| dc.description.abstract | The thesis entitled "Microbiological transformations of terpenes and steroids" is divided into three main chapters.
Chapter I. Microbiological transformations of monoterpenes.
This chapter commences with a review of the significant contributions in the field of microbial degradation of monoterpenes. This is followed by the present studies in which the metabolism of the monoterpene alcohols linalool (1), geraniol (7), nerol (the cis-isomer of geraniol) and the monoterpene hydrocarbon limonene (16) by a soil pseudomonad are described.
The organism, identified as a strain of Pseudomonas incognita, was isolated on linalool (1) as the sole source of carbon in enrichment culture, and was found to grow also on geraniol (7), nerol and limonene (16).
The fermentation of linalool (1) by this organism gave rise to a number of metabolites, among which were 8-hydroxy-linalool (4), linalool-8-carboxylic acid (6), linalool-10-carboxylic acid (2) and oleuropeic acid (3) (Chart 1). The structure of these metabolites showed that there must be at least two different pathways (Chart 1) for the degradation of linalool (1), namely, one involving a stepwise oxidation of the 10-methyl group giving rise to (2) and (3), i.e., pathway A, and another involving a stepwise oxidation of the 8-methyl group giving rise to (4) and (6), i.e., pathway B.
Manometric studies with resting cells were carried out to study the nature of these pathways. From these, it was evident that pathway B, involving the 8-methyl oxidation, is a relatively minor pathway for the metabolism of linalool when compared to pathway A, which is initiated by a 10-methyl oxygenation.
The fermentation of geraniol (7) by this organism yielded a number of metabolites. Among these, the lactone (9), the triol (10), the keto-diol (11), citral (13), geranic acid (14) and the keto-acid (15) (Chart 2) were characterised by various physico-chemical methods. A study of their structures led to the postulation of 3 pathways for geraniol degradation (Chart 2). The existence of pathways B and C was clearly demonstrated by oxygen-uptake studies with resting cells. These studies also revealed the presence of 6-methyl-hept-5-en-2-one (12) in pathway B.
The fermentation of limonene (16) by the linalool strain gave the characteristic perillic acid (17) and 3-isopropenyl pimelic acid (18) among other products. This led to the hypothesis that the molecule is being degraded by the same pathway as that reported for limonene degradation by the PL-strain (Dhavalikar et al., Indian J. Biochem., 58 (1966)). This was further confirmed by manometric studies with the resting cells.
Enzymatic studies were carried out with the cell-free extracts from linalool-grown cells in order to investigate the nature of the enzymes responsible for the degradation of linalool (1). Three enzymes in pathway B (Chart 1) were characterised. The first one, linalool-8-hydroxylase, which is responsible for the formation of 8-hydroxylinalool (4), was found to be associated with the particulate fraction of the cell-free extracts and required NADH and DTT for activity. The second enzyme was the NAD-dependent linalool-8-alcohol dehydrogenase which catalyses the conversion of (4) to (5). The third enzyme of this pathway was the NAD-dependent linalool-8-aldehyde dehydrogenase, responsible for the conversion of (5) to (6). Both these dehydrogenases were associated with the soluble fraction of the cell-free extract and were partially resolved by ammonium sulphate fractionation.
In addition, a cytochrome P-450 hydroxylase enzyme was detected in the soluble fractions. The exact role of this enzyme in the metabolism of linalool could not be unequivocally established, but it is highly probable that it is involved in the 10-methyl hydroxylation leading to pathway A (Chart 1).
Chapter II. Microbiological transformation of caryophyllene.
This chapter begins with a brief review of the microbiological transformations of sesquiterpenes. The studies on the microbial oxidation of the sesquiterpene hydrocarbon caryophyllene (19) are then presented.
A soil microorganism, identified as a strain of Pseudomonas cruciviae, was isolated on caryophyllene as the sole source of carbon. Besides caryophyllene, it was found to grow also on some carbohydrates, salts of organic acids and a few other terpenes. The products of fermentation of caryophyllene (19) by this organism were isolated, and the structure of the major one assigned as the hydroxy-ketone (20) on the basis of spectral and chemical data.
Chapter III. The microbiological degradation of the alkyl side chain in a model substrate and cholesterol.
The microbiological degradation of the alkyl side chain of cholesterol is of great commercial interest in the steroid industry. The modes of biodegradation of steroid side chains by both animal cells and microorganisms are briefly reviewed at the beginning of this chapter.
Most of the cholesterol degrading microorganisms start degrading the molecule from ring A in preference to the side chain. In the present studies, a novel approach to the problem was initiated by screening microorganisms on a model substrate, isooctylcyclopentane (21). This substrate (21) was first synthesised, and an organism, identified as a strain of Pseudomonas convexa, was isolated on it as the sole source of carbon in enrichment culture. Two metabolites, cyclopentanol and cyclopentanone, could be identified among the fermentation products.
The organism, however, failed to grow on cholesterol, but the cells grown on (21) converted cholesterol to dehydroepiandrosterone in poor yield (about 1%). | |
