| In vitro: |
| Applied Microbiology & Biotechnology, 2003, 60(5):534-40. | | Optimization of isonovalal production from alpha-pinene oxide using permeabilized cells of Pseudomonas rhodesiae CIP 107491.[Pubmed: 12536252] |
METHODS AND RESULTS:
Optimization studies on the synthesis of isonovalal from alpha-Pinene oxide by Pseudomonas rhodesiae CIP 107491 operated in a biphasic medium are presented. Three key parameters are identified. The first is the need for a permeabilization of cells by freezing them and then treating the thawed material with an organic solvent such as chloroform, toluene or diethyl ether. This operation allows both enzyme release into the aqueous phase outside the cells and an improvement in the transport properties of both substrate and product across the cell membrane, strongly increasing reaction rates. The second is that the enzyme alpha-Pinene oxide lyase, which exhibits an irreversible inactivation by isonovalal (or a by-product), presents a constant turn-over, i.e., the total product synthesis is proportional to the biomass loading and is close to 108 mmol (16.4 g) isonovalal l(-1) g(-1) biomass. The third phenomenon is that the biphasic system used is not phase-transfer-limited, a feature attributed to the spontaneous formation of an oil-in-water emulsion.
CONCLUSIONS:
It is thus possible to carry out a very efficient process, allowing the recovery of 2.63 mol isonovalal l(-1) (400 g l(-1)) from 25 g biomass l(-1) in 2.5 h, corresponding to an average reaction rate as high as 0.70 mmol min(-1) g(-1) cells (160 g l(-1) h(-1)). | | Appl.environ.microbiol, 1998, 64(9):520-525. | | Effect of selected monoterpenes on methane oxidation, denitrification, and aerobic metabolism by bacteria in pure culture.[Pubmed: 9464387] |
METHODS AND RESULTS:
Selected monoterpenes inhibited methane oxidation by methanotrophs (Methylosinus trichosporium OB3b, Methylobacter luteus), denitrification by environmental isolates, and aerobic metabolism by several heterotrophic pure cultures. Inhibition occurred to various extents and was transient. Complete inhibition of methane oxidation by Methylosinus trichosporium OB3b with 1.1 mM (-)-alpha-pinene lasted for more than 2 days with a culture of optical density of 0.05 before activity resumed. Inhibition was greater under conditions under which particulate methane monooxygenase was expressed. No apparent consumption or conversion of monoterpenes by methanotrophs was detected by gas chromatography, and the reason that transient inhibition occurs is not clear. Aerobic metabolism by several heterotrophs was much less sensitive than methanotrophy was; Escherichia coli (optical density, 0.01), for example, was not affected by up to 7.3 mM (-)-alpha-pinene. The degree of inhibition was monoterpene and species dependent. Denitrification by isolates from a polluted sediment was not inhibited by 3.7 mM (-)-alpha-pinene, gamma-terpinene, or beta-myrcene, whereas 50 to 100% inhibition was observed for isolates from a temperate swamp soil. The inhibitory effect of monoterpenes on methane oxidation was greatest with unsaturated, cyclic hydrocarbon forms [e.g., (-)-alpha-pinene, (S)-(-)-limonene, (R)-(+)-limonene, and gamma-terpinene].
CONCLUSIONS:
Lower levels of inhibition occurred with oxide and alcohol derivatives [(R)-(+)-limonene oxide, alpha-Pinene oxide, linalool, alpha-terpineol] and a noncyclic hydrocarbon (beta-myrcene). Isomers of pinene inhibited activity to different extents. Given their natural sources, monoterpenes may be significant factors affecting bacterial activities in nature. |
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