Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli.
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.
Lawung
R
R
Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund, S-221 00, Sweden.
Prachayasittikul
V
V
Bülow
L
L
D000900:Anti-Bacterial Agents / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006191:Haemophilus ducreyi / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D014169:Transformation, Bacterial; D001618:beta-Lactamases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D047090:beta-Lactams
D000900:Anti-Bacterial Agents; D047090:beta-Lactams; D001618:beta-Lactamases