High-level expression of the 1,3-propanediol oxidoreductase from Klebsiella pneumoniae in Escherichia coli.
As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-beta-D-thiogalac-topyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant enzyme was purified and biochemically characterized. The apparent Km values of the enzyme for 1,3-propanediol and NAD+ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30 degrees C.
Fenghuan
Wang
W
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
Huijin
Qu
Q
He
Huang
H
Tan
Tianwei
T
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001709:Biotechnology; D003001:Cloning, Molecular; D003593:Cytoplasm; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D016680:Genome, Bacterial; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics*; D019897:Periplasm; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C052370:1,3-propanediol dehydrogenase