Heterologous expression and characterization of recombinant glycerol dehydratase from Klebsiella pneumoniae in Escherichia coli.
Glycerol dehydratase (EC 4.2.1.30), as one of the key enzymes in converting glycerol to the valuable intermediate 1,3-propanediol, is important for biochemical industry. The dhaB genes encoding coenzyme B(12)-dependent glycerol dehydratase in Klebsiella pneumoniae were cloned and expressed in Escherichia coli. An effective co-expression system of multiple subunits protein was constructed. Heterologous expression vectors were constructed using the splicing by overlap extension-PCR technique to co-express the three subunits of the glycerol dehydratase. After induction by isopropyl-beta-D-thiogalactopyranoside, SDS-PAGE analysis revealed that: (i) only the alpha subunit of glycerol dehydratase was expressed in direct expression system, (ii) the three subunits of glycerol dehydratase with predicted molecular massess of 64 (agr;), 22 (beta), and 16 kDa (gamma) were expressed simultaneously in co-expression system, and (iii) the fusion expression system expressed the fusion protein of 99 kDa. Enzyme assay showed that the activities of three heterologous expression products were 27.4, 2.3, and 0.2 U/mg. The highest enzyme activity was almost 17 times of that in K. pneumoniae. The recombinant enzyme was purified and biochemically characterized. The apparent Km values of the enzyme for coenzyme B(12) and 1, 2-propanediol were 8.5 nM and 1.2 mM, respectively. The enzyme showed maximum activity at pH 8.5 and 37 degrees C.
Wang
Fenghuan
F
Beijing key lab of bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.
Qu
Huijin
H
Tian
Pingfang
P
Tan
Tianwei
T
D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D006836:Hydro-Lyases; C020671:glycerol dehydratase