Sequence analysis of a pseudomonas fluorescens esterase gene and characterization of the gene product expressed in escherichia coli = Pseudomonas fluorescens esterase 유전자의 sequence와 대장균에서 발현된 산물의 순수 정제 및 특성 분석

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A gene(estB) coding for carboxylesterase (EC, esterase II, of $\mbox{\underline{Pseudomonas}}$ $\mbox{\underline{fluorescens}}$ cloned into $\mbox{\underline{Escherichia}}$ $\mbox{\underline{coli}}$ JM83 was analyzed by nucleotide sequence analysis. DNA sequencing revealed a single open reading frame for esterase II, confirmed by N-terminal amino acid sequence analysis of the purified esterase protein. The promoter-like structure(-10 and -35 regions) and a potential Shine-Dalgarno sequence are followed by the coding sequence of estB gene. The carboxylesterase was purified by means of ionexchnge chromatography and gel filtration. Homogeneity of the purified enzyme was confirmed using SDS-polyacrylamide gel electrophoresis. The molecular weight of the subunit of esterase II determined by SDS-polyacrylamide gel electrophoresis and that of native form determined by gel permeation chromatography were 23,000 and 44,000, respectively, indicating that the enzyme exists as a dimer consisting of two identical subunits. The enzyme was maximally active at 45$^\circ$C and the optimum pH range was found to be from pH 6.0 to 9.0. The enzyme hydrolyzed methyl esters of short chain fatty acids(C2-C6) as well as phenyl acetate. Esterase II activity was strongly inhibited by DFP and PMSF but not by DTNB. The results of the experiments for determining substrate specificity and the inhibitor studies suggest that this enzyme is a carboxylesterase (EC and a serine residue is present at the active site or substrate binding site of the esterase, as for the esterases of animal tissues.
Yoo, Ook-Joonresearcher유욱준researcher
한국과학기술원 : 생물공학과,
Issue Date
67719/325007 / 000891543

학위논문(석사) - 한국과학기술원 : 생물공학과, 1991.2, [ ix, 87 p. ]


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