High performance liquid chromatographic assay of cyclization activity in cell-free systems from Streptomyces...

A thirteen-fold excess of dithiothreitol maintains delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) in its monomeric form under the conditions normally encountered in an ACV cyclization assay system, using Streptomyces clavuligerus. A reversed phase high performance liquid chromatographic (HPLC) system which separates ACV monomer from isopenicillin N, penicillin N and from other cyclization assay components has been developed as follows; mobile phase: 5% methanol-95% KH2PO4 (0.05 M adjusted to pH 4.0 with concentrated H3PO4; stationary phase: muBondapak-C18; flow rate: 2 ml/minute for 5 minutes, 3 ml/minute for the remainder; detection: 220 nm). Under these conditions, authentic samples of isopenicillin N and penicillin N elute with a retention time of 5.25 minutes, which coincides with a peak of newly-formed material observed in cyclization reaction mixtures. The combined concentration of isopenicillin N and penicillin N[(iso)penicillin N] in cyclization reaction mixtures corresponds closely to the concomitant decrease in the ACV monomer. Cyclization reaction mixtures, in which crude cell-free extract from S. clavuligerus NRRL 3585 is the enzyme source, contain (iso)penicillin N at a concentration of 43.3 micrograms/ml after a 1-hour incubation period. Cyclization reaction mixtures, in which salt-precipitated cell-free extract from S. clavuligerus is the enzyme source, contain 39.0 micrograms/ml (iso)penicillin N.