Live Tetrahymena thermophila transforms exogenous cholesterol into 7,22-bis, dehydrocholesterol (DHC) by desaturation at positions C7(8) and C22(23) of the cholesterol moiety. In this first report on expression, isolation, characterization, and reconstitution of Tetrahymena's cholesterol desaturases in cell-free extracts, we describe conditions for increasing the expression of both desaturases based on the addition of specific sterols to the culture medium. Reactions performed in vitro, with isolated microsomes, yield only the mono-unsaturated derivatives, 7-DHC and/or 22-DHC. However, selectivity towards one product can be improved with the addition of specific compounds: beta-mercaptoethanol inhibited C22(23) desaturase activity completely, while ethanol selectively increased this activity. Detergent-solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroyl-phosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b(5). NADH or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b(5) in these reactions.

Cell-free preparations of Tetrahymena thermophila catalyze the direct desaturation of cholesterol to delta7-dehydrocholesterol (provitamin D3). The activity was isolated in the microsomal fraction from Tetrahymena homogenates. Delta7-desaturase activity was stimulated fivefold by the addition of 6 mM ATP. Other cofactors assayed, including NAD, NADP, NADH or NADPH, had no significant effect. The activity was found in microsomes prepared from stationary-phase cultures of the ciliate, grown either with or without added cholesterol, thus indicating that it is constitutively expressed in T. thermophila cells.

The exceptional nutritional values of milk have long been recognized. However, important drawbacks of milk and dairy products are their relatively high cholesterol contents and their low proportion of polyunsaturated fatty acids. Hypersensitivity to milk proteins can also draw the public away from fluid milk consumption. The nonpathogenic ciliate Tetrahymena thermophila has a unique set of enzymes that converts cholesterol into provitamin D3 and related compounds. It also contains delta 12 fatty acid desaturases and releases various hydrolases, including proteases, lipases, and phospholipases. We investigated the potential use of these peculiar traits to improve milk by short-term incubation with live ciliate suspensions. We found that cholesterol can be reduced to 50% of its initial content in whole milk by a 12-h period of incubation with T. thermophila cells, mostly by conversion to delta 7-desaturated cholesterol derivatives, including provitamin D3. In addition, a considerable increase in gamma-linolenate (18:3, n-6) takes place. The protein profile is markedly changed by proteolysis, with a striking reduction of casein and enhancement in total amino acid content. Despite these changes, no marked alterations in flavor or odor were observed at the end of 12 h of incubation. Thus, Tetrahymena appeared ideally suited to correct for milk draw-backs, making this important source of nutrients more acceptable to the health-conscious public.

The potential use of the non-pathogenic ciliate Tetrahymena thermophila for converting cholesterol into pro-vitamin D sterols in egg suspensions was investigated. Conditions screened for efficient conversion included inoculum size and age, egg and egg yolk dilution, initial pH, and aeration. By optimizing these parameters, 55% ± 6 cholesterol reduction was obtained in 24 hours incubation. Tetrahymena converted cholesterol into three unsaturated sterols, D7, 22 bis-dehydrocholesterol, an ergosterol analog (27%), D7-dehydrocholesterol, also known as pro-vitamin D3 (4%), and D22dehydrocholesterol (8%). Despite significant proteolysis, culturing with Tetrahymena did not result in net consumption of total amino acids content, which was in fact increased by 16% ± 2. These findings emphasize the potential use of Tetrahymena in the food industry.