达 纯化

领　　域： [生物学]