Cloning, expression and characterization of a thiolase gene from Clostridium pasteurianum.
A thl gene encoding the thiolase (EC 2.3.1.9) of Clostridium pasteurianum was cloned by thermal asymmetric interlaced (TAIL) PCR. It consists of 1179 bp with 36.8% GC content and encodes 392 amino acids with a deduced molecular mass of 40,954 Da and shows 77% identity and 88% similarity to that of Clostridium tetani E88 and should be classified as a biosynthetic thiolase with three conserved residues Cys89, Cys382 and His352. The gene was over-expressed in Escherichia coli and the thiolase was purified with Ni-NTA agarose column to homogeneity. The K(m) of this thiolase for acetoacetyl-CoA is 0.13 mM with 0.06 mM CoASH at pH 8.2, 25 degrees C and a V(max) value of 46 micromol min(-1) mg(-1).
Meng
Yonghong
Y
Department of Microbiology, State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China.
Li
Jilun
J
D000101:Acetyl-CoA C-Acetyltransferase / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001483:Base Sequence; D002845:Chromatography; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012685:Sepharose / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001426:Bacterial Proteins; D012685:Sepharose; D000101:Acetyl-CoA C-Acetyltransferase; C516372:thl protein, Clostridium pasteurianum