Analysis of penicillin N ring expansion activity from Streptomyces clavuligerus by ion-pair high-pressure liquid chromatography.

An ion-pair, reversed-phase, high-pressure liquid chromatographic method for the analysis of penicillin N ring expansion activity has been developed which allows simultaneous measurement of both substrate and product. The high-pressure liquid chromatography conditions were as follows: stationary phase, C18; flow rate, 2 ml/min; detection, 220 nm. The stationary phase was preconditioned with 4.5 mM tetrabutylammonium bromide in 0.05 M KH2PO4 (pH 4.0)-methanol (85:15, vol/vol) and then equilibrated with 0.06 mM tetrabutylammonium bromide in 0.05 M KH2PO4 (pH 4.0)-methanol (95:5, vol/vol) for analysis of reaction mixtures. These conditions separated authentic samples of penicillin N and desacetoxycephalosporin C and allowed cell-free studies of the ring expansion of penicillin N to desacetoxycephalosporin C by a partially purified enzyme from Streptomyces clavuligerus to be followed conveniently.