Simple assay and extraction of periplasmic penicillinase in Escherichia coli.
Benzylpenicillin was clearly separated from benzylpenicilloic acid by ascending chromatography on a diethylaminoethyl cellulose paper using 0.1 M ammonium acetate as a solvent. Using this chromatographic system, penicillinase was assayed by measuring the formation of [14C]benzylpenicilloic acid from [14C]benzylpenicillin. This assay remedies the lack of specificity of the commonly used iodometric assays. Periplasmic penicillinase was released from Escherichia coli by suspension in a mixture of 1% phenethyl alcohol and 5 mM ethylenediaminetetraacetate (pH 7.0). This simple extraction method not only facilitates the assay of penicillinase in an E. coli culture, but will also be useful for large-scale purification of periplasmic penicillinase.
D003593:Cytoplasm / Q000201:enzymology; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D010400:Penicillin G / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D010405:Penicillinase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D010626:Phenylethyl Alcohol / Q000494:pharmacology; D012997:Solvents; D001618:beta-Lactamases / Q000032:analysis*
D012997:Solvents; D004492:Edetic Acid; D010405:Penicillinase; D001618:beta-Lactamases; D010626:Phenylethyl Alcohol; C030990:benzylpenicilloic acid; D010400:Penicillin G