Résumé
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. 27?mol/min/mg for propionaldehyde and 1. 28mmol/L, 25. 55?mol/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.
Résumé
Based on the principle of the pathway engineering, a novel pathway of producing glycerol was built in E. coli. The gpd1 gene encoding glycerol 3-phosphate dehydrogenase and the hor2 gene encoding glycerol 3-phosphatase were cloned from Saccharomyces cerevisiae, respectively. The two genes were inserted into expression vector pSE380 together. A recombinant plasmid pSE-gpd1-hor2 containing polycistron was constructed under the control of the strong trc promoter. Then it was transformed into E. coli BL21. The result showed the recombinant microorganism GxB-gh could convert glucose to glycerol directly. And the recombinant microorganism GxB-gh was incubated to produce glycerol from D-glucose in the fermentor. The maximal concentration of glycerol was 46.67g/L at 26h. Conversion rate of glucose was 42.87%. The study is about "green" producing glycerol by recombinant microorganism and is also useful for further working in recombining microorganism of producing 1,3-propanediol.