Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy

Abstract

A procedure to enrich microbodies from Penicillium chrysogenum and a method to evaluate the purity and integrity of the microbodies are described. As a P. chrysogenum microbody marker acyltransferase (AT) was used. The P. chrysogenum hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from mycelial debris after 10 000 × g centrifugation. Purified protoplasts were lysed, and the cell homogenate was centrifuged to form a 14 000 × g pellet. After 2 h, 45 000 × g isopycnic centrifugation of the 14 000 × g pellet on a continuous 20–60% nycodenz gradient, ten fractions were collected. The fractions were analyzed for AT containing microbodies by immuno-blotting and immuno-electron microscopy. The results showed that AT-microbodies are enriched in the 38% nycodenz fraction. The microbodies had a diameter of 400 to 500 nm, revealed an intact single membrane and confined AT. The estimated equilibrium density of the P. chrysogenum microbodies was 1.20 g ml −1 as deduced from the 38% (w/v) nycodenz concentration.