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Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.

In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium mutant M-0198, isopenicillin N was converted into a penicillinase-resistant material that behaved like deacetoxycephalosporin C on high-pressure liquid chromatography analysis. This activity was found to be unstable to storage at -80 degrees C; 70-80% of the activity was lost after 1 day.

Laboratory cultures of Trichoderma hamatum produce metabolites that are characterized by an isocyanide functionality. Three such metabolites predominate. One is the known compound trichoviridin (I). The other two, described here for the first time, are 3-(3-isocyano-6-oxabicyclo[3,1,0]hex-2-en-5-yl)acrylic acid (II) and a very unstable compound 3-(3-isocyanocyclopent-2-enylidene-)propionic acid (III). Production of these three metabolites by a random sample of wild isolates of the fungus has been examined. At least one of these isocyanides was isolated from all cultures in which the culture broth inhibited the growth of Micrococcus luteus. The relative amounts of the three isocyanides produced by individual isolates were not the same and cultures were found in which I, II, or III was the main product. The isocyanide III was produced by all wild isolates which had antibiotic activity in their culture broth, and it was present in the concentration range 2-40 mg X L-1.