isonovalal and 2-pinene-oxide

The feasibility of trans-2-methyl-5-isopropylhexa-2,5-dienoic acid (novalic acid) accumulation using the alpha-pinene degradation pathway of Pseudomonas rhodesiae CIP 107491 was studied. This appeared possible by using concentrated living bacterial cells produced under oxygen limitation with alpha-pinene as sole carbon source. The second step of the process, the bioconversion itself, had to be performed without oxygen limitation due to the need for cofactor regeneration. Results showed that a not yet reported cofactor-dependent enzymatic isomerization of isonovalal into novalal was likely to occur and that both aldehyde isomers could be oxidized to the corresponding acid. Precursors tested, alpha-pinene oxide and isonovalal had a strong permeabilization effect on bacterial cells. This effect, which increased from the oxide to the aldehyde, led to an inactivation of the respiratory chain and to acids synthesis stop. Present results allowed to obtain about 12 g/L acids (80% novalic acid) with an average yield close to 50% after 12h reaction in a biphasic system using alpha-pinene oxide as precursor .

Topics: Aldehydes; Bicyclic Monoterpenes; Biomass; Bioreactors; Carbon; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Magnetic Resonance Spectroscopy; Microbial Viability; Monoterpenes; Oxygen; Pseudomonas; Sorbic Acid; Terpenes; Time Factors

In this work the biotransformation of alpha-pinene oxide to isonovalal using resting cells of Pseudomonas fluorescens NCIMB 11671 was evaluated in a membrane bioreactor for biotransformations (MBB). Since the membrane area required to obtain optimum productivities was calculated to be very large (1,000 m2 m(-3)), and not possible to fit into the laboratory reactor used, we initially evaluated performance with lower membrane areas (71 m2 m(-3)) in a batch system with both the substrate and product in the organic phase. This resulted in low productivities due to mass transfer limitations, so an optimum feeding rate of 0.1 g alpha-pinene oxide h(-1) g(cells) (-1) added directly to the reactor contents was determined in batch culture to minimise inhibition. The MBB was then operated continuously for the production of isonovalal, and a final concentration of 108 g l(-1) was obtained in the organic reservoir after nearly 400 h of operation (0.32 g-isonovalal l(-1) h(-1)), and the reaction was found not to be mass transfer limited. Finally, the relative viability of the cells was measured using fluorescent probes, and their half-life was found to be almost 2 months, confirming the ability of the MBB to facilitate biotransformations with inhibitory substrates and products.

Topics: Aldehydes; Bicyclic Monoterpenes; Bioreactors; Biotransformation; Fermentation; Industrial Microbiology; Membranes, Artificial; Micropore Filters; Monoterpenes; Pseudomonas fluorescens; Terpenes

When submerged cultured Pseudomonas fluorescens NCIMB 11761 was fed-batch supplemented with alpha-pinene oxide, a rapid formation of 2,6-dimethyl-5-methylene-hept-(2Z)-enal (I) (isonovalal) was observed. Biotransformation and isomerisation of (I) to the (2E)-isomer (II) (novalal) were enhanced by Lewatit OC 1064, a macroporous polystyrene adsorbent. Accelerated isomerisation in the presence of an amino donor (glycine) at pH 7.3 pointed to a merely chemical mechanism. A maximum yield of 48 g of aldehydesl(-1) was achieved, but quantitative analysis of the volatile fraction showed that the molar conversion of the pinene oxide substrate reached no more than 67%. To fill this gap of the mass balance, the acidic fraction was isolated. It contained several compounds which suggested a beta-oxidation-like catabolism starting from 2,6-dimethyl-5-methylene-hept-(2E)-enoic acid (III) (novalic acid). Using [2H7]-2,5,6-dimethyl-hept-(2E)-enoic acid as a conversion substrate and gas chromatography coupled to atomic emission detection and mass spectrometry a degradation pathway via labelled 3,4-dimethylpentenoic and methylpropanoic acids was evidenced. This pathway may play a predominant role in isoprenoid degradation by soil bacteria.

Topics: Aldehydes; Bicyclic Monoterpenes; Chromatography, High Pressure Liquid; Fatty Acids, Monounsaturated; Isomerism; Molecular Structure; Monoterpenes; Oxidation-Reduction; Pentanoic Acids; Pseudomonas fluorescens; Terpenes

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. 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)).

Topics: Aldehydes; Bicyclic Monoterpenes; Biomass; Biotransformation; Cell Membrane Permeability; Models, Molecular; Monoterpenes; Osmolar Concentration; Pseudomonas; Terpenes