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pounds is accomplished by means of a biosynthetic pathway which closely parallels the corresponding pathway to cephalosporin C in Cephalosporium acremonium. Two of the enzymes involved in this pathway, deacetoxycephalosporin C (DAOC) synthetase and DAOC hydroxylase, carry out sequential reactions in which penicillin N undergoes ring expansion to DAOC and then DAOC is hydroxylated at the C3 methyl group to give deacetylcephalosporin C (DAC). These two enzymes are intermolecular dioxygenases which require molecular oxygen, iron, ascorbate and a-ketoglutarate for activity in both C. acremonium1~4) and S. clavuligerus5~6) • In view of the similar cofactor requirements of DAOC synthetase and DAOC hydroxylase, SCHEIDEGGER et al.7) recently undertook a purification and comparison of the two enzymes in cell-free extract from C. acremonium. They were unable to separate the two enzyme activities by any of a variety of protein purification techniques. On this basis, they have suggested that DAOC synthetase and DAOC hydroxylase activities may both reside in a single bifunctional enzyme in C. acremonium. In previous studies with S. clavuligerus8), it was found that DAOC hydroxylase activity was absent from DAOC synthetase purified by anion exchange chromatography. The separate existence of DAOC hydroxylase was not determined because of the lack of an assay procedure for this activity. Such a procedure has now been developed, and it has been found that the synthetase and hydroxylase activities reside in different enzymes. Cell-free extract was prepared from S. clavuligerus and partially purified by streptomycin sulfate and ammonium sulfate precipitation as previously described8). Partially-purified enzyme from 1 liter of original culture was then applied to a DEAE-trisacryl column (1.6 x 30 cm) which had been equilibrated with TDE buffer (0.05 M Tris-HCl (pH 7.0) 1.0 mM dithiothreitol 0.01 mM EDTA). The column was washed with 50 ml of TDE buffer and eluted with a linear gradient of 200 ml each of normal TDE buffer and TDE buffer containing 0.2 M Tris-HCl. Fractions of 50 drops (2.5 ml) were collected and monitored for UV-absorption at 280 nm. DAOC synthetase and DAOC hydroxylase were measured in reaction mixtures containing 2.8 mM sodium ascorbate, 0.045 mM FeSO4 1 mM aketoglutarate, 7.5 mM KCl, 7.5 mM MgSO4 0.05 M Tris-HCl (pH 7.0) and 0.03 ml of enzyme in a final volume of 0.04 ml. Each reaction mixture also contained 4leg of either penicillin N (DAOC synthetase) or DAOC (DAOC hydroxylase). Reactions were incubated at 20'C

Deacetoxycephalosporin C synthetase and deacetoxycephalosporin C hydroxylase are two separate enzymes in Streptomyces clavuligerus.