Characterization of Pseudomonas fluorescens carboxylesterase: cloning and expression of the esterase gene in Escherichia coli.
The Pseudomonas fluorescens gene (estB) that encodes a novel esterase (esterase II) was cloned into Escherichia coli JM83. DNA sequencing found a single open reading frame of 654 nucleotides. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the esterase protein. A potential Shine-Dalgarno sequence is followed by the coding sequence of the estB gene. The amino acid sequence deduced from the nucleotide sequence contains the consensus active site sequence, G-X-S-X-G, of serine esterases. The enzyme expressed in an E. coli clone was purified by ion-exchange chromatography and gel filtration. Homogeneity of the purified enzyme was confirmed using SDS-polyacrylamide gel electrophoresis. The native enzyme exists as a dimer consisting of two identical subunits, each with a molecular weight of 23,000. The results of the experiments for identifying 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 esterase, as in the esterases of animal tissues.
Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, Seoul.
K H
KH
Hong
W H
WH
Jang
K D
KD
Choi
O J
OJ
Yoo
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D002265:Carboxylic Ester Hydrolases