Mycobacterium fortuitum NRRL B-8 1 19 transformes sterols into 9 alpha-hydroxy-androsta-4-ene-3,17-dione (90H-AD) at high efficieny. Cells strongly aggregate to the surface of the sterol particles forming stable agglomerates ("substrate immobilized cells"). Substrate uptake and product formation were studied as function of the size of the sterol particles. Using particle sizes comparable with the size of the mycobacterial cells ( < 5pm) highest rates were found for both the substrate uptake and the product formation. After mechanical reinforcement of the contact between mycobacterial cells and the sterol substrate by co-grinding of the components, a significantly increased product formation was observed. Associated cells and cell-sterol agglomerates were investigated by freeze-fracture electron microscopy. The micrographs obtained demonstrate that cells of M . fortuitum are growing into the sterol microcrystallite. The uptake of the sterol substrate is assumed to take place via direct contact between cells and the substrate particles. To understand the transport mechanism a model is proposed that includes a flexible multicomponent mesophase (FMCM) which is placed between cells and particles and which mediates the sterol uptake. The putative FMCM is assumed to be composed of mycobacterial glycolipids, extracellular biolipids, synthetical detergents, sterol and water.Many bacterial species of different genera are capable of utilizing hydrophobic sterol substrates as the only carbon and energy sources in a remarkably high transformation rate. There are a large number of commercially operated processes to cleave selectively the side chain of the sterol molecule at position C-27, yielding C,, of C,, steroid intermediates which are important for the manufacture of steroid drugs. Microbial strains mostly used in fermentation processes are members of the genus Mycobacterium. Many reviews dealing with the biotechnology and biochemistry of these processes have been published during the last decade (ARIMA 1980, MARTIN 1977, 1984, SCHOMER and MARTIN 1980, NOMINE 1980, KIESLICH 1980, HESSELINK 1988, CHEN 1985. Enzymatic mechanisms of both the side chain cleavage and the degradation or modification of the steroid ring skeleton are well elucidated (SCHUBERT et al. , FUJUMOTO et a(. 1982, CHEN 1985. However, elementary mechanisms of the uptake of the hydrophobic solid sterol substrate occurring in fermentation processes are not clearly understood.Since more than ten years two basic mechanisms have been discussed: a) the substrate uptake via solubilized (molecular dispersed) or via pseudosolubilized (micellar) structures independent on the reason for solubilization or pseudosolubilization, and b) the substrate uptake caused by the direct contact between the microbial surface and the surface of the solid substrate microcrystallite (GOSHWAMI et al. 1983, CAMEOTRA, et al. 1983, HOSEL 1982, HOSEL et al. 1981.Elementary steps actually taking place seem to be a quite more complex process influenced by the physiological state of the gro...

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The steroid 1-dehydrogenase of Rhodococcus erythropolis IMET 7030, an active steroid-transforming strain, was localized by immunogold labelling both in cells induced with 17-alpha-methyl-testosterone and in noninduced cells. The labelling intensity was much higher in induced cells than in noninduced cells, indicating increased enzyme production in the case of induction. Using the postembedding procedure, the main portion of the enzyme was found in the peripheral region of the cytoplasm. A considerable amount was bound in clusters to the inner side of the cytoplasmic membrane. The enzyme was also detected in channels connecting the cytoplasm with the cell surface. By means of the preembedding labelling, a few gold clusters could be detected on the cell surface. The significance of this observation was discussed.