Open AccessPurification and characterization of a new beta-lactamase from Clostridium butyricum
Author(s) -
Tadataka Kesado,
L Lindqvist,
Maria Hedberg,
K Tunér,
Carl Erik Nord
Publication year - 1989
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.33.8.1302
Subject(s) - chemistry , gel electrophoresis , isoelectric point , chromatography , isoelectric focusing , biochemistry , sodium dodecyl sulfate , enzyme
A highly penicillin-resistant beta-lactamase-producing strain of Clostridium butyricum, strain NBL 3, was isolated. The specific activity of the unpurified beta-lactamase was 0.29 U/mg of protein. The enzyme was purified 1,130-fold by QAE Zetaprep, Sephacryl S-300, and Mono Q column passages. The purified enzyme was judged homogeneous by sodium dodecyl sulfate gradient gel electrophoresis and fast protein liquid chromatography. The enzyme hydrolyzed phenoxymethylpenicillin, benzylpenicillin, ampicillin, and carbenicillin more rapidly than piperacillin and cephaloridine. Cephalothin, cephalexin, cefoxitin, moxalactam, cefotaxime, and imipenem were only slightly hydrolyzed, at less than 1% the rate for phenoxymethylpenicillin. The enzyme was inhibited by clavulanic acid, sulbactam, tazobactam, and p-chloromercuribenzoate. The molecular weight was determined by gel filtration and sodium dodecyl sulfate gradient gel electrophoresis to be 32,000. The isoelectric point was 4.4. Aspartic acid-asparagine, glutamic acid-glutamine, leucine, lysine, alanine, and serine dominated the amino acid composition.