Sterol aide chain cleavage by micro-organisms

Abstract

Isolation, maintenance and identification of microorganisms are described. A novel method of strain selection is adopted using a model compound isooctylcyclopentane (I). Garden soil was treated first with cholesterol and enrichment was carried out in shake flasks containing cholesterol and isooctylcyclopentane separately. After few weeks of enrichment on isooctylcyclopentane, cholesterol was used for further enrichment. Two organisms were isolated from the enriched cultures which had the capability of degrading cholesterol. The organism isolated on cholesterol was identified to be a Pseudomonas sp. The other organism isolated on isooctylcyclopentane–cholesterol was found to belong to the family Neisseriaceae and to the genus Moraxella. The fermentation of cholesterol (IIIa), 3,5?cyclocholestane? 6??ol (i?cholesterol) (IIIa), 6??methoxy?3,5? cyclocholestane (IIIB), 3,5?cyclocholestane?6?one (i?choles? tanone) (IIIC), 3??methoxy?5?cholestene (IIb), cholesteryl chloride (IIc), 3??hydroxy?5??cholestane (cholestanol) (IV), 19?hydroxy?3??acetoxy?5?cholestene (V), potassium cholesteryl sulphate (IId), cholesteryl acetate (IIe) and 3??methoxy?5? stigmastene (VI) by both Pseudomonas sp. and Moraxella sp. is described. The Pseudomonas sp. failed to degrade any of the substrates described above except cholesterol and its acetate. With Moraxella sp. two novel transformations were observed. i?Cholesterol was isomerized back to cholesterol and 6??methoxy?3,5?cyclocholestane was demethylated to give i?cholesterol followed by isomerization to cholesterol. All the other modified substrates were degraded to the corresponding 17?keto compounds with varying yields. The advantage of the strain selection method and the dependence of the side?chain degradability of the microorganism on the modification of ring structure are discussed here. Study of cell?free enzymatic isomerization of i?cholesterol is the subject of the fourth chapter, "Partial purification and properties of i?cholesterol isomerase." The novel transformation of i?cholesterol to cholesterol, observed in the fermentation of i?cholesterol, has been studied at the cell?free level. The enzyme responsible for this isomerization has been partially purified by ammonium sulphate fractionation. Various properties of this enzyme are studied. The fifth and last chapter entitled "Immobilization of microbial whole cells" describes the fermentation of 19?hydroxycholesteryl acetate (V) and potassium cholesteryl sulphate (IId) by immobilized whole cells of Moraxella sp. This chapter starts with an introduction on general methods of immobilization. Various steroid transformations by immobilized microorganisms reported to date are reviewed. Though immobilized cell transformation is known for more than a decade, relatively few transformations of steroids are known through immobilized systems. Reports on side? chain degradation of sterols by immobilized microorganisms are extremely rare. Two methods of immobilization, i.e. entrapment and chemical binding on different supports, have been described. The fermentation of 19?hydroxycholesteryl acetate with the immobilized cells of Moraxella sp. resulted in the formation of estrone and potassium cholesteryl sulphate in dehydroepiandrosterone sulphate, in a continuous column reactor. Of the various supports studied agar was found to be the most suitable with more or less total retention of activity of the free cells.