Biotransformation of monoterpenes by Oocystis pusilla

The biotransformation of several monoterpenes by the locally isolated unicellular microalga, Oocystis pusilla was investigated. The metabolites were identified by thin layer chromatography and GC/MS. The results showed that O. pusilla had the ability to reduce the C=C double bond in (+)-carvone to yield trans-dihydrocarvone and traces of cis-dihydrocarvone. O. pusilla also converted (+)-limonene to trans-carveol, as the main product, and yielded carvone and trans-limonene oxide. Furthermore, (−)-linalool was converted to trans-furanoid and trans-pyranoid linalool oxide, thymol was converted to thymoquinone, (−)-carveol was converted to carvone and trans-dihydrocarvone, (−)-menthone and (+)-pulegone were converted to menthol, (L)-citronellal was converted to citronellol, and (+)-β-pinene was converted to trans-pinocarveol.     

Identification of plant volatiles activating single receptor neurons in the pine weevil (Hylobius abietis)

Naturally produced plant volatiles, eliciting responses of single olfactory receptor neurons in the pine weevil, have been identified by gas chromatography linked with mass spectrometry. The receptor neurons (n = 72) were classified in 30 types, according to the compound which elicited the strongest response in each neuron, 20 of which compounds were identified. Most potent for 14 types of neurons (n = 50) were monoterpenes, including bicyclic (e.g. α-pinene, camphor and myrtenal) for 8 types (n = 32), monocyclic (limonene, carvone, α-terpinene) for 3 types (n = 12) and acyclic (e.g. β-myrcene and linalool) for 3 types (n = 6). Other compounds eliciting strongest responses of a neuron were five sesquiterpenes, including α-copaene and a farnesene-isomer, and an anethole type which has no biosynthetic relationship with terpenes. Within one type, receptor neurons with quite selective responses to the most potent compound as well as neurons with additional responses to several, structurally similar compounds were found, indicating that the neurons may have the same functional types of membrane receptors, but different sensitivities. Response spectra of neurons within the bicyclic-, mono-cyclic and acyclic types showed more overlapping than across the neuron types. Minimal overlapping response spectra was found between monoterpene and sesquiterpene neurons. The results suggest that this structure-activity relationship is significant for encoding plant odour information in the pipe weevil. Accepted: 6 January 1997     

Monoterpene biotransformation by <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Objective

To investigate the biocatalytic potential of Colletotrichum acutatum and Colletotrichum nymphaeae for monoterpene biotransformation.

Results

C. acutatum and C. nymphaeae used limonene, α-pinene, β-pinene, farnesene, citronellol, linalool, geraniol, perillyl alcohol, and carveol as sole carbon and energy sources. Both species biotransformed limonene and linalool, accumulating limonene-1,2-diol and linalool oxides, respectively. α-Pinene was only biotransformed by C. nymphaeae producing campholenic aldehyde, pinanone and verbenone. The biotransformation of limonene by C. nymphaeae yielded 3.34–4.01 g limonene-1,2-diol l^?1, depending on the substrate (R-(+)-limonene, S-(?)-limonene or citrus terpene (an agro-industrial by-product). This is among the highest concentrations already reported for this product.

Conclusions

This is the first report on the biotransformation of these terpenes by Colletotrichum spp. and the biotransformation of limonene to limonene-1,2-diol possibly involves enzymes similar to those found in Grosmannia clavigera.

     

Biocatalytic reduction of (+)-carvone and (−)-carvone in submerged cultures of the fungi Penicillium citrinum and Fusarium oxysporium

Abstract

Biocatalytic transformation represents a green approach to the asymmetric hydrogenation of activated alkenes. This paper details catabolic events after the addition of (?)-carvone or (+)-carvone to submerged cultures of Penicillium citrinum and Fusarium oxysporium. These microorganisms were shown to biotransform the isomers of carvone, leading to the formation of a diastereoisomeric excess of derivatives of carvone and reduced carveols, and also to isomerize both dihydrocarvone, and their derivatives dihydrocarveols.     

Changes in monoterpene composition of Mentha aquatica produced by gene substitution from a high limonene strain of M. Citrata

The dominant gene Lm that causes 60–90% limonene/cineole was substituted into M. aquatica by four convergent backcrosses. The natural strain of M. aquatica has 7·7% cineole, 4·9% limonene, traces of terpinolene and pulegone, 0·1% menthone, 0.2% menthol, and 66·4% menthofuran. The two modified hybrid strains with dominant gene Lm have 53·8 and 78·7% limonene/cineole and a total of only 1·0-3·8% 3-oxygenated compounds in contrast to a total of 66·7% found in the natural strain. The postulate is made that the Lm gene largely prevents either the conversion of a-terpineol → terpinolene or of limonene → isopiperitenone and that in these strains the recessive cc genotype largely but not completely prevents the conversion of limonene → carvone resulting in limonene accumulation. Mentha species almost invariably have either 2-oxygenated or 3-oxygenated compounds, not both. Close coupling phase linkage of the genes Lm and C explains why the self-pollinated progeny of M. spicata or M. crispa C-Lm/c-lm have a ratio of 3 carvone/dihydrocarvone: 1 pulegone/menthone rather than a ratio of 9 carvone : 3 limonene : 3 carvone and menthone: 1 menthone which would be expected if the genes Lm and C were independently inherited     

Studies on Leaf Oils of Citrus Species

Leaf oil samples of four Japanese citrus species were analysed by gas chromatography to determine the detailed composition of each leaf oil. The following components were identified: α-pinene, α-thujene, camphene, β-pinene, sabinene, β-myrcene, α-terpinene, limonene, β-phellandrene, trans-2-hexen-1-al, γ-terpinene, p-cymene, terpinolene, cis-2-penten-1-ol, n-hexyl alcohol, cis-3-hexen-1-ol, trans-2-hexen-1-ol, p-α-dimethylstyrene linalool, linalyl acetate, β-elemene, terpinen-4-ol, caryophyllene, humulene, α-terpineol, neryl acetate, geranyl acetate, β-selinene, geraniol and thymol. Most components were contained in common in leaf oils of the four citrus species, but relative contents of some of the components; such as γ-terpinene, linalyl acetate, and thymol differed from species to species. For example, γ-terpinene was the major component (33.8%) of Hassaku, whereas it was only a minor component in Daidai. Daidai is characterized by a very high content of linalyl acetate (35%) which is only a trace in the other three species. Kishu-mikan is characterized by a high content of thymol (15%).     

P450<Subscript>BM-3</Subscript>-catalyzed whole-cell biotransformation of α-pinene with recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> in an aqueous–organic two-phase system

A recombinant Escherichia coli BL21 (DE3) strain overexpressing a variant of P450_BM-3 (V26T/R47F/A74G/F87V/L188K; abbreviated: BL21 (P450_BM-3 QM)) oxyfunctionalizes the bicyclic monoterpene α-pinene to α-pinene oxide, verbenol, and myrtenol. To address the low water solubility and the toxicity of terpenoids, an aqueous–organic two-phase bioprocess was developed. Diisononyl phthalate was selected as a biocompatible organic carrier solvent capable of masking the toxic effects mediated by α-pinene and of efficiently extracting the products enabling scale-up to the bioreactor. With an aqueous to organic phase ratio of 3:2 and 30% (v/v) of α-pinene in the organic phase, a biocatalytic product formation period of more than 4 h was achieved. A comparison of the biotransformation performance of BL21 (P450_BM-3 QM) and a strain with an additional heterologous NADPH regeneration system comprising glucose facilitator and dehydrogenase, but only expressing half the amount of P450_BM-3 QM, shows comparable product concentrations of 1,020 ± 144 and 800 ± 61 mg l_Aq⁻¹, respectively. The total product yields Y _P/P450 (μmol μmol_P450⁻¹) were 80% higher when the strain with the cofactor regeneration system was used. A total product concentration of over 1 g l_Aq⁻¹, corresponding to the highest value reported for microbial α-pinene oxyfunctionalization so far, marks a promising step forward toward a future application of recombinant microorganisms for the selective oxidation of terpenoids to value-added products.     

POLLINATION OF DALECHAMPIA MAGNOLIIFOLIA (EUPHORBIACEAE) BY MALE EUGLOSSINE BEES

Flowers of Dalechampia magnoliifolia in eastern Peru were pollinated primarily by male Eulaema meriana and E. cingulata, which collected fragrance from the secretory gland borne in the staminate cymule. The fragrance contains carvone oxide, benzyl acetate, limonene, α-pinene, myrcine, β-pinene/camphene, and carvone. Trigona cf. pallens was the most abundant visitor, but was primarily a pollen thief. The rate of contact with the stigmas by Trigona, and hence the bee's efficacy as a pollinator, was highly variable and was determined by variation in the separation of anthers and stigmas. Dalechampia magnoliifolia co-occurred with a congeneric species, D. cissifolia, but did not share pollinators with it.     

Olfactory discrimination ability of South African fur seals (Arctocephalus pusillus) for enantiomers

Using a food-rewarded two-choice instrumental conditioning paradigm we assessed the ability of South African fur seals, Arctocephalus pusillus, to discriminate between 12 enantiomeric odor pairs. The results demonstrate that the fur seals as a group were able to discriminate between the optical isomers of carvone, dihydrocarvone, dihydrocarveol, menthol, limonene oxide, α-pinene, fenchone (all p < 0.01), and β-citronellol (p < 0.05), whereas they failed to distinguish between the (+)- and (?)-forms of limonene, isopulegol, rose oxide, and camphor (all p > 0.05). An analysis of odor structure–activity relationships suggests that a combination of molecular structural properties rather than a single molecular feature may be responsible for the discriminability of enantiomeric odor pairs. A comparison between the discrimination performance of the fur seals and that of other species tested previously on the same set of enantiomers (or subsets thereof) suggests that the olfactory discrimination capabilities of this marine mammal are surprisingly well developed and not generally inferior to that of terrestrial mammals such as human subjects and non-human primates. Further, comparisons suggest that neither the relative nor the absolute size of the olfactory bulbs appear to be reliable predictors of between-species differences in olfactory discrimination capabilities. Taken together, the results of the present study support the notion that the sense of smell may play an important and hitherto underestimated role in regulating the behavior of fur seals.     

Screening of antibacterial activities of twenty-one oxygenated monoterpenes

Plant essential oils are widely used as fragrances and flavours in the cosmetic, perfume, drug and food industries. Oxygenated monoterpenes are widespread components of the essential oils, usually occurring in high amount. In this paper, the antibacterial activities of twenty-one oxygenated monoterpenes (borneol, borneol acetate, camphor, carvone, 1,8-cineole, citronellal, beta-citronellol, dihydrocarvone, fenchol, fenchone, geraniol acetate, isomenthol, limonene oxide, linalool, linalool acetate, nerol, nerol acetate, terpinen-4-ol, alpha-terpineol, menthol and menthone) and penicillin (standard antibiotic) were determined using a disc diffusion method (in vitro) against 63 bacterial strains, belonging to 37 different genera and 54 species (plant, food and clinic origins). The results showed that the oxygenated monoterpenes exhibited a variable degree of antibacterial activities. These compounds also inhibited the growth of bacterial strains by producing a weak zone of inhibition from 7 to 11 mm in diameter, depending on the susceptibility of the tested bacteria. Among the tested compounds, nerol, linalool alpha-terpineol, fenchol and terpinen-4-ol showed antibacterial activity at a broad spectrum. However, their antibacterial activities were lower than those of penicillin. In contrast to these compounds, camphor and 1,8-cineole exhibited no inhibition effects on the growth of all tested bacteria.     

Studies on Leaf Oils of Citrus Species

Leaf oils from 6 domestic citrus species were analysed by gas chromatography and 32 compounds were identified as constituents of one or more oils. Most components present were common to all 6 citrus leaf oils, but the percent composition of some components, i.e. β-pinene, limonene, β-phellandrene, γ-terpinene, p-cymene, p-α-dimethylstyrene, citronellal, linalool and thymol methyl ether, differed considerably from species to species. For example, γ-terpinene is a main component (36.5%) of Shiikuwasha (Citrus depressa Hayata), but is a trace in leaf oils from Kawabata-mikan (C. aurea Tanaka) and Otaheite-orange (C. limonia Osbeck var. otaitensis Tanaka). Yuzu (C. Junos Sieb. ex Tanaka) is characterized by relatively high contents of β-phellandrene (11.2%) and p-α-dimethylstyrene (6.7%), which are minor constituents in the other 5 citrus leaf oils. Otaheite-orange is characterized by high contents of limonene (39.7%) and citronellal (10.0%).     

Olfactory discrimination ability of human subjects for enantiomers with an isopropenyl group at the chiral center

The ability of 20 human subjects to distinguish between nine enantiomeric odor pairs sharing an isopropenyl group at the chiral center was tested in a forced-choice triangular test procedure. I found (i). that as a group, the subjects were only able to significantly discriminate the optical isomers of limonene, carvone, dihydrocarvone, dihydrocarveol and dihydrocarvyl acetate, whereas they failed to distinguish between the (+)- and (-)-forms of perillaalcohol, perillaaldehyde, isopulegol and limonene oxide; (ii). marked interindividual differences in discrimination performance, ranging from subjects who were able to significantly discriminate between eight of the nine odor pairs to subjects who failed to do so with six of the nine tasks; and (iii). that with none of the nine odor pairs the antipodes were reported to differ significantly in subjective intensity when presented at equal concentrations. Additional tests of the chemesthetic potency and threshold measurements of the optical isomers of dihydrocarvone, dihydrocarveol, and dihydrocarvyl acetate suggest that the discriminability of these three enantiomeric odor pairs is indeed due to differences in odor quality. Analysis of structure-activity relationships suggest that the combined presence of (i). an isopropenyl group at the chiral center; (ii). a methyl group at the para-position; and/or (iii). an oxygen-containing group at the meta-position allows for the discrimination of enantiomeric odor pairs.     

Bioconversion of 2-cyanopyrazine to 5-hydroxypyrazine-2-carboxylic acid with Agrobacterium sp. DSM 6336

5-Hydroxypyrazine-2-carboxylic acid, a versatile building block for the synthesis of new antituberculous agents, was prepared by whole-cell biotransformation from 2-cyanopyrazine via pyrazinecarboxylic acid using Agrobacterium sp. DSM 6336. By developing a fermentation process for this two-enzyme-step bioconversion, a product concentration of 286 mM (40 g/l) was obtained. After the isolation method had been optimized the total yield was 80%. Received: 28 February 1997 / Received revision: 28 April 1997 / Accepted: 4 May 1997     

Formation of benzaldehyde by Pseudomonas putida ATCC 12633

Aromatic and heterocyclic aldehydes may be produced by the mandelate pathway of Pseudomonas putida ATCC 12633 via the biotransformation of benzoyl formate and substrate analogues. Under optimised biotransformation conditions (37 °C, pH 5.4) and with benzoyl formate as a substrate, benzaldehyde may be accumulated with yields above 85%. Benzaldehyde is toxic to P. putida ATCC 12633; levels above 0.5 g/l (5 mM) reduce the biotransformation activity. Total activity loss occurs at an aldehyde concentration of 2.1 g/l (20 mM). To overcome this limitation, the rapid removal of the aldehyde is desirable via in situ product removal. The biotransformation of benzoyl formate (working volume 1 l) without in situ product removal accumulates 2.1 g/l benzaldehyde. Benzaldehyde removal by gas stripping produces a total of 3.5 g/l before inhibition. However, the most efficient method is solid-phase adsorption using activated charcoal as the sorbant, this allows the production of over 4.1 g/l benzaldehyde. Addition of bisulphite as a complexing agent causes inhibition of the biotransformation and bisulphite is therefore is not suitable for in situ product removal. Received: 16 March 1998 / Received revision: 20 May 1998 / Accepted: 21 May 1998     

Biotransformation of selected monoterpenes under nitrate-reducing conditions

Batch experiments were conducted to assess both the biotransformation potentials of one hydrocarbon (α-pinene) and four alcohol monoterpenes (arbanol, linalool, plinol, and α-terpineol) under nitrate-reducing conditions at 23 °C, as well as their effects on the nitrate-reducing process. A mixed, nitrate-reducing culture developed from a forest-soil extract was enriched using ethanol as the electron donor and used in this study. α-Pinene was not biotransformed under the conditions of this study and inhibited both ethanol and nitrate utilization. Partial transformation of the alcohol monoterpenes was observed and resulted in inhibition of the nitrate-reducing process and cessation of further utilization of the added monoterpenes. Accumulation of biotransformation products – mainly hydrocarbon monoterpenes such as camphene, β-myrcene, and d-limonene – was observed. The hydrocarbon monoterpenes formed may have been responsible for the observed inhibition of the nitrate-reducing process and lack of complete utilization of the alcohol monoterpenes. These results have significant implications for the expected rate and extent of biotransformation of monoterpenes under anoxic conditions as well as their effect on the nitrate-reducing process in both engineered and natural systems. Received: 8 December 1998 / Received revision: 9 June 1999 / Accepted: 27 June 1999     

Repellent Efficacy of Caraway and Grapefruit Oils for Sitophilus oryzae (Coleoptera: Curculionidae)

This study examined the repellent efficacy of six essential oils extracted from caraway, clary sage, grapefruit, strawberry, thyme white, ylangylang, and their related volatile constituents against the adult rice weevil, Sitophilus oryzae using an olfactometer. The caraway and grapefruit oil showed the highest repellent efficacy against the rice weevil at a dose of 10μl. Gas Chromatography-Mass Spectrophotometer analysis revealed caraway oil to be rich in carvone and limonene, and grapefruit oil to be rich in limonene, β-myrcene and α-pinene. When the monoterpene was mixed in equal parts with the caraway and grapefruit essential oils, carvone with limonene in caraway oil demonstrated the highest repellent efficacy (96.7%). Limonene with α-pinene and β-myrcene in grapefruit showed strong repellent efficacy (86.4%) with synergistic effects on the S. oryzae.A mixture of caraway and grapefruit oils, as well as carvone and limonene, can be potent repellents that may be useful for controlling S. oryzae.     

Biotransformation of geraniol, nerol and citral by sporulated surface cultures of Aspergillus niger and Penicillium sp

The biotransformation of geraniol, nerol and citral by Aspergillus niger was studied. A comparison was made between submerged liquid, sporulated surface cultures and spore suspensions. This bioconversion was also carried out with surface cultures of Penicillium sp. The main bioconversion products obtained from geraniol and nerol by liquid cultures of A. niger were linalool and alpha-terpineol. Linalool, alpha-terpineol and limonene were the main products obtained from nerol and citral by sporulated surface cultures, whereas geraniol was converted predominantly to linalool, also resulting in higher yields. Bioconversion of nerol with Penicillium chrysogenum yielded mainly alpha-terpineol and some unidentified compounds. With P. rugulosum the major bioconversion product from nerol and citral was linalool. The bioconversion of nerol to alpha-terpineol and linalool by spore suspensions of A. niger was also investigated. Finally the biotransformation with sporulated surface cultures was also monitored by solid phase microextraction (SPME). It was found that SPME is a very fast and efficient screening technique for biotransformation experiments.     

Improved monoterpene biotransformation with <Emphasis Type="Italic">Penicillium</Emphasis> sp. by use of a closed gas loop bioreactor

A closed gas loop bioprocess was developed to improve fungal biotransformation of monoterpenes. By circulating monoterpene-saturated process gas, the evaporative loss of the volatile precursor from the medium during the biotransformation was avoided. Penicillium solitum, isolated from kiwi, turned out to be highly tolerant towards monoterpenes and to convert α-pinene to a range of products including verbenone, a valuable aroma compound. The gas loop was mandatory to reproduce the production of 35 mg L⁻¹ verbenone obtained in shake flasks and also in the bioreactor. Penicillium digitatum DSM 62840 regioselectively converted (+)-limonene to the aroma compound α-terpineol, but shake flask cultures revealed a pronounced growth inhibition when initial concentrations exceeded 1.9 mM. In the bioreactor, toxic effects on P. digitatum during biotransformation were alleviated by starting a sequential feeding of non-toxic limonene portions after a preceding growth phase. Closing the precursor-saturated gas loop during the biotransformation allowed for an additional replenishment of limonene via the gas phase. The gas loop system led to a maximum α-terpineol concentration of 1,009 mg L⁻¹ and an average productivity of 8–9 mg L⁻¹ h⁻¹ which represents a doubling of the respective values previously reported. Furthermore, a molar conversion yield of up to 63% was achieved. M. Pescheck and M. A. Mirata have contributed equally to this work.     

Cell adaptation to solvent, substrate and product: a successful strategy to overcome product inhibition in a bioconversion system

Carvone has previously been found to highly inhibit its own production at concentrations above 50 mM during conversion of a diastereomeric mixture of (−)-carveol by whole cells of Rhodococcus erythropolis. Adaptation of the cells to the presence of increasing concentrations of carveol and carvone in n-dodecane prior to biotransformation proved successful in overcoming carvone inhibition. By adapting R. erythropolis cells for 197 h, an 8.3-fold increase in carvone production rate compared to non-adapted cells was achieved in an air-driven column reactor. After an incubation period of 268 h, a final carvone concentration of 1.03 M could be attained, together with high productivity [0.19 mg carvone h⁻¹ (ml organic phase)⁻¹] and high yield (0.96 g carvone g carveol⁻¹).     

Bioremediation of polychlorinated biphenyl-contaminated soil using carvone and surfactant-grown bacteria

Partial bioremediation of polychlorinated biphenyl (PCB)-contaminated soil was achieved by repeated applications of PCB-degrading bacteria and a surfactant applied 34 times over an 18-week period. Two bacterial species, Arthrobacter sp. strain B1B and Ralstonia eutrophus H850, were induced for PCB degradation by carvone and salicylic acid, respectively, and were complementary for the removal of different PCB congeners. A variety of application strategies was examined utilizing a surfactant, sorbitan trioleate, which served both as a carbon substrate for the inoculum and as a detergent for the mobilization of PCBs. In soil containing 100 μg Aroclor 1242 g⁻¹ soil, bioaugmentation resulted in 55–59% PCB removal after 34 applications. However, most PCB removal occurred within the first 9 weeks. In contrast, repeated addition of surfactant and carvone to non-inoculated soil resulted in 30–36% PCB removal by the indigenous soil bacteria. The results suggest that bioaugmentation with surfactant-grown, carvone-induced, PCB-degrading bacteria may provide an effective treatment for partial decontamination of PCB-contaminated soils. Received: 9 March 2000 / Received revision: 27 June 2000 / Accepted: 16 July 2000