Purification of beta-lactamases by affinity chromatography on phenylboronic acid-agarose.
Several beta-lactamases, enzymes that play an important part in antibiotic resistance, have been purified by affinity chromatography on boronic acid gels. The procedure is rapid, appears to be selective for beta-lactamases, and allows a one-step purification of large amounts of enzyme from crude cell extracts. We have found the method useful for any beta-lactamase that is inhibited by boronic acids. Two kinds of boronic acid column have been prepared, the more hydrophobic one being reserved for those beta-lactamases that bind boronic acids relatively weakly. beta-Lactamase I from Bacillus cereus, P99 beta-lactamase and K 1 beta-lactamase from Gram-negative bacteria are among the better-known beta-lactamases that have been purified by this method. The procedure has also been used to purify a novel beta-lactamase from Pseudomonas maltophilia in high yield; the enzyme has an exceptionally broad substrate profile and hydrolyses monocyclic beta-lactams such as azthreonam and desthiobenzylpenicillin.
D002846:Chromatography, Affinity / Q000379:methods*; D007525:Isoelectric Focusing; D011549:Pseudomonas / Q000201:enzymology; D012685:Sepharose / Q000031:analogs & derivatives*; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000302:isolation & purification*
C035597:phenylboronic acid-sepharose; D012685:Sepharose; D001618:beta-Lactamases