biodegration of monoterpenes by pseudomonas incognita

Abstract

Terpenoids are ubiquitous in nature. Some soil microorganisms, especially species belonging to Pseudomonas, have the ability to utilize terpenes as the sole source of carbon and energy. This process of mineralization helps recycle the carbon of terpene molecules. However, studies on microbial degradation of terpenes are fewer compared with those on steroids and aromatic compounds.

In the present thesis, the metabolism of some monoterpenes by a versatile bacterium, Pseudomonas incognita, and the enzymes involved in their biodegradation have been studied. P. incognita was originally isolated from soil using linalool as the sole carbon source. Metabolic pathways have been proposed based on:

Identification of metabolites from the culture medium.

Growth and manometric studies with isolated or synthesized probable intermediates.

Demonstration of enzyme systems involved in the degradative pathways.

Linalyl acetate (1) is metabolized by three different pathways:

One route involves oxygenation of the C-8 methyl group, forming 8-hydroxy linalyl acetate (2), which undergoes progressive oxidation with the acetoxy group intact. Cleavage of the 6,7-double bond of linalyl acetate-8-carboxylic acid (3) yields 4-acetoxy-4-methyl-hex-5-enoic acid (4).

The second pathway begins with hydrolysis of linalyl acetate (1) to linalool (5). Metabolism of linalool is initiated by oxidation at the C-8 position, forming linalool-8-carboxylic acid (7).

The third pathway involves cyclization of linalool to ?-terpineol (8), which is metabolized through oleuropeic acid (9).

P. incognita was also tested for its ability to metabolize structurally modified acyclic monoterpenes such as 1,2-dihydrolinalyl acetate, tetrahydrolinalyl acetate, 2,6-dimethyl octa-2,6-diene, 2,6-dimethyl oct-2-ene, and 2,6-dimethyl octane. The 6,7-double bond was found essential for an acyclic monoterpene to act as a substrate. Reduction of the 1,2-double bond did not affect suitability. Among analogs tested, metabolism of 1,2-dihydrolinalyl acetate (10) was studied in detail, showing two routes: one retaining the acetoxy group, the other involving hydrolysis to 1,2-dihydrolinalool (11).

Metabolism of geranyl acetate (12) yielded geraniol (13), geranic acid (14), and 3-hydroxy citronellic acid (15). Neryl acetate (16) was degraded to nerol (17), nerol-2,3-epoxide (18), neranic acid (19), and 3-hydroxy citronellic acid (15). The isolation of 3-hydroxy citronellic acid was particularly intriguing, as it had not been implicated in terpene degradation before.

?-Terpineol (8), a cyclic monoterpene alcohol with antimicrobial activity, was accepted by P. incognita as the sole carbon source. Its metabolism produced compounds such as limonene (20), p-cymene-8-ol (24), oleuropeic acid (9), cumic acid (27), 8-hydroxy cumic acid (25), p-isopropenyl benzoic acid (26), p-isopropyl pimelic acid (23), and 1-hydroxy-4-isopropenyl cyclohexane carboxylic acid (22). Pathways included aromatization of ?-terpineol to p-cymene-8-ol, oxidation at C-7 to oleuropeic acid, and subsequent ring cleavage.

Enzyme systems involved in initial metabolism were investigated. The 10,000 × g supernatant of cell-free extracts from linalyl acetate-adapted cells converted linalyl acetate to linalyl acetate-8-carboxylic acid and linalool-8-carboxylic acid, demonstrating the presence of:

Linalyl acetate-8-hydroxylase

Linalyl acetate-8-alcohol dehydrogenase

Linalyl acetate-8-aldehyde dehydrogenase

Hydroxylase activity was inhibited by carbon monoxide and metyrapone, indicating involvement of a cytochrome P-450 dependent monooxygenase system. Binding studies showed linalool and 1,2-dihydrolinalool bound tightly to cytochrome P-450.

The second enzyme, linalyl acetate-8-alcohol dehydrogenase, was partially purified. It is NAD-specific (inactive with NADP), has a broad pH optimum (8.0–8.8), and is strongly inhibited by p-hydroxy mercuribenzoate and N-methyl maleimide. Substrate specificity studies showed acyclic monoterpenes with a C-8 hydroxyl group are suitable substrates, while cyclic monoterpenes with C-7 hydroxyl groups are dehydrogenated more slowly.