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

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Abstract
A 13-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 S. 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 was developed as follows: mobile phase, 5% methanol -95% KH2PO4 (0.05 M adjusted to pH 4.0 with concentrated H3PO4); stationary phase, .mu.Bondapak-C18; flow rate, 2 ml/min for 5 min, 3 ml/min 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 min, 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 .mu.g/ml after a 1-h incubation period. Cyclization reaction mixtures, in which salt-precipitated cell-free extract from S. clavuligerus is the enzyme source, contain 39.0 .mu.g/ml (iso)penicillin N.

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