Hydroperoxide Lyase Research Articles

Increased straight-chain esters content after ultra low oxygen storage and its relation to the lipoxygenase system in ‘Golden Reinders®’ apples

‘Golden’ is the most cultivated apple group in Europe. In recent years, new mutants have emerged which improve both aspects of production and sensory attributes. In this work, emission of volatile compounds and the activity of lipoxygenase system–related enzymes in ‘Golden Reinders®’ apples were analysed after 19 and 30 weeks under ultra low oxygen atmosphere (ULO) or under ULO plus different periods in cold air atmosphere and after remaining 1 and 7 days at 20 °C. This study examined modifications in the capacity for volatile ester production, specifically focusing on modification in lipoxygenase and hydroperoxide lyase activity. Volatile compound emission reached a maximum after 30 weeks of cold storage plus 7 days at 20 °C. Straight-chain esters were closely related to lipoxygenase activity in the flesh tissue, leading, suggesting that lipoxygenase enzyme plays an important role in the emission of straight-chain esters: the most characteristic volatile type associated with this apple variety.

Purification and characterization of hydroperoxide lyase from amaranth tricolor (Amaranthus mangostanus L.) leaves

Hydroperoxide lyase (HPL) was extracted from amaranth tricolor leaves using Triton X-100, and purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography. The purified HPL preparation consisted of a single band and spot with a molecular mass of about 55 kDa as shown in SDS–PAGE and 2-D PAGE, respectively; the isoelectric point was found to be about 5.4. The maximum activity of the enzyme was observed at pH 6.0 and 25 °C, respectively. The HPL showed higher activity against 13-hydroperoxy-linolenic acid compared to 13-hydroperoxy-linoleic acid. Km value for 13-hydroperoxy-linolenic acid was 62.7 μM, and the corresponding Vmax was 178.5 μM min−1. The activity of HPL was significantly inhibited by nordihydroguaiaretic acid, HgCl2 and 2(E)-hexenal but not by EDTA and hexanal.

The reaction mechanism of allene oxide synthase: Interplay of theoretical QM/MM calculations and experimental investigations

A combined theoretical and experimental study highlights the reaction mechanism of allene oxide synthase (AOS) and its possible link to hydroperoxide lyase (HPL) pathway. A previously published study (Lee et al., Nature 455 (2008) 363) has shown that the F137 residue is of central importance in differentiating between the AOS and HPL pathways after initial identical steps. In the experimental part of this study, we show that wild-type AOS from Arabidopsis or rice in fact produces both AOS and HPL products in a ratio of about 80:15, something that was found only in trace amounts before. Theoretical calculations successfully map the whole AOS pathway with 13( S)-hydroperoxy linolenic and linoleic acid as substrates. Subsequent calculations investigated the effects of in silico F137L mutation at the suggested diverging point of the two pathways. The results show that QM/MM calculations can reasonably reproduce three out of four experimentally available cases, and confirm that the pathways are energetically very close to each other, thus making a switch from one path to other plausible under different circumstances.

Lipoxygenase and hydroperoxide lyase activities in two olive varieties from Northern Italy

AbstractLipoxygenase (LOX) and hydroperoxide lyase (HPL) activities were characterised in olive pulp microsomes from two cultivars (Ghiacciolo and Nostrana di Brisighella) from Northern Italy. LOX activity exhibited a maximum at pH 5.5 in both cultivars, and the highest value was found in cv Ghiacciolo. This was correlated with the higher total antioxidant capacity detected in cv Nostrana, rather than by a different LOX expression, as demonstrated by immunoblotting assay. Furthermore, isomer characterisation showed the predominance of 13‐hydroperoxides of linoleic acid (HPOD) in both cultivars, suggesting the occurrence of a 13‐LOX. HPL activity exhibited an optimum at pH 7.5 in the two cultivars, but was higher in cv Nostrana, as confirmed by the increased amount of hexanal production after 13‐HPOD addition. In addition, the constitutive aldehyde profile in cv Nostrana fruits were shown to be enriched in aldehydes too when compared to cv Ghiacciolo. These results suggest that the different features in the two olive cultivars are mainly due to genetic rather than environmental factors.Practical applications: In this paper we studied the lipoxygenase (LOX) and hydroperoxide lyase (HPL) activities, and the formation of their products (hydroperoxides and aldehydes) in microsomes extracted from olive pulp of two Italian cultivars. The study of LOX and HPL, belonging to the so‐called ‘oxylipin pathway’, has important implications on the aroma development in both fruit and oil. Thus, a better knowledge of the properties of these enzymes can be useful to improve the organoleptic features of the oil. In fact, ‘LOX pathway’ may play a positive role in the formation of pleasant flavours during the technological processes. The preliminary study of the activity of the LOX/HPL enzymes could represent an important tool to facilitate the varietal choice in order to improve oil quality.

Up-regulated expression of lipoxygenase and divinyl ether synthase genes in pepper leaves inoculated with Tobamoviruses

To evaluate the importance of the oxylipin pathway in plant defense against viruses we studied the induction of this pathway in a compatible and an incompatible virus–pepper interaction. Obuda pepper virus (ObPV) inoculation led to the appearance of hypersensitive necrotic lesions (incompatible interaction), to a marked induction of lipoxygenase (LOX) enzyme activity, and to elevated transcription of three LOX genes in leaves of the pepper cultivar TL 1791 harbouring the L 3 resistance gene. In the same pepper cultivar Pepper mild mottle virus (PMMoV) inoculation caused very mild chlorotic symptoms (compatible interaction), and exerted only negligible effects on LOX activity and gene expression. The transcription of an allene oxide synthase ( CaAOS) and a hydroperoxide lyase ( CaHPL) gene was not modified significantly by any virus inoculation. The expression of a novel divinyl ether synthase ( CaDES) gene was massively induced in ObPV-inoculated leaves, but only very slightly by PMMoV inoculation. Our results showed that LOX and DES genes may be specifically involved in resistance responses against virus infections.

Expression of Genes Associated with Aroma Formation Derived from the Fatty Acid Pathway during Peach Fruit Ripening

Changes in characteristic aroma volatiles, levels of fatty acids as aroma precursors, and expression patterns of related genes, including lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), alcohol acyltransferase (AAT), and fatty acid desaturase (FAD), were studied in peach ( Prunus persica L. Batsch., cv. Yulu) fruit during postharvest ripening at 20 degrees C. Concentrations of n-hexanal, (E)-2-hexenal, (E)-2-hexenol, and (Z)-3-hexenol decreased, whereas the production of (Z)-3-hexenyl acetate, gamma-hexalactone, gamma-octalactone, gamma-decalactone, and delta-decalactone increased with fruit ripening. Lactones showed a clear pattern concomitant with the climacteric rise in ethylene production, with gamma-decalactone being the principal volatile compound at the late ripening stage. Of the LOX family genes, PpLOX2 and PpLOX3 had relatively high transcript levels initially followed by a decline with fruit ripening, while levels of PpLOX1 and PpLOX4 transcripts were upregulated by accumulated ethylene production. Expression of PpHPL1, PpADH1, PpADH2, and PpADH3 showed similar decreasing patterns during ripening. Expression levels of PpAAT1 showed a rapid increase during the first 2 days of postharvest ripening followed by a gradual decrease. Contents of polyunsaturated linoleic and linolenic acids increased, and saturated palmitic acid levels tended to decline as the fruit ripened. The increased levels of unsaturated fatty acids closely paralleled increasing expression of PpFAD1 and PpFAD2. The significance of gene expression changes in relation to aroma volatile production is discussed.

Volatile ester-synthesising capacity in ‘Tardibelle’ peach fruit in response to controlled atmosphere and 1-MCP treatment

Peach fruit is highly perishable, which drastically restricts storage potential and marketing possibilities. Although aroma is a very important attribute for sensory quality of peach, post-harvest procedures, aimed at extending commercial availability of fruit, have focused preferentially on other quality aspects. In this work, we were interested in assessing the effect of 1-methylcyclopropene (1-MCP) treatment and controlled atmosphere storage on the post-storage production of volatile esters, important aroma-contributing compounds, by fruit of the late season cultivar ‘Tardibelle’. Results indicate that the supply of alcohol and acyl-CoA precursors was altered as a consequence of treatments considered, leading to significant changes in the emission of some volatile esters, particularly of the straight-chain type. Some enzyme activities involved in the production of volatile esters from fatty acids were partially inhibited in 1-MCP-treated fruit, suggesting that they are under ethylene regulation, although tissue-specific differences were also observed. Lipoxygenase and hydroperoxide lyase activities were particularly relevant for these modifications.

Differential colonization and defence responses of resistant and susceptible melon lines infected by Fusarium oxysporum race 1·2

A green fluorescent protein (GFP)‐expressing strain of Fusarium oxysporum f.sp. melonis race 1·2 (FOM 1·2‐GFP) was used to visualize infection of a susceptible melon cultivar, Ein Dor (ED). At 1–2 days post‐inoculation (d.p.i.), the fungus grew on the root epidermis and adhered to epidermal cell borders. By day 4, the mycelium crossed the cortex and endodermis through narrow pores in cell walls and reached xylem vessels, where it sporulated and produced secondary hyphae that grew upwards. Colonization dynamics of ED seedlings were compared with those of a genetically resistant line, BIZ. FOM 1·2‐GFP colonized the resistant plant’s vascular system, but the incidence of seedling infection was lower than in ED, suggesting stronger defence responses in BIZ expressed at the pre‐xylem stage of infection. Infection of the vascular system of BIZ was slower: at 11 d.p.i., FOM 1·2‐GFP only colonized the lower hypocotyl sections of BIZ, whilst the upper hypocotyls of ED were already infected, indicating that BIZ also restricted FOM 1·2 movement in the xylem. The expression patterns of three defence genes were compared between the resistant and susceptible genotypes using real‐time PCR. Transcript levels of phenylalanine ammonia lyase (PAL), chitinase (CHI) and hydroperoxide lyase (HPL) were induced to a greater extent in BIZ than in ED. A constitutive two‐ to fourfold difference between BIZ and ED in the basal levels of all three transcripts was also apparent. Both the constitutive and inducible defence responses could contribute to reduced vascular colonization of the resistant genotype.

Plant volatiles

Plant volatiles are the metabolites that plants release into the air. The quantities released are not trivial. Almost one-fifth of the atmospheric CO2 fixed by land plants is released back into the air each day as volatiles. Plants are champion synthetic chemists; they take advantage of their anabolic prowess to produce volatiles, which they use to protect themselves against biotic and abiotic stresses and to provide information - and potentially disinformation - to mutualists and competitors alike. As transferors of information, volatiles have provided plants with solutions to the challenges associated with being rooted in the ground and immobile.

Volatile compounds and changes in flavour-related enzymes during cold storage of high-intensity pulsed electric field- and heat-processed tomato juices

The effects of high-intensity pulsed electric field (HIPEF) processing (35 kV cm(-1) for 1500 micros, using 4 micros bipolar pulses at 100 Hz) on the production of volatile compounds and flavour-related enzymes in tomato juice were investigated and compared with those of thermal processing (90 degrees C for 30 or 60 s). Tomato juice treated by HIPEF showed lower residual lipoxygenase (LOX) activity (70.2%) than juice heated at 90 degrees C for 60 s (80.1%) or 30 s (93.2%). In contrast, hydroperoxide lyase (HPL) was almost completely inactivated when the juice was subjected to 90 degrees C for 60 s, whereas roughly 50% of the control tomato juice was depleted after HIPEF treatment or thermal processing at 90 degrees C for 30 s. A slight decrease was observed in the initial LOX activity of treated and untreated samples during storage, whereas initial HPL activity was strongly affected over time. HIPEF-treated juice exhibited higher levels of compounds contributing to tomato aroma than untreated and heat-treated juices throughout storage. Thus HIPEF processing can preserve flavour quality and stability of tomato juice compared with conventional thermal treatments.

Overexpression of hydroperoxide lyase gene in Nicotiana benthamiana using a viral vector system

13-Lipoxygenase (13-LOX) and 13-hydroperoxide lyases (13-HPL) are the key enzymes for the production of the 'green note' compounds hexanal, (3Z)- and (2E)-hexenal in plant tissues. To produce high levels of 13-LOX and 13-HPL enzymatic activities for a biocatalytic process to generate C(6)-aldehydes on a large scale, soya bean 13-LOX (GmVLXC) and watermelon 13-HPL (ClHPL) genes were expressed in Nicotiana benthamiana using a viral vector system mediated by agroinfiltration. The N. benthamiana leaves produced high activity of watermelon HPL, but not GmVLXC 13-LOX. In addition, all leaves treated with bacterial suspension displayed a high activity of 9-LOX, indicating that the internal tobacco 9-LOX gene was highly induced through agroinfiltration because of wounding. GmVLXC and ClHPL transcripts could be detected in the corresponding transformed tobacco leaves by real-time RT-PCR analysis but the expression level of ClHPL was 24-fold higher than that of GmVLXC. Western blot analysis showed that LOX was present in all tobacco leaves which were treated with bacterial suspensions, but not in the untreated wild-type control. This result confirms that internal 9-LOX was highly induced by agroinfiltration. The highest levels of ClHPL activity under optimal infiltration conditions were 80 times the HPL activity of wild-type plants or plants transformed with control vector. A large amount of hexanal was formed when linoleic acid was incubated with extracts from N. benthamiana leaves over-expressing ClHPL in combination with GmVLXC-expressing yeast extracts. One gram of ClHPL-expressing N. benthamiana leaves (fresh weight) could produce 17 +/- 0.4 mg hexanal from 50 mg linoleic acid after 30 min.

Isolation, Expression, and Characterization of a 13-Hydroperoxide Lyase Gene from Olive Fruit Related to the Biosynthesis of the Main Virgin Olive Oil Aroma Compounds

A full-length cDNA clone (OepHPL) coding for hydroperoxide lyase was isolated from olive fruit ( Olea europaea cv. Picual). The deduced amino acid sequence shows significant similarity to known plant hydroperoxide lyases and contains a N-terminal sequence that displays structural features of a chloroplast transit peptide. Genomic Southern blot analysis indicates that at least one copy of OepHPL is present in the olive genome. The recombinant hydroperoxide lyase was specific for 13-hydroperoxide derivatives of linolenic and linoleic acids but did not use 9-hydroperoxy isomers as substrates. Analyses of reaction products revealed that this enzyme produces primarily (Z)-hex-3-enal, which partially isomerizes to (E)-hex-2-enal, from 13-hydroperoxylinolenic acid and hexanal from 13-hydroperoxylinoleic acid. Expression levels were measured in different tissues of Picual and Arbequina varieties, including mesocarp and seed during development and ripening of olive fruits. The involvement of this olive hydroperoxide lyase gene in the biosynthesis of virgin olive oil aroma compounds is discussed.

Stability of hydroperoxide lyase activity from Amaranthus tricolor (Amaranthus mangostanus L.) leaves: influence of selected additives

Hydroperoxide lyase (HPL) has potential value for the flavour additive industry. Currently, the production and application of HPL suffer from stability problems. The objective of this study was to investigate the stabilisation of HPL preparation from Amaranthus tricolor leaves by the addition of selected chemical additives. Amaranthus tricolor leaves were identified as a particularly rich source of 13-HPL activity. The addition of 100 g L(-1) sucrose and trehalose to microsomal HPL prior to lyophilisation could retain nearly 100% enzymatic activity, compared to only 20% for the lyophilised control. The lyophilised microsomal HPL containing sucrose maintained full activity for even 40 days storage at -20 degrees C. For HPL solution, glycerol was effective for long-term stability at -20 degrees C. Moreover, poyols (sucrose and trehalose) and amino acid (glycine) enhanced the thermostability of HPL, while KCl and polyol mannitol decreased the thermostability of HPL. The flavour-producing enzyme HPL, found in the leaves of Amaranthus tricolor, was stabilised by the addition of chemical additives.

Predicted secondary structure of hydroperoxide lyase from green bell pepper cloned in the yeast Yarrowia lipolytica

Fatty acid hydroperoxide lyase (HPL) is a member of the cytochrome P450 family acting on fatty acid hydroperoxides in many organisms. The active green bell pepper HPL, cloned and expressed in the yeast Yarrowia lipolytica, was purified by immobilized metal-ion affinity chromatography (IMAC) in the presence of 2% of Triton X-100R. The secondary structure prediction by bioinformatics servers of HPL was realized by ANTHEPROT software, using the GOR, DPM and Predator methods. The theoretical results which are average values obtained from three different calculation methods showed 33% α-helix, 18% β-sheet, 7% turn and 42% coil. On the other hand, the secondary structure approach of the purified active HPL (specific activity of 2.94 U/mg protein) was realized by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy, and showed 13% α-helix, 29% β-sheet, 5% turn and 53% random coil.

CLONING AND SEQUENCING OF THE HYDROPEROXIDE LYASE (HPL) GENE AND GENETIC TRANSFORMATION IN GUAVA

The guava tree (Psidium guajava L.) is a crop species of great economic interest in tropical and subtropical regions of the world. The fruit is characterized by a high content of vitamin C, fiber and β-carotenes, all of which are desirable traits for genetic improvement programs. On the other hand, the progress in the knowledge of the mechanisms and functions of genes in plants, using molecular biology techniques, contribute to increase our understanding of the role played by them and their products in plant development. The enzyme hydroperoxide lyase (HPL) is widely distributed in plants and is involved in the biosynthesis of volatile aldehydes and alcohols that are important constituents in the characteristic flavour of fruits, vegetables, and leaves. The products of the HPL reaction have commercial relevance due to their use by the food industry, but they also play an important role in plant defense mechanisms against pathogens and wounds by injuries. The guava enzyme shows a high similarity to known HPL from other plant species. Prior work with the guava HPL confirmed that the enzyme shares homology with cytochrome P450, placing it in the cytochrome P45074B subfamily as CYP74B5. However, the catalytic mechanism of HPL is still poorly understood. For further characterization, the guava HPL gene was cloned and sequenced. The gene proved to be polymorphic, as observed in a subset from a 100 specimen collection of guavas sampled in different regions of the country. One guava HPL gene was sub-cloned into a plant transformation vector, and a transformation protocol was performed in order to analyze the effects of HPL gene silencing in guava transformants.

FLOUR BLENDING BASED ON CRUDE FAT IS MORE CRUCIAL THAN ASH CONTENT IN VIEW OF PROLONGED QUALITY ASSURRANCE

ABSTRACT Conventionally, the ash content has been a quality control index for flour grading and blending. However, commercially, such a criteria is often found unreliable. In the present study, no significant correlation between the ash- and crude fat contents was found. Conversely, significant correlation was identified between the lipoxygenase activity and the crude fat. Furthermore, from the confirmatory experiment using the standardized synthetic formula, significant relationships were found among the crude fat content, the lipid peroxidative enzymes including the lipoxygenase, hydroperoxide lyase and peroxidase, which apparently could account for the progress of flour off-odor. In contrast, only poor correlation was found with the ash content. In addition, the sensory evaluation in parallel also supported such a result. Conclusively, the crude fat content could be a more reliable parameter than ash content to serve as a novel indicator for flour grading and blending. PRACTICAL APPLICATIONS Identification of effective selection criteria for flour grading and blending is highly desirable by the industry. The new criteria developed in the present study, which signifies the role of crude fat distribution instead of ash content in blending, is expected to provide the industry with a rational tool to ensure the quality of flour and subsequent products.

Role of hydroperoxide lyase in white-backed planthopper (Sogatella furciferaHorváth)-induced resistance to bacterial blight in rice,Oryza sativaL.

A pre-infestation of the white-backed planthopper (WBPH), Sogatella furcifera Horváth, conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) in rice (Oryza sativa L.) under both laboratory and field conditions. The infestation of another planthopper species, the brown planthopper (BPH) Nilaparvata lugens Stål, did not significantly reduce the incidence of bacterial blight symptoms. A large-scale screening using a rice DNA microarray and quantitative RT-PCR revealed that WBPH infestation caused the upregulation of more defence-related genes than did BPH infestation. Hydroperoxide lyase 2 (OsHPL2), an enzyme for producing C(6) volatiles, was upregulated by WBPH infestation, but not by BPH infestation. One C(6) volatile, (E)-2-hexenal, accumulated in rice after WBPH infestation, but not after BPH infestation. A direct application of (E)-2-hexenal to a liquid culture of Xoo inhibited the growth of the bacterium. Furthermore, a vapour treatment of rice plants with (E)-2-hexenal induced resistance to bacterial blight. OsHPL2-overexpressing transgenic rice plants exhibited increased resistance to bacterial blight. Based on these data, we conclude that OsHPL2 and its derived (E)-2-hexenal play some role in WBPH-induced resistance in rice.

Effect of Temperature on Lipid-Related Volatile Production in Tomato Puree

Lipid-related volatiles were measured in real time after the blending of grape tomatoes, using selected ion flow tube mass spectrometry (SIFT-MS). Measurements were made at 4, 23, or 37 degrees C. The volatiles in the headspace of the tomatoes, other than hexanal, increased with increasing temperature. The concentration of hexanal in the headspace increased from 4 to 23 degrees C, but decreased at 37 degrees C. The activity of hexanal-specific hydroperoxide lyase decreases at 37 degrees C. Moreover, precursors of hexanal may go through alternative pathways to form trans-2-heptenal and trans-2-octenal. The increase in concentration in the headspace for most volatiles can be explained by the increase in volatility, except for trans-2-heptenal, trans-2-octenal, and trans-2-pentenal. These three volatiles appear to be generated at a much higher rate at 37 degrees C due to the dominance of alternate pathways at this temperature. Temperature did not affect the time to peak level for most volatiles, except the time for hexanal was shorter with increasing temperature. A temperature-dependent lipoxygenase pathway was postulated.

Effects of High‐Intensity Pulsed Electric Fields on Lipoxygenase and Hydroperoxide Lyase Activities in Tomato Juice

The influence of high-intensity pulsed electric field (HIPEF) parameters, namely, pulse frequency, pulse width, and polarity on tomato juice lipoxygenase (LOX) and hydroperoxide lyase (HPL) activities was studied using a response surface methodology. Samples were subjected to square-shaped pulses of 35 kV/cm for 1000 micros, with pulse width ranging from 1 to 7 micros at frequencies from 50 to 250 Hz, either in monopolar or bipolar mode. Tomato LOX was more resistant to HIPEF than HPL within the range of assayed conditions. An increase in frequency or pulse width resulted in a decrease of both residual LOX (RA(LOX)) and HPL (RA(HPL)) activities. The lowest RA(LOX) (81%) was observed when tomato juice was treated at 250 Hz for 7 mus in bipolar mode. Moreover, the same conditions led to the highest HPL activity reduction (RA(HPL) = 10%). A validation of the predictive models determined that 2nd-order expressions were accurate enough to fit the experimental results.

Evidence for an Ionic Intermediate in the Transformation of Fatty Acid Hydroperoxide by a Catalase-related Allene Oxide Synthase from the Cyanobacterium Acaryochloris marina

Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4omega3 and 18.3omega3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted approximately 70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only approximately 30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H2(18)O indicated that approximately 72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.