| dc.description.abstract | The thesis entitled “Microbiological Transformations of Terpenes and Steroids” is divided into three chapters.
Chapter I — Microbiological Transformations of Monoterpenes
This chapter opens by emphasizing microbiology as a fundamental principle underlying much of current biological thinking and as an important tool in the hands of the organic chemist. Significant contributions to the area of microbial degradation of monoterpenes have been reviewed.
Certain bacteria belonging to the genus Pseudomonas grow on a large number of monoterpenes. A strain of Pseudomonas incognita, isolated on the monoterpene alcohol linalool (11), was found to grow on ??phellandrene and ??terpineol.
Fermentation of ??phellandrene (1) by P. incognita produced several metabolites. The neutral metabolites corresponded to auto?oxidized products of ??phellandrene. From the acidic fraction, phellandrene?7?carboxylic acid (2), cumic acid (3), 2,3?dihydroxycumic acid (4), and p?isopropylpyruvic acid (5) were identified by various physicochemical methods. Based on these identified metabolites, a probable pathway for ??phellandrene degradation was postulated (Chart I).
After the formation of cumic acid, the pathway converges with the known degradation pathway of p?cymene by Pseudomonas putida arvilla (PL strain). Manometric studies with resting cells were used to elucidate the nature of this pathway.
??Terpineol (6), an important intermediate in the biosynthesis of various monoterpenes and a key metabolite of linalool in P. incognita, was degraded to several metabolites, including oleuropeic acid (7), a dicarboxylic acid (8), p?mentha?1,8?diol?2?one (9), and a mixture of isomeric alcohols (10a, 10b), in which allylic hydroxylation had occurred. The acids (7), (8), and neutral metabolite (9) showed oxygen uptake in manometric studies using ??terpineol?grown cells. Probable degradation pathways were postulated (Chart II). One pathway parallels limonene degradation by P. putida (PL strain) and P. incognita. The metabolism of the dihydroxy ketone remains uncertain.
Substrate Specificity of the Cytochrome P?450 System
The substrate specificity of the soluble cytochrome P?450 obtained from cell?free extracts of linalool?grown P. incognita was studied. This hydroxylase cross?reacted with substrates such as ??phellandrene, ??terpineol, limonene (12), p?cymene (13), ??pinene (14), and geraniol (15). The P?450 system was induced when the organism was grown on each of these substrates, as shown by the identical spectral behaviour of the CO?complex of the dithionite?reduced enzyme, exhibiting the characteristic absorption at 447 nm.
In each case, the oxidation products were isolated and characterized. Crude P?450 fraction converted linalool into linalool?8?carboxylic acid (16) and oleuropeic acid (7). Due to alcohol and aldehyde dehydrogenases present in the preparation, only carboxylic acids appeared as end products.
The hydroxylase behaved like a typical P?450 monooxygenase: inhibited by carbon monoxide and cytochrome c, but not by cyanide. No product formation was observed with toluene, p?xylene, ethylbenzene, p?ethylmethylbenzene, cumene, or menth?5?ene.
Protonation Enzyme
The presence of a protonation enzyme, responsible for the conversion of linalool to oleuropeic acid, was demonstrated under anaerobic conditions. Both intact cells and cell?free sonicates, when incubated with linalool under nitrogen, yielded ??terpineol. This suggests that oleuropeic acid formation may proceed via ??terpineol (Chart 3).
Chapter II — Microbiological Transformation of Longifolene
This chapter begins with a brief review of microbial transformations of sesquiterpenes. The present study concerns the microbial oxidation of longifolene (17), a sesquiterpene with a novel tricyclic skeleton.
Two soil organisms, identified as strains of P. liranogenii and P. alcaligenes, were isolated using longifolene as the sole carbon source. A strain of P. cruciviae, originally isolated on caryophyllene, also degraded longifolene. P. alcaligenes could also grow on several other terpenic substrates.
The major metabolite (C??H??O?) was assigned structure (18) based on IR, PMR, CMR, and mass spectral data. This appeared to be a key intermediate in all three strains.
Chapter III — Microbiological Degradation of the Alkyl Side Chain in a Model Substrate and Cholesterol
Microbial degradation of the side chain of sterols is important for synthesizing corticosteroids and sex hormones. A model?substrate strategy was used to screen organisms capable of cleaving sterol side chains. Enrichment cultures using cholestane (19) yielded two bacteria (Micrococcus and Pseudomonas stutzeri) and one fungus.
These organisms were then tested for degradation of cholestane, cholesterol, and sitosterol. All produced the same metabolites. One cholestane metabolite had IR and NMR spectra identical to androstan?17?one (20). The strains grew well on cholesterol, but initial attack occurred on ring A yielding cholest?4?en?3?one.
However, in the presence of the chelating agent ?,???bipyridyl, the side?chain–cleaved product androsta?4?ene?3,17?dione (21) was obtained from both cholesterol and sitosterol. The corresponding dehydroepiandrosterone (22) was obtained in very poor yield. | |
