CEPHALOSPORIN FORMATION BY CELL-FREE EXTRACTS FROM STREPTOMYCES CLAVULIGERUS

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Cell-free extracts of Streptomyces clavuligerus convert δ-L-(α-aminoadipyl)-L-cysteinyl-D-valine (ACV) into an antibiotic product which is 30-50% penicillinase-insensitive. Thin-layer chromatography resolves this antibiotic product into one major penicillinase-sensitive component and one major and one minor penicillinase-resistant component. The major and minor penicillinase-resistant antibiotics co-chromatograph with deacetoxycephalosporin C and deacetylcephalosporin C, respectively. Ring expansion of a penicillin intermediate, as evidenced by the production of penicillinase-resistant antibiotic, shows an absolute requirement for α-ketoglutarate, while ATP, K⁺ and Mg²⁺ have lesser effects. Ring expansion activity is not sedimented by high speed centrifugation and is unaffected by membrane-disrupting treatments. Penicillin N and ACV (presumably via penicillin N) are the only substrates so far accepted by the ring expanding enzyme. New syntheses of penicillin N and isopenicillin N are described.