Optimization of <Emphasis Type="Italic">R</Emphasis>-(+)-α-terpineol production by the biotransformation of <Emphasis Type="Italic">R</Emphasis>-(+)-limonene

R-(+)-limonene is an abundant and non-expensive by-product of the citrus industry and is, therefore, a suitable starting material for the production of natural flavor and fragrance compounds. The biotransformation of R-(+)-limonene to R-(+)-alpha-terpineol by Fusarium oxysporum 152b has already been reported, although the influence of the main process parameters on the production has not yet been evaluated. In this paper, a Plackett-Burman screening design was used to define the effects of the medium composition (glucose, peptone, yeast extract, malt extract and pH), the presence of a co-substrate (biosurfactant), the cultivation conditions (temperature, agitation), the substrate concentration and the inoculum/culture medium ratio on the absolute amount of R-(+)-alpha-terpineol resulting from this biotransformation. The process conditions were further optimized applying response surface methodology (RSM). The volatiles were extracted using a SPME device and were subsequently quantified by GC-FID and identified by GC-MS. The best results were obtained using 0.5% (v/m) R-(+)-limonene in pure distilled water as the culture medium with an inoculum/culture medium ratio of 0.25 (m/m) and 72 h cultivation at 26 degrees C/240 rpm. Under these conditions the concentration of R-(+)-alpha-terpineol in the culture medium reached 2.4 g L(-1), a production almost six times greater than in earlier trials. The presence of a biosurfactant (0-500 mg L(-1)) did not significantly increase the yield.     

Asymmetric hydrolysis of R-(−),S(+)-2-methylbutyronitrile by Rhodococcus rhodochrous NCIMB 11216

Whole cells and cell-free extracts derived from Rhodococcus rhodochrous NCIMB 11216 were shown to hydrolyse both aliphatic and aromatic nitriles, when the organism had been grown on either propionitrile or benzonitrile as the source of carbon and nitrogen. Whole cell suspensions and cell-free extracts derived from bacteria grown on either substrate were able to biotransform R-(-),S-(+)-2-methylbutyronitrile. The S-(+) enantiomer was biotransformed more rapidly than the the R-(-) enantiomer. For whole cell biotransformations at 30°C, the maximum enantiomeric excess (ee) of the remaining R-(-)-2-methylbutyronitrile was 93% when 70% of the R-(-) enantiomer had been converted to the product, 2-methylbutyric acid. For the corresponding biotransformation at 4°C, there was an ee of 93% for the residual R-(-) enantiomer of the substrate when only 60% of it had been converted to product. For biotransformations by cell-free extracts at 30°C the 2-methylbutyric acid product had an ee of 17% for the S-(+) enantiomer at the time of optimal ee for the remaining R-(-) enantiomer of the substrate. In contrast, when the reaction was carried out by whole cells, the ee for the product acid was 0.36%. This was probably due to further, non-selective metabolism of the acid, which was especially significant at the beginning of the reaction. At both temperatures, the ee for the S-(+) enantiomer of 2-methylbutyric acid was at a maximum in the early stage of the biotransformation; for example, at 4°C the maximum detectable ee was 100% when the yield was 11%.Abbreviations EDTA Ethylenediaminetetraacetic acid - ee enantiomeric excess - FID flame ionisation detector - GC gas chromatography - ¹HNMR H nuclear magnetic resonance - K _m Michaelis constant - NCIMB National Collection of Industrial and Marine Bacteria - td doubling time - V _max Maximum velocity     

Enantioseparation of chiral alcohols by complex formation and subsequent supercritical fluid extraction

The very first application of supercritical fluid extraction (SFE) on enantioseparation of alcohols is discussed. Resolution of three chiral alcohols (trans-2-chloro-cyclohexanol, trans-2-bromo-cyclohexanol, and trans-2-iodo-cyclohexanol) were performed by partial complexation with (-)-O,O'-dibenzoyl-(2R,3R)-tartaric acid monohydrate (DBTA). DBTA formed diastereomeric complexes with all S,S-enantiomers stable enough to extract the unreacted alcohols with supercritical carbon dioxide. Resolution efficiency increased with the size of halogen substituents, and by the proper selection of molar ratio, pure (-)-R,R-trans-2-iodo-cyclohexanol (ee > 99%, yield: 39%) or (+)-S,S-trans-2-iodo-cyclohexanol (ee = 98%, yield: 8%) were prepared in one process step. Achieved resolution efficiency values were much higher in all resolution procedures than in any other known enantioseparation of these racemic compounds. The developed method offers an environmentally friendly, efficient alternative of currently applied resolution processes, also on a preparative scale.     

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.     

Biotransformation of (R)-(+)- and (S)-(-)-limonene by fungi and the use of solid phase microextraction for screening

The biotransformation of (R)-(-)- and (S)-(-)-limonene by fungi was investigated. More than 60 fungal cultures were screened for their ability to bioconvert the substrate, using solid phase microextraction as the monitoring technique. After screening, the best fungal strains were selected for further study and were grown as sporulated surface cultures in conical flasks and as submerged liquid cultures. It was found that (+)- and (-)-limonene were converted by Penicillium digitatum to alpha-terpineol (main metabolite), cis- and trans-p-menth-2-en-1-ol, neodihydrocarveol and limonene oxide (minor metabolites) using liquid cultures. The bioconversion of (R)-(-)- and (S)-(-)-limonene by Corwespora cassiicola yielded (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol respectively. The bioconversions by liquid cultures were also monitored by solid phase microextraction as a function of time. The optimum conversion of limonene to alpha-terpineol by Penicillium digitatum was obtained after 8 hours (yield up to 100%). Since an important pH-decrease was noticed in some liquid broths, the stability of limonene under acidic conditions was investigated. No acid catalysed conversion products were recovered after 8 days from control flasks at pH 3.5 containing limonene.     

Biotransformation of (+)limonene and (-)piperitone by yeasts and yeast-like fungi

Arxula adeninivorans and Yarrowia lipolytica converted (+)limonene to perillic acid (0.06 and 1.0 g/l; yield 3% and 50%) and (-)piperitone to 7-hydroxy-piperitone (0.06 and 0.04 g/l; yield 12% and 8%). Two unclassified strains of the basidiomycetes, Trichosporon, transformed (+)limonene to isopiperitenone (0.05 and 0.4 g/l; yield 2% and 20%) and trans-1,2-dihydroxy-limonene (0.6 g/l; yield 30%) and (-)piperitone to trans-6-hydroxy-piperitone (0.1 and 0.2 g/l; yield 20% and 40%) and 2-isopropyl-5-methyl-hydroquinone (0.8 g/l; yield 16%).     

Bioconversion of limonene to α-terpineol by immobilized Penicillium digitatum

Bioconversion of (4R)-(+)-limonene to (4R)-(+)-α-terpineol by immobilized fungal mycelia of Penicillium digitatum was investigated in batch, repeated-batch and continuously fed systems. The fungi were immobilized in calcium alginate beads. These beads remained active for at least 14 days when they were stored at 4 °C. Three different aeration rates were tested. The highest yield was obtained at a dissolved oxygen level of 50.0 μmol/l. α-Terpineol production by this fungus was 12.83 mg (g beads)⁻¹ day⁻¹, producing a 45.81% bioconversion of substrate. Repeated-batch bioconversion showed yield decreases in the second and the third cycles. Regeneration with nutrient media after the third cycle improved the bioconversion yields. With continuous bioconversion, the half-life was dependent on the aeration. The optimum conditions with a continuous reactor were at an aeration rate of 0.3 standard l/min and a dilution rate of 0.0144 h⁻¹. Received: 10 June 1997 / Received revision: 18 August 1997 / Accepted: 11 September 1997     

Monoterpene biosynthesis pathway construction in Escherichia coli

Four genes encoding sequential steps for the biosynthesis of the spearmint monoterpene ketone (-)-carvone from the C(5) isoprenoid presursors isopentenyl diphosphate and dimethylallyl diphosphate were installed in Escherichia coli. Inducible overexpression of these genes in the bacterial host allowed production of nearly 5 mg/l of the pathway intermediate (-)-limonene, which was mostly excreted to the medium such that products of the downstream steps, (-)-carveol and (-)-carvone, were not detected. Assay of pathway enzymes and intermediates indicated that flux through the initial steps catalyzed by geranyl diphosphate synthase and limonene synthase was severely limited by the availability of C(5) isoprenoid precursors in the host. Feeding studies with (-)-limonene, to overcome the flux deficiency, demonstrated the functional capability of limonene-6-hydroxylase and carveol dehydrogenase to produce the end-product carvone; however, uptake and trafficking restrictions greatly compromised the efficiency of these conversions.     

Hydroxylation of limonene enantiomers and analogs by recombinant (-)-limonene 3- and 6-hydroxylases from mint (Mentha) species: evidence for catalysis within sterically constrained active sites

Limonene enantiomers and substrate analogs, including specifically fluorinated derivatives, were utilized to probe active site interactions with recombinant (-)-(4S)-limonene-3-hydroxylase (CYP71D13) and (-)-(4S)-limonene-6-hydroxylase (CYP71D18) from mint (Mentha) species. (-)-(4S)-Limonene is hydroxylated by both enzymes at the designated C3- and C6-allylic positions, with strict regio- and stereospecificity and without detectable allylic rearrangement, to give the corresponding products (-)-trans-isopiperitenol and (-)-trans-carveol. CYP71D13-catalyzed hydroxylation of (+)-(4R)-limonene also yields the corresponding trans-3-hydroxylated product ((+)-transisopiperitenol); however, the C6-hydroxylase converts (+)-(4R)-limonene to a completely different product profile dominated by the enantiopure cis-6-hydroxylated product (+)-cis-carveol along with several minor products, including both enantiomers of the trans-6-hydroxylated product ((+/-)-trans-carveol), indicating allylic rearrangement during catalysis. These results demonstrate that the regiospecificity and facial stereochemistry of oxygen insertion is dictated by the absolute configuration of the substrate. Fluorinated limonene analogs are also tightly bound by both enzymes and hydroxylated at the topologically congruent positions in spite of the polarizing effect of the fluorine atom on substrate reactivity. This strict retention of oxygenation geometry suggests a rigid substrate orientation imposed by multiple hydrophobic active site contacts. Structurally simplified substrate analogs are hydroxylated at slower rates and with substantial loss of regiospecificity, consistent with a loss of active site complementarity. Evaluation of the product profiles generated allowed assessment of the role of hydrophobic contacts in orienting the substrate relative to the activated oxygen species.     

Hydroxylation of specifically deuterated limonene enantiomers by cytochrome p450 limonene-6-hydroxylase reveals the mechanism of multiple product formation

The regiochemistry and facial stereochemistry of the limonene-6-hydroxylase- (CYP71D18-) mediated hydroxylation of the monoterpene olefin limonene are determined by the absolute configuration of the substrate. (-)-(4S)-Limonene is hydroxylated at the C6 allylic position to give (-)-trans-carveol as the only product, whereas (+)-(4R)-limonene yields multiple hydroxylation products with (+)-cis-carveol predominating. Specifically deuterated limonene enantiomers were prepared to investigate the net stereospecificity of hydroxylation at C6 and the mechanism of multiple product formation. The results of isotopically sensitive branching experiments of competitive and noncompetitive design were consistent with a nondissociative kinetic mechanism, indicating that (4R)-limonene has sufficient freedom of motion within the active site of CYP71D18 to allow formation of either the trans-3- or cis-6-hydroxylated product. However, the kinetic isotope effects resulting from deuterium abstraction were significantly smaller than expected for an allylic hydroxylation, and they did not approach the intrinsic isotope effect. (4S)-Limonene is oxygenated with almost complete stereospecificity for hydrogen abstraction from the trans-6-position, demonstrating rigid orientation during hydrogen abstraction and hydroxyl delivery. The oxygenation of (4R)-limonene leading to the formation of (+/-)-trans-carveol is accompanied by considerable allylic rearrangement and stereochemical scrambling, whereas the formation of (+)-cis-carveol proceeds without allylic rearrangement and with nearly complete stereospecificity for hydrogen abstraction from the cis-6-position. These results demonstrate that a single cytochrome P450 enzyme catalyzes the hydroxylation of small antipodal substrates with distinct stereochemistries and reveal that substrate-dependent positional motion of the intermediate carbon radical (and, therefore, hydroxylation stereospecificity) is determined by active-site binding complementarity. Thus, epimerization and allylic rearrangement are not inherent features of these reactions but occur when loss of active-site complementarity allows increased substrate mobility.     

Solubilization of insoluble inorganic phosphates by a novel salt- and pH-tolerant Pantoea agglomerans R-42 isolated from soybean rhizosphere

To develop environment-friendly biofertilizer solubilizing insoluble phosphates, salt- and pH-tolerant, insoluble inorganic phosphate-solubilizing bacterium was isolated from soybean rhizosphere. On the basis of its physiological characteristics and Vitek analysis, this bacterium was identified as Pantoea agglomerans. The optimal medium composition and cultural conditions for the solubilization of insoluble phosphate by P. agglomerans R-42 were 3% (w/v) of glucose, 0.1% (w/v) of NH4NO3, 0.02% (w/v) of MgSO4 x 7H2O, and 0.06% (w/v) of CaCl2 x 2H2O along with initial pH 7.5 at 30 degree C. The soluble phosphate production under optimal condition was around 900 mg/l, which was approximately 4.6-fold higher than the yield in the MPVK medium. The solubilization of insoluble phosphate was associated with a drop in the pH of the culture medium. P. agglomerans R-42 showed resistance against different environmental stresses like 5-45 degrees C temperature, 1-5% salt concentration and 3-11 pH range. Insoluble phosphate solubilization was highest from CaHPO4 (1367 mg/l), hydroxyapatite (1357 mg/l) and Ca3(PO4)2 (1312 mg/l). However, the strain produced soluble phosphate to the culture broth with the concentrations of 28 mg/l against FePO4, and 19 mg/l against AlPO4, respectively.     

On the kinetics of the enzyme-catalyzed hydrolysis of axial chiral alkyl allenecarboxylates: preparation of optically active R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid and its optically pure S-(+)-methylester

Pig liver esterase (PLE) was used for the preparation of optically active alkyl allenecarboxylates with axial chirality. Free and immobilized enzymes were used as biocatalysts for the kinetic resolution of racemic ester substrates. Whereas the biotransformations using the free biocatalyst resulted in moderately to high enantiomeric ratios, the immobilization significantly decreased the E-value. The reaction conditions were optimized with respect to the enantiomeric ratio and scaled up. The enantiomeric ratio (E-value) was thereby enhanced by a factor of four to E=60. Under optimized conditions (free enzyme, addition of acetone as a cosolvent and Triton X-100 as an emulgator) in a preparative scale biotransformation, 282 mg of optically pure S-(+)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid methylester (96% ee, 82% yield) and 257 mg of R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid (83% ee, 80% yield) could be synthesized from the racemic substrate.     

Effects of rhamnolipid on the cellulase and xylanase in hydrolysis of wheat straw

The effects of biosurfactant rhamnolipid (RL) and chemical surfactant Triton X-100 on the production of cellulases and xylanase from Penicillium expansum (P. expansum) in untreated, acid- and alkali-pretreated wheat straw submerged fermentations were studied, and the influences on the activity and stability of Cellulase R-10 were also investigated. The results showed that RL and Triton X-100 enhanced the activities of cellulases and xylanase to different extents and the stimulatory effects of RL were superior to those of Triton X-100. During the peak enzyme production phase, RL (60 RE mg/l) increased cellulases activities by 25.5-102.9%, in which the raise of the same enzyme in acid-pretreated straw broths was the most. It was found that the reducing sugars by hydrolyzing wheat straw with Cellulase R-100 were not visibly increased after adding RL. However, it distinctly protected Cellulase R-10 from degradation or inactivation, keeping the reducing sugars yield at about 17%.     

Oxidation of d-Limonene with Selenium Dioxide-hydrogen Peroxide

Oxidation of d-Iimonene with selenium dioxide-hydrogen peroxide affords (+)-l-hydroxyneodihydrocarveol as the major product formed via cis- and trans-limonene epoxide. Hydrolysis of the former epoxide is much faster than that of the latter, which can therefore be obtained in almost quantitative yield on acid hydrolysis of a mixture of cis- and trans-limonene epoxide (1:1) under mild condition.

Minor significance of oxygenation in an allylic position to a trisubstituted double bond and the difference of accessibility of an allylic position to di- and trisubstituted double bond toward the oxidant were also observed.     

Oxidation of 6,7-dihydro-5H-benzocycloheptene by bacterial strains expressing naphthalene dioxygenase, biphenyl dioxygenase, and toluene dioxygenase yields homochiral monol or cis-diol enantiomers as major products.

Bacterial strains expressing naphthalene, biphenyl, and toluene dioxygenase were examined for their abilities to oxidize 6,7-dihydro-5H-benzocycloheptene (benzocyclohept-1-ene). The major oxidation products were isolated, and their absolute configurations were determined by chiral 1H nuclear magnetic resonance analysis of diastereomeric boronate esters, chiral stationary-phase high-pressure liquid chromatography, and stereo-chemical correlation. Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae (formerly identified as a Beijerinckia sp.) B8/36 expressing naphthalene and biphenyl dioxygenases, respectively, oxidized benzocyclohept-1-ene to a major product identified as (-)-(1R,2S)-cis-dihydroxybenzocycloheptane (> 98% enantiomeric excess [ee], 50 and 90% yield, respectively). In contrast, Pseudomonas putida F39/D expressing toluene dioxygenase oxidized benzocyclohept-1-ene to (+)-(5R)-hydroxybenzocyclohept-1-ene (> 98% ee, 90% yield) as the major metabolite and to the "opposite" diol, (+)-(1S,2R)-cis-dihydroxybenzocycloheptane (> 98% ee, 10% yield). The results indicate that, for benzocyclohept-1-ene, the major reaction catalyzed by naphthalene and biphenyl dioxygenases is dioxygenation whereas toluene dioxygenase catalyzes mainly R-stereospecific benzylic monooxygenation. Although the type of reaction catalyzed by each organism was not predictable, the absolute configuration of the diol and monol products formed by naphthalene and toluene dioxygenases are consistent with the stereochemistry of the products formed by these enzymes from other benzocycloalkene substrates.     

Enantioselective Dynamic Process Reduction of α- and β-Tetralone and Stereoinversion of Resulting Alcohols in a Selected Strain Culture

α-Tetralone and β-tetralone were subjected to biotransformation by 14 fungal strains. Enantiomeric purity of the products depended on the reaction time. 3-Day transformation of α-tetralone in Absidia cylindrospora culture gave S-(+)-1,2,3,4-tetrahydro-1-naftol of 92 % ee, whereas longer biotransformation time resulted in decrease of ee value. 3-Day transformation of β-tetralone by the same strain gave predominantly S-(-)-1,2,3,4-tetrahydro-2-naftol, whereas after 9 days of the reaction, the R-enantiomer with 85 % ee was isolated. Transformation of β-tetralone by Chaetomium sp. KCh 6651 gave pure (S)-(-)-1,2,3,4-tetrahydro-2-naftol in high yield at the concentration of 1 g/l. In this process, a non-selective carbonyl reduction was observed, followed by a selective oxidation of the R-alcohol.     

Synthesis of 2-(3-(3,5-bis(trifluoromethyl)phenyl)thioureido)-3-((dimethylamino)methyl)camphor organocatalysts

In a stereo-divergent synthesis, three novel camphor-derived bifunctional thiourea organocatalysts 7-9 have been prepared in five steps starting from (+)-camphor. In addition, borneol-derived bifunctional thiourea organocatalysts 19/19' have been prepared in three steps from (1S)-(+)-camphorquinone. Novel organocatalysts 7-9, 19/19' have been evaluated in a model reaction of Michael addition of dimethyl malonate to trans-β-nitrostyrene with low to moderate enantioselectivities (20%-60% ee). Configuration of all novel compounds has been meticulously determined using nuclear magnetic resonance (NMR) techniques.     

A novel enantioselective synthesis of (S)-(-)- and (R)-(+)-nornicotine via alkylation of a chiral 2-hydroxy-3-pinanone ketimine template

An asymmetric synthesis of the optically pure isomers of the minor tobacco alkaloid and CNS nicotine metabolite, nornicotine, has been achieved with moderately high optical purity. The synthetic pathway involves alkylation of a chiral ketimine, prepared from either 1R,2R,5R-(+)- or 1S,2S,5S-(-)-2-hydroxy-3-pinanone and 3-(aminomethyl)pyridine with 3-bromopropan-1-ol. After cleavage of the respective C-alkylated ketimines with NH2OH.HCl, and treatment of the resulting amino alcohols with HBr, followed by base-catalyzed intramolecular ring closure, (S)-(-)-nornicotine and (R)-(+)-nornicotine were obtained with ee values of 91% and 81%, respectively.     

Improved enantioselective hydrolysis of racemic ethyl-2,2-dimethylcyclopropanecarboxylate catalyzed by modified Novozyme 435

S-(+)-2,2-dimethylcyclopropanecarboxylic acid (S-(+)-DMCPA) is a key chiral intermediate for the synthesis of Cilastatin. The enzymatic preparation of S-(+)-DMCPA has attracted much attention. In order to improve the activity and stability of Novozyme 435 for enzymatic preparation of S-(+)-DMCPA from 2,2-dimethylcyclopropane carboxylate (DMCPE), the glutaraldehyde modification for Novozyme 435 was investigated and the glutaraldehydemodified Novozyme 435 was used as biocatalyst for the synthesis of S-(+)-DMCPA. The results showed that the modified Novozyme 435 had a better reusing merit than unmodified enzyme. The maximum specific activity was obtained by modification Novozyme 435 with 1.5% glutaraldehyde solution under the conditions of shaking at 200 rpm and 30°C for 45 min. The optimal enzymatic hydrolysis conditions for glutaraldehyde-modified Novozyme 435 were also confirmed. The optimized hydrolytic reaction mixture contained 10 mL potassium phosphate buffer (1.0 mol/L, pH 7.6), 90 mg of DMCPE and 160 mg of glutaraldehyde-modified enzyme, and the reaction was performed at 30oC and 200 rpm for 52 h. The reusing efficiency of modified Novozyme 435 was further evaluated. Under the optimal conditions, the modified enzyme remained 76.0% of its original yield after 10 times reuse, but the optical purity of the product kept intact; whereas the yield of unmodified enzyme reduced to 20.8% of its initial value and the ee value of product decreased a lot to 90.7% after 7 times recycle. These results showed that the modified Novozyme 435 was more cost-effective for the preparation of S-(+)-DMCPA in industrial application.     

Increased EAG responses of tortricid moths after prolonged exposure to plant volatiles: evidence for octopamine-mediated sensitization

As measured by electroantennograms (EAG), both male and female obliquebanded leafrollers, Choristoneura rosaceana (Harris), and redbanded leafrollers, Argyrotaeniavelutinana (Walker), were similarly sensitive to host-related plant volatiles: trans-2-hexenal, benzaldehyde, 1-hexenol, cis-3-hexen-1-ol, geraniol, linalool, (+)-limonene, hexenal and trans-2-hexenol. Females of both species were similarly sensitive to the shared major component of their sex-attractant pheromone ((Z)11-14:Ac). Continuous 60 min pre-exposure of male and female C. rosaceana and A. velutinana to successively higher concentrations of a mixture of the nine plant volatiles in Teflon chambers with continuous air exchange caused a dosage-dependent increase in subsequent responsiveness (sensitization) to green leaf volatiles, as measured by EAGs. In addition, 60 min of pre-exposure of male C. rosaceana to certain individual volatiles ((+)-limonene, geraniol, benzaldehyde) increased EAGs nearly as much as did the mixture of nine volatiles. Pre-exposures to the nine plant-volatile mixture at concentrations achieved by 100 microg and 1 mg loading dosages in 100 microl of mineral oil significantly increased EAG depolarization to pheromone (cross-sensitization) in males but not females of both moth species. Antennae of male C. rosaceana pre-injected with 100 microg of octopamine (OA) without volatile pre-exposure exhibited sensitization nearly identical to that induced by pre-exposing moths to sensitizing concentrations of the plant-volatile mixture. Moreover, injection of the OA antagonist chlorpromazine (CP) blocked sensitization by the plant-volatile pre-exposure. Collectively, these findings suggest that exposures of tortricid moths to certain host-plant related volatiles may modulate subsequent olfactory sensitivity to behaviorally relevant chemical cues and that plant-volatile induced sensitization may be octopamine mediated.