Limonene Degradation Research Articles

Evaluation of toxic effect of monoterpene compounds on anaerobic digestion

Monoterpenes are antimicrobial compounds widely distributed in vegetable biomass, whose inhibitory potential for anaerobic digestion is underestimated. In this research, the toxic effect of limonene and fenchone, two of the main monoterpenes present in vegetable biomasses, and those of 4-terpineol, α-terpineol, and p-cymene, compounds described as main metabolites of limonene degradation, have been assessed. Methane production was totally inhibited at dosed of 1000 mg L−1 of fenchone and limonene and at 600 mg L−1 of p-cymene and 4-terpineol. Based on the methane production rate, the inhibition followed the next trend: α-terpineol < < fenchone < limonene ≈ p-cymene < 4-terpineol. Regardless of dosed concentration, monoterpenes were mostly degraded at the end of the experiment (>85%), except p-cymene at 600 mg L−1. Therefore, monoterpenes could entail a high risk of inhibition that can be aggravated by the difficulty to accurately follow their concentration and by the scarce information on their effect on anaerobic process.

Physiological and Proteomic Analysis Responsive Mechanisms for Salt Stress in Oat.

Oat is considered as a moderately salt-tolerant crop that can be used to improve saline and alkaline soils. Previous studies have focused on short-term salt stress exposure, and the molecular mechanisms of salt tolerance in oat have not yet been elucidated. In this study, the salt-tolerant oat cultivar Vao-9 and the salt-sensitive oat cultivar Bai5 were treated with 6 days of 0 and 150 mmol L−1 salt stress (nNaCl:nNa2SO4 = 1:1). Label-Free technology was then used to analyze the differentially expressed proteins in leaves under 0 and 150 mmol L−1 salt stress. The obtained results indicated that total of 2,631 proteins were identified by mass spectrometry in the four samples. The salt-tolerant cultivar Vao-9 mainly enhances its carbohydrate and energy metabolism through the pentose and glucuronate interconversions, and carbon fixation pathways in prokaryotes, thereby reducing the damage caused by salt stress. In addition, the down-regulation of ribosomes expression and the up-regulated expression of HSPs and CRT are all through the regulation of protein synthesis in response to salt stress. However, GABA metabolism presents a different synthesis pattern in Bai5 and Vao-9. The main KEGG function of differential expressed protein (DEP) in Bai5 is classified into protein processing in the endoplasmic reticulum, estrogen signaling pathway, antigen processing and presentation, longevity regulating pathway-multiple species, arginine and proline metabolism, beta-alanine metabolism, vitamin B6 metabolism, salmonella infection, chloroalkane and chloroalkene degradation, and limonene and pinene degradation. Moreover, the main KEGG functions of DEP in Vao-9 are classified as ribosome and carbon fixation pathways in prokaryotes, pentose and glucuronate interconversions, GABA ergic synapse, and taurine and hypotaurine metabolism. The results obtained in this study provide an important basis for further research on the underlying mechanisms of salt response and tolerance in oat and other plant species.

Experimental Evaluations of the Impact of an Additive Oxidizing Electronic Air Cleaner on Particles and Gases

Electronic air cleaning (EAC) technologies have garnered significant attention for use in buildings. Many EAC technologies rely on the addition of reactive constituents to indoor air to react with gas-phase compounds, enhance particle deposition, and/or inactivate microorganisms. However, limited data are available on the efficacy of many EAC technologies and their potential to form chemical byproducts during operation. Here we experimentally evaluate the indoor air quality impacts, specifically targeting particles and gases but not microbial constituents, of a commercially available additive oxidizing EAC that generates positive and negative ions and hydrogen peroxide (H2O2). Tests were conducted in a large unoccupied test chamber in Chicago, IL and an unoccupied laboratory in Portland, OR under a combination of natural conditions (i.e., without pollutant injection) and perturbation conditions (i.e., with pollutant injection and decay). A combination of integrated and time-resolved measurements was used across both test locations. Chamber tests at lower airflow rates demonstrated that operation of the EAC: (i) had no discernible impact on particle concentrations or particle loss rates, with estimated clean air delivery rates (CADRs) for various particle measures less than ±10 m3/h, (ii) was associated with apparent decreases in some volatile organic compounds (VOCs) and increases in other VOCs and aldehydes, especially acetaldehyde, although a combination of high propagated uncertainty, limitations in test methods (e.g., lack of replicates), and variability between repeated tests limit what quantitative conclusions can be drawn regarding gas-phase organics; (iii) did generate H2O2, assessed using a crude measure, and (iv) did not generate ozone (O3). Laboratory tests at higher airflow rates, which involved injection and decay of particles and a single VOC (limonene), both simultaneously and separately, demonstrated that: (i) pollutant loss rates for both particles and limonene were slightly lower with the EAC on compared to off, yielding slightly negative pollutant removal efficiencies (albeit largely within propagated uncertainty) and (ii) there was a change in observed concentrations of one potential limonene degradation product, m/z 59 (putatively identified as acetone), with steady-state levels increasing from 10 ppb (air cleaner off) to 15 ppb (air cleaner on). No increases or decreases beyond measurement uncertainty were observed for other analyzed gaseous limonene degradation products. Overall, both chamber and laboratory tests demonstrated negligible effectiveness of this device at the test conditions described herein for removing particles and mixed results for VOCs, including decreases in some VOCs, no discernible differences in other VOCs, and apparent increases in other compounds, especially lower molecular weight aldehydes including acetaldehyde.

De novo transcriptome and phytochemical analyses reveal differentially expressed genes and characteristic secondary metabolites in the original oolong tea (Camellia sinensis) cultivar \u2018Tieguanyin\u2019 compared with cultivar \u2018Benshan\u2019

BackgroundThe two original plants of the oolong tea cultivar (‘Tieguanyin’) are “Wei shuo” ‘Tieguanyin’—TGY (Wei) and “Wang shuo” ‘Tieguanyin’—TGY (Wang). Another cultivar, ‘Benshan’ (BS), is similar to TGY in its aroma, taste, and genetic make-up, but it lacks the “Yin Rhyme” flavor. We aimed to identify differences in biochemical characteristics and gene expression among these tea plants.ResultsThe results of spectrophotometric, high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS) analyses revealed that TGY (Wei) and TGY (Wang) had deeper purple-colored leaves and higher contents of anthocyanin, catechins, caffeine, and limonene compared with BS. Analyses of transcriptome data revealed 12,420 differentially expressed genes (DEGs) among the cultivars. According to a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the flavonoid, caffeine, and limonene metabolic pathways were highly enriched. The transcript levels of the genes involved in these three metabolic pathways were not significantly different between TGY (Wei) and TGY (Wang), except for two unigenes encoding IMPDH and SAMS, which are involved in caffeine metabolism. The comparison of TGY vs. BS revealed eight up-regulated genes (PAL, C4H, CHS, F3’H, F3H, DFR, ANS, and ANR) and two down-regulated genes (FLS and CCR) in flavonoid metabolism, four up-regulated genes (AMPD, IMPDH, SAMS, and 5′-Nase) and one down-regulated XDH gene in caffeine metabolism; and two down-regulated genes (ALDH and HIBADH) in limonene degradation. In addition, the expression levels of the transcription factor (TF) PAP1 were significantly higher in TGY than in BS. Therefore, high accumulation of flavonoids, caffeine, and limonene metabolites and the expression patterns of their related genes in TGY might be beneficial for the formation of the “Yin Rhyme” flavor.ConclusionsTranscriptomic, HPLC, and GC-MS analyses of TGY (Wei), TGY (Wang), and BS indicated that the expression levels of genes related to secondary metabolism and high contents of catechins, anthocyanin, caffeine, and limonene may contribute to the formation of the “Yin Rhyme” flavor in TGY. These findings provide new insights into the relationship between the accumulation of secondary metabolites and sensory quality, and the molecular mechanisms underlying the formation of the unique flavor “Yin Rhyme” in TGY.

Effect of zein-based microencapsules on the release and oxidation of loaded limonene

Limonene is a kind of widely used flavor oil, susceptible to degradation with the loss of lemon-like odors and the formation of off-flavors. This study aimed to develop a novel structurally well defined zein-based core-shell microencapsules loaded with limonene for limonene protection and controllable release. The microencapsules were fabricated using a two-step heterogeneous precipitation process, with the shell being formed by direct zein precipitation on the oil-water interface and growing along with the water addition driven by hydrophobic interaction. The formation of core-shell microencapsules occurred at the limonene/zein ratio around 2.0 with D4,3 close to 10 μm and shell thickness about 25 nm, while the smaller self-assembled zein particles could be formed at the ratio of 0.1 and 5.0, suggesting that the mass ratio of core to shell affected the formation of encapsulation and the shell thickness could be controlled. An interior rough and porous shell of the microencapsules was also observed. Sodium caseinate (SC) was adsorbed onto hydrophobic zein surface by electrostatic interaction, contributing to a hydrophilic interface and improving the re-dispersibility of the freeze-dried powder in water. The release rate of limonene in the burst release phase from zein-SC microencapsules was significantly lower than that of whey protein isolate (WPI)-stabilized emulsion, and tended to be gradual in the sustained phase, followed the zero order kinetics. The limonene retention rate of the microencapsules was higher than that of the WPI-stabilized emulsion during an acceleration storage condition, attributing to the solid barrier of the zein-SC shell of the microencapsules. High linear relationship (R2 > 0.96) between limonene retention rate and storage time in both microencapsules and WPI-stabilized emulsion was observed, suggesting potential controllability of limonene degradation. The ability to design the natural biopolymer-based colloidal microcapsules with slow-release and oxidation prevention of the unstable flavor oils opens a new opportunity in a stable dispersions in aqueous foods.

Biomethanization of citrus waste: Effect of waste characteristics and of storage on treatability and evaluation of limonene degradation

This study proposes the evaluation of the suitability of mesophilic anaerobic digestion as a simple technology for the treatment of the citrus waste produced by small-medium agro-industrial enterprises involved in the transformation of Citrus fruits. Two different stocks of citrus peel waste were used (i.e., fresh and stored citrus peel waste), to evaluate the influence of waste composition (variability in the type of processed Citrus fruits) and of storage (potentially necessary to operate the anaerobic digester continuously over the whole year due to the seasonality of the production) on anaerobic degradation treatability. A thorough characterization of the two waste types has been performed, showing that the fresh one has a higher solid and organic content, and that, in spite of the similar values of oil fraction amounts, the two stocks are significantly different in the composition of essential oils (43% of limonene and 34% of linalyl acetate in the fresh citrus waste and 20% of limonene and 74% of linalyl acetate in the stored citrus waste). Contrarily to what observed in previous studies, anaerobic digestion was successful and no reactor acidification occurred. No inhibition by limonene and linalyl acetate even at the maximum applied organic load value (i.e., 2.72 gCODwaste/gVSinoculum) was observed in the treatment of the stored waste, with limonene and linalyl acetate concentrations of 104 mg/l and 385 mg/l, respectively. On the contrary, some inhibition was detected with fresh citrus peel waste when the organic load increased from 2.21 to 2.88 gCODwaste/gVSinoculum, ascribable to limonene at initial concentration higher than 150 mg/l. A good conversion into methane was observed with fresh peel waste, up to 0.33 lCH4/gCODremoved at the highest organic load, very close to the maximum theoretical value of 0.35 lCH4/gCODremoved, while a lower efficiency was achieved with stored peel waste, with a reduction down to 0.24 lCH4/gCODremoved at the highest organic load.

Gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle-associated pathogen Grosmannia clavigera.

To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.

EFFECT OF ULTRA HIGH HYDROSTATIC PRESSURE ON CONCENTRATIONS OF LIMONENE, Α‐TERPINEOL AND CARVONE IN NAVEL ORANGE JUICE

ABSTRACT Response surface methodology (RSM) was used to compare the effects of the selected variables on the changes of concentrations of limonene,α‐terpineol and carvone by ultra‐high hydrostatic pressure (UHP) treatment. A Box–Behnken experimental design was used with control variables of pressure (100–500 MPa), temperature (20–40C) and treatment time (10–20 min). Pressure, temperature and treatment time were found to be critical factors influencing changes of concentrations. Both pressure and temperature could cause limonene degradation, resulting in significant increase ofα‐terpineol and carvone concentrations. Limonene degradation was increased with increasing processing pressure or temperature. An interaction effect of pressure and temperature on limonene degradation was also found. This study showed that Box–Behnken design and RSM could be used to model the changes in concentrations of limonene,α‐terpineol and carvone in orange juice by UHP treatment.PRACTICAL APPLICATIONSAs we know, ultra‐high hydrostatic pressure (UHP) processing has been an attractive way to replace conventional heat treatments for the preservation of foods. In our study, models of changes in the key aroma compounds in orange juice under UHP process were established. The changes of these aroma compounds in orange juice under UHP process could be understood further by these models. An improved understanding of the effect of UHP on critical aroma quality compounds of orange juices was helpful for us to facilitate industrial adoption of this technology. An effective research way to study the effect on aroma compounds by UHP process was also offered. Base on the data, there was a reference for us to establish an establishment of UHP processing criteria for commercial production of high‐aroma‐quality orange juice.

Microbial Oxidation of (-)-α-pinene to Verbenol Production by Newly Isolated Strains

Verbenol is a bicyclicbicycle secondary allylic alcohol, with pronounced camphor and mint flavor notes, mainly used as food flavoring. This compound is also used to control harmful insects, and hence has potential for using in agriculture, and is an intermediate in the synthesis of valuable perfume and medicinal substances. This work is focused on the microbial oxidation of (-)-α-pinene to verbenol production. To carry out the present study, 405 microorganisms were tested for their ability to bioconvert the substrate. From the isolated microorganisms, 193 were selected in the pre-screening using mineral medium for limonene degradation. At the screening step, 31 strains were able to convert (-)-α-pinene in verbenol. The highest concentration in verbenol from (-)-α-pinene was about 125.6mg/L for yeast isolated from orange juice industrial residue.

Isolation and Screening of Microorganisms for R-(+)-Limonene and (−)-β-Pinene Biotransformation

This work is focused on the biotransformation of R-(+)-limonene and (-)-beta-pinene to bioflavor production. To carry out the present study, 405 microorganisms were tested for their ability to bioconvert the substrates. From the isolated microorganisms, 193 were selected in the prescreening using mineral medium for limonene degradation. At the screening step, eight strains were able to convert R-(+)-limonene and 15 to transform (-)-beta-pinene, both in alpha-terpineol. The highest concentration in alpha-terpineol from R-(+)-limonene was about 3,450 mg/L for Penicillium sp. isolated from eucalyptus steam. From (-)-beta-pinene, the highest product concentration of 675.5 mg/L was achieved using an Aspergillus sp. strain isolated from orange tree stem.

Chemical and Physical Stability of Protein and Gum Arabic‐Stabilized Oil‐in‐Water Emulsions Containing Limonene

An important flavor component of citrus oils is limonene. Since limonene is lipid soluble, it is often added to foods as an oil-in-water emulsion. However, limonene-containing oil-in-water emulsions are susceptible to both physical instability and oxidative degradation, leading to loss of aroma and formation of off-flavors. Proteins have been found to produce both oxidatively and physically stable emulsions containing triacylglycerols. The objective of this research was to determine if whey protein isolate (WPI) could protect limonene in oil-in-water emulsion droplets more effectively than gum arabic (GA). Limonene degradation and formation of the limonene oxidation products, limonene oxide and carvone, were less in the WPI- than GA-stabilized emulsions at both pHs 3.0 and 7.0. These data suggest that WPI was able to inhibit the oxidative deterioration of limonene in oil-in-water emulsions. The ability of WPI to decrease oxidative reactions could be due to the formation of a cationic emulsion droplet interface at pH 3.0, which can repel prooxidative metals, and/or the ability of amino acids in WPI to scavenge free radical and chelate prooxidative metals.

Chemical and Physical Stability of Citral and Limonene in Sodium Dodecyl Sulfate−Chitosan and Gum Arabic-Stabilized Oil-in-Water Emulsions

Citral and limonene are the major flavor components of citrus oils. Both of these compounds can undergo chemical degradation leading to loss of flavor and the formation of undesirable off-flavors. Engineering the interface of emulsion droplets with emulsifiers that inhibit chemical reactions could provide a novel technique to stabilize citral and limonene. At present, emulsified flavor oils are usually stabilized by gum arabic (GA), which is a naturally occurring polysaccharide-protein complex. The objective of this study was to examine if citral and limonene were more stable in emulsions stabilized with a sodium dodecyl sulfate (SDS)-chitosan complex than GA. Citral degraded less in GA-stabilized than in SDS-chitosan-stabilized emulsions at pH 3.0. However, SDS-chitosan-stabilized emulsions were more effective at retarding the formation of the citral oxidation product, p-cymene, than GA-stabilized emulsions. Limonene degradation and the formation of limonene oxidation products, limonene oxide and carvone, were lower in the SDS-chitosan- than GA-stabilized emulsions at pH 3.0. The ability of an SDS-chitosan multilayer emulsifier system to inhibit the oxidative deterioration of citral and limonene could be due to the formation of a cationic and thick emulsion droplet interface that could repel prooxidative metals, thus decreasing prooxidant-lipid interactions.

Catalytic Mechanism of Limonene Epoxide Hydrolase, a Theoretical Study

The catalytic mechanism of limonene epoxide hydrolase (LEH) was investigated theoretically using the density functional theory method B3LYP. LEH is part of a novel limonene degradation pathway found in Rhodococcus erythropolis DCL14, where it catalyzes the hydrolysis of limonene-1,2-epoxide to give limonene-1,2-diol. The recent crystal structure of LEH was used to build a model of the LEH active site composed of five amino acids and a crystallographically observed water molecule. With this model, hydrolysis of different substrates was investigated. It is concluded that LEH employs a concerted general acid/general base-catalyzed reaction mechanism involving protonation of the substrate by Asp101, nucleophilic attack by water on the epoxide, and abstraction of a proton from water by Asp132. Furthermore, we provide an explanation for the experimentally observed regioselective hydrolysis of the four stereoisomers of limonene-1,2-epoxide.

Terpene Compounds as Possible Precursors of 1,8-Cineole in Red Grapes and Wines

While the contribution of 1,8-cineole to the aroma of wine has been reported, it is a matter of controversy that the vineyards producing such wines are surrounded by Eucalyptus trees, which may contribute their essence to the grapes. However, experimental information presented in this paper suggests that 1,8-cineole can be produced by chemical transformation of limonene and alpha-terpineol, and this process may be responsible for the occurrence of Eucalyptus-like aroma in Tannat wines from vines not grown in the vicinity of Eucalyptus trees. A mechanism for the chemical transformation of these aroma compounds is proposed.

Functional Characterization of a Catabolic Plasmid from Polychlorinated- Biphenyl-Degrading Rhodococcus sp. Strain RHA1

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.

Metabolism of carveol and dihydrocarveol in Rhodococcus erythropolis DCL14.

Rhodococcus erythropolis DCL14 assimilates all stereoisomers of carveol and dihydrocarveol as sole source of carbon and energy. Induction experiments with carveol- or dihydrocarveol-grown cells showed high oxygen consumption rates with these two compounds and with carvone and dihydrocarvone. (Dihydro)carveol-grown cells of R. erythropolis DCL14 contained the following enzymic activities involved in the carveol and dihydrocarveol degradation pathways of this micro-organism: (dihydro)carveol dehydrogenase (both NAD+- and dichlorophenolindophenol-dependent activities), an unknown cofactor-dependent carvone reductase, (iso-)dihydrocarvone isomerase activity, NADPH-dependent dihydrocarvone monooxygenase (Baeyer-Villiger monooxygenase), epsilon-lactone hydrolase and an NAD+-dependent 6-hydroxy-3-isopropenylheptanoate dehydrogenase. Product accumulation studies identified (4R)-carvone, (1R,4R)-dihydrocarvone, (4R,7R)-4-isopropenyl-7-methyl-2-oxo-oxepanone, (3R)-6-hydroxy-3-isopropenylheptanoate, (3R)-3-isopropenyl-6-oxoheptanoate, (3S,6R)-6-isopropenyl-3-methyl-2-oxooxepanone and (5R)-6-hydroxy-5-isopropenyl-2-methylhexanoate as intermediates in the (4R)-carveol degradation pathway. The opposite stereoisomers of these compounds were identified in the (4S)-carveol degradation pathway. With dihydrocarveol, the same intermediates are involved except that carvone was absent. These results show that R. erythropolis DCL14 metabolizes all four diastereomers of carveol via oxidation to carvone, which is subsequently stereospecifically reduced to (1R)-(iso-) dihydrocarvone. At this point also dihydrocarveol enters the pathway, and this compound is directly oxidized to (iso-)dihydrocarvone. Cell extracts contained both (1R)-(iso-)dihydrocarvone 1,2-monooxygenase and (1S)-(iso)-dihydrocarvone 2,3-monooxygenase activity, resulting in a branch point of the degradation pathway; (1R)-(iso-)dihydrocarvone was converted to 4-isopropenyl-7-methyl-2-oxo-oxepanone, while (1S)-(iso)-dihydrocarvone, which in vivo is isomerized to (1R)-(iso-)dihydrocarvone, was converted to 6-isopropenyl-3-methyl-2-oxo-oxepanone. 4-Isopropenyl-7-methyl-2-oxooxepanone is hydrolysed to 6-hydroxy-3-isopropenylheptanoate, which is subsequently oxidized to 3-isopropenyl-6-oxoheptanoate, thereby linking the (dihydro)carveol degradation pathways to the limonene degradation pathway of this micro-organism. 6-Isopropenyl-3-methyl-2-oxo-oxepanone is, in vitro, hydrolysed to 6-hydroxy-5-isopropenyl-2-methylhexanoate, which is thought to be a dead-end metabolite.

Substrate specificity and stereospecificity of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14; an enzyme showing sequential and enantioconvergent substrate conversion

Limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene degradation pathway of this microlorganism, has a narrow substrate specificity. Of the compounds tested, the natural substrate, limonene-1,2-epoxide, and several alicyclic and 2-methyl-1,2-epoxides (e.g. 1-methylcyclohexene oxide and indene oxide), were substrates for the enzyme. When LEH was incubated with a diastereomeric mixture of limonene-1,2-epoxide, the sequential hydrolysis of first the (1R,2S)- and then the (1S,2R)-isomer was observed. The hydrolysis of (4R)- and (4S)-limonene-1,2-epoxide resulted in, respectively, (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol as the sole product with a diastereomeric excess of over 98%. With all other substrates, LEH showed moderate to low enantioselectivities (E ratios between 34 and 3).

Stereoselective Carveol Dehydrogenase from Rhodococcus erythropolis DCL14: A NOVEL NICOTINOPROTEIN BELONGING TO THE SHORT CHAIN DEHYDROGENASE/REDUCTASE SUPERFAMILY

A novel nicotinoprotein, catalyzing the dichlorophenolindophenol-dependent oxidation of carveol to carvone, was purified to homogeneity from Rhodococcus erythropolis DCL14. The enzyme is specifically induced after growth on limonene and carveol. Dichlorophenolindophenol-dependent carveol dehydrogenase (CDH) is a homotetramer of 120 kDa with each subunit containing a tightly bound NAD(H) molecule. The enzyme is optimally active at pH 5.5 and 50 degrees C and displays a broad substrate specificity with a preference for substituted cyclohexanols. When incubated with a diastereomeric mixture of (4R)- or (4S)-carveol, CDH stereoselectively catalyzes the conversion of the (6S)-carveol stereoisomers only. Kinetic studies with pure stereoisomers showed that this is due to large differences in V(max)/K(m) values and simultaneous product inhibition by (R)- or (S)-carvone. The R. erythropolis CDH gene (limC) was identified in an operon encoding the enzymes involved in limonene degradation. The CDH nucleotide sequence revealed an open reading frame of 831 base pairs encoding a 277-amino acid protein with a deduced mass of 29,531 Da. The CDH primary structure shares 10-30% sequence identity with members of the short chain dehydrogenase/reductase superfamily. Structure homology modeling with trihydroxynaphthalene reductase from Magnaporthe grisea suggests that CDH from R. erythropolis DCL14 is an alpha/beta one-domain protein with an extra loop insertion involved in NAD binding and a flexible C-terminal part involved in monoterpene binding.

Rhodococcus erythropolis DCL14 contains a novel degradation pathway for limonene.

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (-)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1, 2-monooxygenase activity, a cofactor-independent limonene-1, 2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S, 4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R, 4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the beta-oxidation pathway.

The Rhodococcus erythropolis DCL14 limonene-1,2-epoxide hydrolase gene encodes an enzyme belonging to a novel class of epoxide hydrolases

Recently, we reported the purification of the novel enzyme limonene-1,2-epoxide hydrolase involved in limonene degradation by Rhodococcus erythropolis DCL14. The N-terminal amino acid sequence of the purified enzyme was used to design two degenerate primers at the beginning and the end of the 50 amino acids long stretch. Subsequently, the complete limonene-1,2-epoxide hydrolase gene ( limA) was isolated from a genomic library of R. erythropolis DCL14 using a combination of PCR and colony hybridization. The limA gene encoded a 149-residue polypeptide with a deduced molecular mass of 16.5 kDa. It was functionally expressed in Escherichia coli. The amino acid sequence of limA contains neither any of the conserved regions of the α,β-hydrolase fold enzymes, to which most of the previously reported epoxide hydrolases belong, nor any of the conserved motifs present in leukotriene A 4 hydrolase. The structural data presented in this paper confirm previous physical and biochemical findings [van der Werf et al. (1998) J. Bacteriol. 180, 5052–5057] that limonene-1,2-epoxide hydrolase is the first member of a new class of epoxide hydrolases.