Microbial transformation of α-pinene

Summary A bacterium capable of utilizing -pinene as a sole carbon and energy source was isolated from soil. This strain, named strain S201-1, which was identified as Pseudomonas maltophilia on the basis of its taxonomical properties, accumulated limonene, borneol, camphor, perillic acid, and 2-(4-methyl-3-cyclohexenylidene) propionic acid from -pinene in the culture broth. It was demonstrated that -pinene, -pinene, borneol, camphor, and a number of p-menthane derivatives were oxidized by this strain. Relations between the protonation of -pinene and the formation of the products by the microbe are discussed.     

Thermophilic biofiltration of methanol and α-pinene

Biofiltration systems utilizing thermophilic (55C) bacteria were constructed and tested for the removal of methanol and α-pinene — two important volatile organic compounds (VOCs) in the forest products industry. Thermophilic bacterial mixtures that can degrade both methanol and α-pinene were obtained via enrichment techniques. Two bench-scale thermophilic biofiltration systems (1085 and 1824 cm³) were used to examine compound removals at different residence times, with influent concentrations of 110 ppmv methanol and 15 ppmv α-pinene. At a residence time of 10.85 min, the smaller system had removal efficiencies of >98% for methanol, but only 23% for α-pinene. The larger system was operated with the same parameters to evaluate residence time and surfactant effects on compound removals. At a residence time of 18.24 min, both methanol and α-pinene removal rates were ≥95%. However, α-pinene removal dropped to 26% at a residence time of 6.08 min; methanol removal was not affected. Subsequent addition of a surfactant mixture increased α-pinene removal to 94% at the shortest residence time. No residual α-pinene was detected with the support medium Celite R-635, indicating that the surfactant may increase mass transfer of α-pinene. Journal of Industrial Microbiology & Biotechnology (2001) 26, 127–133. Received 06 June 2000/ Accepted in revised form 09 November 2000     

Biotransformation of α- and β-pinene into flavor compounds

Products that bear the label “natural” have gained more attention in the marketplace. In this approach, the production of aroma compounds through biotransformation or bioconversion has been receiving more incentives in economic and research fields. Among the substrates used in these processes, terpenes can be highlighted for their versatility and low cost; some examples are limonene, α-pinene, and β-pinene. This work focused on the biotransformation of the two bicyclic monoterpenes, α-pinene and β-pinene; the use of different biocatalysts; the products obtained; and the conditions employed in the process.

     

Pheromones of Dendroctonus: Origin of α-pinene oxidation products present in emergent adults

Experiments showed that larvae and adults of the bark beetles Dendroctonus terebrans and Dendroctonus frontalis are capable of metabolizing α-pinene, a component of the oleoresin of their host Pinus taeda, to produce large quantities of oxidation products such as trans-verbenol, whereas the pupae do not. The results suggest that the pupae conjugate some form of the terpene molecule with an unknown compound and this conjugate is later metabolized by the young adult to yield the previously identified oxidation products found in emergent beetles. Only adult males of D. frontalis produced large quantities of the ketone verbenone. This compound was not detectable in the hindguts until after the adult maturation period and its production by emergent males could be related to the exposure of the pupae to α-pinene vapours. D. frontalis males are also capable of producing verbenone from α-pinene taken up in the adult stage. It is suggested that the production of verbenone by the males represents a specialization in the evolution of chemical communication in bark beetles. On the basis of this and earlier work, it is considered likely that other terpenes are metabolized in the same manner and that the same or a very similar system of terpene metabolism exists in other Dendroctonus species and closely related genera.     

A Study on the Process of Lipase-catalyzed Synthesis of α-pinene Oxide in Organic Solvents

The synthesis of α-pinene oxide was studied in a three-phase system where immobilized Candida antarctica lipase B (Novozyme 435) was used to catalyze the formation of peroxyoctanoic acid from the parent carboxylic acid and hydrogen peroxide in toluene. The peroxycarboxylic acid formed was then used in situ for the oxidation of α-pinene to the corresponding epoxide. When hydrogen peroxide was added in the reaction mixture gradually over 6 h, conversions increased up to 31.6%. Initial rates of α-pinene oxidation increased from 85 to 708 mmol L^?1 h^?1 when the amount of H₂O₂ increased from 5 to 60 mmol. When the lipase was exposed to 75 mmol H₂O₂ for 0.5 h before its addition in the reaction mixture, its activity decreased to about 50%. The reusability of lipase was studied in five reaction cycles and was found to depend on the concentration of the hydrogen peroxide used.     

Effect of oil concentration and residence time on the biodegradation of α-pinene vapours in two-liquid phase suspended-growth bioreactors

Recently, research on the use of binary aqueous-organic liquid phase systems for the treatment of polluted air has significantly increased. This paper reports the removal of α-pinene from a waste air stream in a continuous stirred tank bioreactor (CSTB), using either a single-liquid aqueous phase or a mixed aqueous-organic liquid phase. The influence of gas flow rate, load and pollutant concentration was evaluated as well as the effect of the organic to aqueous phase ratio. Continuous experiments were carried out at different inlet α-pinene concentrations, ranging between 0.03 and 25.1 g m?3 and at four different flow rates, corresponding to residence times (RTs) of 120 s, 60 s, 36 s and 26 s. The maximum elimination capacities (ECs) reached in the CSTB were 382 g m?3 h?1 (without silicone oil) and 608 g m?3 h?1 (with 5%v/v silicone oil), corresponding to a 1.6-fold improvement using an aqueous-organic liquid phase. During shock-loads experiments, the performance and stability of the CSTB were enhanced with 5% silicone oil, quickly recovering almost 100% removal efficiency (RE), when pre-shock conditions were restored. The addition of silicone oil acted as a buffer for high α-pinene loads, showing a more stable behaviour in the case of two-liquid-phase systems.     

A membrane bioreactor for the biotransformation of α-pinene oxide to isonovalal by Pseudomonas fluorescens NCIMB 11671

In this work the biotransformation of α-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 α-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.     

A systematic selection of the non-aqueous phase in a bacterial two liquid phase bioreactor treating α-pinene

A systematic evaluation of the selection criteria of non-aqueous phases in two liquid phase bioreactors (TLPBs), also named two-phase partitioning bioreactors (TPPBs), was carried out using the biodegradation of α-pinene by Pseudomonas fluorescens NCIMB 11671 as a model process. A preliminary solvent screening was thus carried out among the most common non-aqueous phases reported in literature for volatile organic contaminants biodegradation in TLPBs: silicon oil, paraffin oil, hexadecane, diethyl sebacate, dibutyl-phtalate, FC 40, 1,1,1,3,5,5,5-heptamethyltrisiloxane (HMS), and 2,2,4,4,6,8,8-heptamethylnonane (HMN). FC 40, silicone oil, HMS, and HMN were first selected based on its biocompatibility, resistance to microbial attack, and α-pinene mass transport characteristics. FC 40, HMS, HMN, and silicone oil at 10% (v/v) enhanced α-pinene mass transport from the gas to the liquid phase by a factor of 3.8, 14.8, 11.4, and 8.6, respectively, compared to a single-phase aqueous system. FC 40 and HMN were finally compared for their ability to enhance α-pinene biodegradation in a mechanically agitated bioreactor. The use of FC 40 or HMN (both at 10% v/v) sustained non-steady state removal efficiencies (RE) and elimination capacities (EC) approximately 7 and 12 times higher than those achieved in the system without an organic phase, respectively. In addition, preliminary results showed that P fluorescens could uptake and mineralize α-pinene directly from the non aqueous phase.     

Gas chromatographic-mass spectrometric identification of metabolites from α-pinene in human urine after occupational exposure to sawing fumes

Three metabolites from α-pinene (two diols and an alcohol with an aldehyde group) have been identified in human urine after occupational exposure to sawing fumes from pine. Urine was enzymatically hydrolyzed, cleaned up on a C₁₈ micro-column and the metabolites were identified by GC-MS using electron impact (70 eV) and chemical ionization with ammonia or isobutane as the reagent gas. Analysis of underivatized metabolites was performed using a capillary column with a semi-polar phase and trimethylsilylated derivatives were analyzed using a non-polar phase.     

Pseudomonas rhodesiae PF1: A New and Efficient Biocatalyst for Production of Isonovalal from α-pinene Oxide

A new bacterial strain, identified as Pseudomonas rhodesiae PF1 and deposited under the accession number CIP 107491, is presented. It is very active for the production of the acyclic compound Z-2-methyl-5-isopropyl-hexa-2,5-dien-1-al (isonovalal) from &#102 -pinene oxide. Enzyme synthesis is induced by culturing cells on &#102 -pinene; growing bacteria also have the ability to synthesize the epoxide derivative of &#102 -pinene. Isonovalal production was performed without aeration and with concentrated resting cells previously frozen at &#109 20°C, and subsequently thawed in a water-organic solvent, two-phase system. The organic layer was hexadecane, the volume ratio being 1:1. The best results achieved allowed recovery of c.a. 60 g/l organic solvent of isonovalal in 2.5 h operation, which is the most efficient process to date in the area of terpene biotransformations.     

New metabolites of α-pinene produced by the mountain pine beetle,Dendroctonus ponderosae (Coleoptera: Scolytidae)

Emergent females of the mountain pine beetle, Dendroctonus ponderosae, contained five previously undetected volatiles: toluene, 4-methylene-6,6-dimethylbicyclohept-2-ene (verbenene), p-mentha-1,5,8-triene, o- and p-cymene. Exposure of wild or axenically reared beetles to protio- and deuterio-α-pinene or protio- and deuterio-trans-verbenol indicated that all compounds except toluene were produced from α-pinene, with trans-verbenol as a probable intermediate. The ratio between these α-pinene metabolites was insensitive to the level of α-pinene to which the beetles were exposed, suggesting a tightly regulated enzymatic and/or acid-catalyzed conversion of α-pinene. Exposure of females to either enantiomer of α-pinene or to the same amount of (±)-α-pinene indicated that female mountain pine beetles possess two enantiospecific enzyme systems for processing α-pinene. Production of p-cymene constitutes the first record in an insect of an aromatic volatile produced from a monoterpene hydrocarbon.     

Synthesis of new α-heterocyclic α-aminoesters

alpha-Heterocyclic alpha-aminoesters were obtained in good yields by reaction of a glycine cation equivalent and different heterocyclic nucleophiles; diastereoselectivity using a carbohydrate (galactopyranose) as N-protecting group was modest.     

Mechanism of the cyanide-catalyzed oxidation of α-ketoaldehydes and α-ketoalcohols

Cyanide catalyzes the reduction of dioxygen or of ferricytochrome c by dihydroxyacetone phosphate. The rapid initial phase of these reactions, but not the subsequent slow phase, was augmented by incubating the triose phosphate aerobically or anaerobically at pH 9.0 prior to adding the cyanide. The aerobic incubation, which was most effective, was associated with a decline in enediol, whereas the less effective anaerobic incubation was accompanied by an increase in enediol content. This suggested that the α-ketoaldehyde product of autoxidation of the enediol, rather than the enediol itself, was responsible for the rapid phase reaction which followed addition of cyanide. This was confirmed by exploring the cyanide-catalyzed oxidation of the α-ketoaldehyde, phenylglyoxal. The inhibitory effect of the manganese-containing superoxide dismutase indicated that O₂⁻ was a kinetically important intermediate of the rapid phase reaction. A reaction mechanism is proposed which is consistent with the results presented.     

Photo-oxygenation of α-Ionone

Photo-oxygenation of α-ionone was studied to clarify the relationship between the maturity of aroma and photo-oxygenative change of α-ionone. α-Ionone was converted to oxygenated derivatives which were identified as 2,3-epoxy-β-ionone, 3,4-epoxy-α-ionone, 4-keto-β-ionone (trans- and cis-form), 5-keto-α-ionone and 3,4-dihydroxy-α-ionone.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

The Effect of α-pinene from Pinus densiflora S. and a Polysaccharide from Angelica gigas Nakai on Differentiation and Proliferation of Human Embryonic Stem Cells

The leukemia inhibitory factor (LIF), which is a very expensive reagent, can be used to efficiently control the differentiation of human embryonic stem (ES) cells at concentrations >1000 units/ml for 6–7 days. However, in supplement <500 units/ml, most ES cells differentiate within 3–4 days in in vitro cultures. α-Pinene from Pinus densiflora S. and a polysaccharide (MW 25 kDa) from A. gigas Nakai showed promising results as a substitute for LIF in cultivating ES cells. By adding both 0.5 (μg/ml) of α-pinene and the polysaccharide, most of the ES cells could be maintained under undifferentiated conditions after adding only 100 units/ml of LIF. It was found that α-pinene can play a role in preventing the ES cells from differentiating and the polysaccharide can be used to grow the ES cells. The results suggest that human ES cells can be maintained under undifferentiated conditions by supplementing both plant extracts, which can result in a reduction in the amount of LIF needed.     

Syntheses of α-D-glucosyl-D-fructoses by use of a reversed hydrolysis activity of α-glucosidase

Summary -D-Glucosyl-D-fructoses were synthesized by use of a reversed hydrolysis activity of -glucosidase fromSaccharomyces sp. Although -D-glucosyl-(1–1)-D-fructose was synthesized predominantly by the incubation of D-glucose solution in the presence of -glucosidase (batch method reaction), -(1–4)-linked disaccharide was a major product in a procedure by use of an immobilized -glucosidase column and an activated carbon column (column method reaction).