Cloning of Pseudomonas fluorescens carboxylesterase gene and characterization of its product expressed in Escherichia coli.
A gene (estC) coding for an esterase (esterase III) of Pseudomonas fluorescens was cloned into Escherichia coli JM83. DNA sequencing showed a single open reading frame with GTG as a translation initiation codon for esterase III. This was confirmed by N-terminal amino acid sequence analysis of the purified esterase III protein from an E. coli clone. The promoter sequence and a potential Shine-Dalgarno sequence were followed by the coding sequence of the estC gene. The amino acid sequence deduced from the nucleotide sequence contains the consensus active site sequence, G-X-S-X-G, of serine esterase. The esterase III expressed in an E. coli clone was purified by ion-exchange chromatography and gel filtration. The native form of the enzyme was a monomer with a molecular weight of 41,000. The results of substrate specificity and the inhibitor studies suggest that this enzyme is a carboxylesterase (EC 3.1.1.1) and a serine residue is present at the active site of the enzyme.
Kim
Y S
YS
Department of Life Science, Korea Advanced Institute of Science and Technology, Kusung-dong Yusung-gu, Taejon.
Lee
H B
HB
Choi
K D
KD
Park
S
S
Yoo
O J
OJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase