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Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli

Lawung, R LU ; Prachayasittikul, V and Bülow, L LU (2001) In Protein Expression and Purification 23(1). p.8-151
Abstract

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by... (More)

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.

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organization
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type
Contribution to journal
publication status
published
subject
keywords
Anti-Bacterial Agents, Base Sequence, Escherichia coli, Haemophilus ducreyi, Kinetics, Molecular Sequence Data, Transformation, Bacterial, beta-Lactamases, beta-Lactams
in
Protein Expression and Purification
volume
23
issue
1
pages
8 pages
publisher
Academic Press
external identifiers
  • scopus:0034805919
ISSN
1046-5928
DOI
language
English
LU publication?
yes
id
daa9d47e-baff-4527-9ee2-a495618a0525
date added to LUP
2016-04-18 15:55:53
date last changed
2018-05-29 12:03:32
@article{daa9d47e-baff-4527-9ee2-a495618a0525,
  abstract     = {<p>A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.</p>},
  author       = {Lawung, R and Prachayasittikul, V and Bülow, L},
  issn         = {1046-5928},
  keyword      = {Anti-Bacterial Agents,Base Sequence,Escherichia coli,Haemophilus ducreyi,Kinetics,Molecular Sequence Data,Transformation, Bacterial,beta-Lactamases,beta-Lactams},
  language     = {eng},
  number       = {1},
  pages        = {8--151},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli},
  url          = {http://dx.doi.org/},
  volume       = {23},
  year         = {2001},
}