Spheroplast formation and partial purification of microbodies from hydrocarbon-grown cells ofCladosporium resinae

Cells of Cladosporium resinae form greater numbers of microbodies when grown on n-alkanes than when grown on glucose.To facilitate isolation of microbodies, hydrocarbon-grown cells were spheroplasted.Of four spheroplasting agents and five osmotic supports examined, best results were obtained after a 4-h incubation with Novozym 234 plus chitinase and with 0.8 M sorbitol as osmotic support.Equal numbers of spheroplasts were obtained at pH 5.8 and at pH 7.0.Catalase was used as a marker for microbodies and cytochrome-c oxidase as a marker for mitochondria.Urate oxidase, a second marker for microbodies, was not detected in cell extracts.Microbodies were extremely fragile; of eight spheroplast disruption techniques attempted, the best yield of microbodies was obtained using a Teflon homogenizer for 5 min.Microbodies were partially purified by differential and density gradient centrifugation.Best results were obtained with discontinuous Percoll gradients which yielded a fraction enriched in microbodies and one enriched in mitochondria.