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.
Publisher
JAPAN SOC BIOSCI BIOTECHN AGROCHEM
Issue Date
1991-11
Language
ENG
Keywords

UNDECYL ACETATE ESTERASE; INTRACELLULAR ESTERASE; NUCLEOTIDE-SEQUENCE; MOLECULAR-CLONING; LIPASE GENE; PURIFICATION; PROTEINS; 2-TRIDECANONE; HYDROLASE; MEMBRANE

Citation

AGRICULTURAL AND BIOLOGICAL CHEMISTRY, v.55, no.11, pp.2839 - 2845

ISSN
0002-1369
URI
http://hdl.handle.net/10203/6153
Appears in Collection
MSE-Journal Papers(저널논문)
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