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

Summary Cells ofCladosporium resinae form greater numbers of microbodies when grown onn-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.