C6 Volatiles Research Articles

Flavour quality critical production steps from fruit to extra-virgin olive oil at consumption

The production of olive oil comprises a number of production steps where the quality of the oil may be controlled through an understanding of how the production step influences key compounds such as volatiles and biophenols. In this study, critical production steps and significant inter-relationships between isolated production steps from fruit to oil-at-consumption were identified with the application of multivariate statistics. Having identified the key steps and relationships, sensory attributes associated with volatile and compounds may be enhanced and maintained during production and through to consumption. Our study showed that flavour compounds (e.g. oleuropein and derivates) could be controlled through olive fruit properties whereas transfer of the best sensory attributes from the fruit (e.g. arising from C5 and C6 volatiles) was critically controlled during oil extraction. Once olive oil was produced, maintenance of quality was critically controlled through storage conditions. Consequently, quality attributes from phenolic and volatile compounds could be targeted for maximum transfer from the olive fruit to oil while taking into account the impact on aroma, bitterness, and pungency of fresh oils and the subsequent loss in flavour quality during storage and consumption.

The abundance of pink-pigmented facultative methylotrophs in the root zone of plant species in invaded coastal sage scrub habitat.

Pink-pigmented facultative methylotrophic bacteria (PPFMs) are associated with the roots, leaves and seeds of most terrestrial plants and utilize volatile C1 compounds such as methanol generated by growing plants during cell division. PPFMs have been well studied in agricultural systems due to their importance in crop seed germination, yield, pathogen resistance and drought stress tolerance. In contrast, little is known about the PPFM abundance and diversity in natural ecosystems, let alone their interactions with non-crop species. Here we surveyed PPFM abundance in the root zone soil of 5 native and 5 invasive plant species along ten invasion gradients in Southern California coastal sage scrub habitat. PPFMs were present in every soil sample and ranged in abundance from 102 to 105 CFU/g dry soil. This abundance varied significantly among plant species. PPFM abundance was 50% higher in the root zones of annual or biennial species (many invasives) than perennial species (all natives). Further, PPFM abundance appears to be influenced by the plant community beyond the root zone; pure stands of either native or invasive species had 50% more PPFMs than mixed species stands. In sum, PPFM abundance in the root zone of coastal sage scrub plants is influenced by both the immediate and surrounding plant communities. The results also suggest that PPFMs are a good target for future work on plant-microorganism feedbacks in natural ecosystems.

Isolation and characterization of two hydroperoxide lyase genes from grape berries

C6 compounds are the major fraction of the volatile profiles of grape berries, contributing the typical 'green' aroma to the grape and wine. Hydroperoxide lyase (HPL) catalyzes the cleavage of fatty acid hydroperoxides to produce C6 compounds. Two hypothetical genes, VvHPL1 and VvHPL2 were cloned from grape berries (Vitis vinifera L. Cabernet Sauvignon). Bioinformatics analysis revealed that the proteins encoded by these two genes both belong to subfamily of cytochrome P450 and contain typical conserved domains of HPLs, and have high identity with HPLs from other plants. Prokaryotically-expressed VvHPL1 and VvHPL2 with thioredoxin-6xHis-fusion partner were confirmed to have enzymatic activity. VvHPL1 is specific for 13-HPOD (T) producing C6 aldehydes with relatively higher activity and VvHPL2 catalyzes the cleavage of both 9- and 13-hydroperoxides producing C6 aldehydes and C9 aldehydes respectively. Analysis of real time-PCR showed that VvHPL2 was highly expressed in the leaves and the flowers of the grapes, while relatively low transcript abundance was detected in the berries, tendril and stems; VvHPL1 had high expression in all detected tissues. During grape berry development, the expression of these two isogenes presented similar trends with a rapid increase after veraison and a decrease at full-ripen stage, which roughly corresponded to the accumulation of their volatile products. These data lay an essential foundation for further study on the accumulation and control of C6 volatiles in grape berries.

Effects of Sedimentation Plus Racking Process in the Extra Virgin Olive Oil Aroma Fingerprint Obtained by DHS–TD/GC–MS

Dynamic headspace–thermal desorption/gas chromatography–mass spectrometry was employed to characterize the volatile profile of extra virgin olive oils before and after the process of sedimentation plus racking. Several oils produced in the provinces of Ourense (San Cibrao das Vinas) and Lugo (Quiroga) (northwestern Spain) and in that of Cordoba (southwestern Spain) were selected for such a purpose. There are no data on the effect of racking on the headspace composition of EVOOs. The volatile components were identified, and the differences found were highlighted in terms of the process but also in terms of different varieties and origins. The results suggest that besides the genetic factor, environmental conditions and process treatments influence the volatile formation. After racking, there is an increase of fruity vs. green notes (C6 alcohols vs. C6 aldehydes) and an apple-like character vs. a banana-like nuance (C6 linoleic acid route vs. C6 linolenic acid route) but also a more bitter character (C5 vs. C6 volatiles), with whiskey, spicy and malty nuances (by the slight increase of minor alcohols and aldehydes).

The Activity of Healthy Olive Microbiota during Virgin Olive Oil Extraction Influences Oil Chemical Composition

The activity of olive microbiota during the oil extraction process could be a critical point for virgin olive oil quality. With the aim to evaluate the role of microbiological activity during the virgin olive oil extraction process, just before oil extraction freshly collected healthy olive fruits were immersed in contaminated water from an olive mill washing tank. The oils extracted were then compared with control samples from the same batch of hand-picked olives. The presence of lactic and enteric bacteria, fungi and Pseudomonas on the surface of olives was proved to be much higher in washed than in control olives, with increments in cfu/g between 2 and 3 orders of magnitude. The biogenesis of volatile compounds and the extraction of olive polyphenols and pigments were significantly influenced by the microbiological profile of olives even without any previous storage. In most cases the effect of olive microbiota on oil characteristics was greater than the effect exerted by malaxation time and temperature. Oils from microbiologically contaminated olives showed lower amounts of C5 volatiles and higher levels of C6 volatiles from the lipoxygenase pathway and some fermentation products. On the other hand, a decrease of chlorophylls, pheophytins, xanthophylls and the ratio chlorophyll/pheophytin was observed in these oils. Likewise, the microbiological activity during oil extraction led to significantly lower amounts of polyphenols, in particular of oleuropein derivatives. These differences in olive oil chemical composition were reflected in oil sensory characteristics by the decrease of the green and bitter attributes and by the modification of the oil color chromatic ordinates.

Chilling and heating may regulate C6 volatile aroma production by different mechanisms in tomato (Solanum lycopersicum) fruit

Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicum L.) volatile aroma compounds derived from oxygenation of unsaturated fatty acids. Chilling and heating may suppress production of these C6 volatiles. The objective of this research was to determine the effects of chilling or heating on gene expression and enzyme activity of lipoxygenase (LOX), hydroperoxide lyase (HPL), and alcohol dehydrogenase (ADH), which catalyze key steps in C6 volatile production via the oxylipin pathway. ‘Tasti-Lee’ and ‘Sanibel’ tomatoes, harvested at different stages of fruit development, were ripened to full (red) ripe stage at 20°C. Fruit were then treated by either chilling (5°C for 5d) or heating (52°C hot water for 15min), then cooled with 23°C tap water to 25°C and placed at 20°C for 4d, or held continuously at 20°C as the control. Both chilling and heating reduced C6 aldehyde and alcohol aroma volatiles immediately after treatment, and the levels of aldehydes did not fully recover after 4d at 20°C. Chilling down-regulated expression of TomloxA, B, and C, but not D; however, it increased total LOX activity. Chilling also down-regulated HPL and ADH expression immediately after treatment, but, after 4d at 20°C, both genes were up-regulated compared to the control. HPL activity in chilled tomatoes was reduced, but recovered to control levels after 4d at 20°C. ADH activity in chilled fruit decreased after 4d at 20°C. On the other hand, heating greatly up-regulated TomloxB and C expression, even after 4d at 20°C, and slightly down-regulated TomloxA and D, while increasing overall LOX activity. Heating up-regulated both HPL and ADH, and that effect persisted for 4d at 20°C. However, heating reduced the activities of HPL for 4d at 20°C and ADH immediately after treatment. The results indicate that heating and chilling regulate C6 volatile production by different mechanisms. Chilling-induced inhibition of C6 volatile production may be due to down-regulation of gene expression, and subsequent reduction of HPL and ADH enzyme activities in the oxylipin pathway. Heating-inhibition of C6 volatile production, however, does not appear to be due to down-regulation of gene expression, but HPL and ADH activities were briefly suppressed.

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.

Survey of volatile oxylipins and their biosynthetic precursors in bryophytes

Oxylipins are metabolites which are derived from the oxidative fragmentation of polyunsaturated fatty acids. These metabolites play central roles in plant hormonal regulation and defense. Here we survey the production of volatile oxylipins in bryophytes and report the production of a high structural variety of C5, C6, C8 and C9 volatiles of mosses. In liverworts and hornworts oxylipin production was not as pronounced as in the 23 screened mosses. A biosynthetic investigation revealed that both, C18 and C20 fatty acids serve as precursors for the volatile oxylipins that are mainly produced after mechanical wounding of the green tissue of mosses.

Aroma potential of three autochthonous grapevine varieties from Tunisia

<p style="text-align: justify;"><strong>Aims</strong>: The aim of the present work is to characterize the aromatic profiles of three Tunisian autochthonous grapevine (Vitis vinifera L.) varieties (Asli, Beldi and Ferrani).</p><p style="text-align: justify;"><strong>Methods and results</strong>: The GC-MS analysis of the autochthonous grapevine varieties studied allowed identifying 38 volatile compounds in the free fraction and 36 glycosidically bound aroma compounds. Volatile C6 compounds were typical for the studied varieties especially for Asli variety. The analysis of the liberated aglycones through enzymatic hydrolysis show that a-terpineol and cis linalool oxide pyran that did not appear as free aroma compounds were found with significantly highest concentration in Asli grape juice. Also, 3-oxo-a-ionol and 3-hydroxy-7,8-dihydro-b-ionol were found only under their glycosidically bound forms with significantly higher concentrations in Asli and Ferrani than in Beldi grape juices.</p><p style="text-align: justify;"><strong>Conclusion</strong>: The results obtained in the present work show qualitative and quantitative differences in the aroma profiles among the studied varieties. However, non-terpenyl compounds were the most abundant aroma substances in the three studied varieties.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: The present work characterize, for the first time, the aromatic profiles of three Tunisian autochthonous grapevine varieties (Asli, Beldi and Ferrani) and constitutes a major contribution to the aroma chemistry of these three grape varieties.</p>

Wounding and pathogen infection induce a chloroplast-targeted lipoxygenase in the common bean (Phaseolus vulgaris L.)

Chloroplastic LOXs are implicated in the biosynthesis of oxylipins like jasmonic acid and C6 volatiles among others. In this study, we isolated the cDNA of a novel chloroplast-targeted Phaseolus vulgaris LOX, (PvLOX6). This gene is highly induced after wounding, non-host pathogen infection, and by signaling molecules as H2O2, SA, ethylene and MeJA. The phylogenetic analysis of PvLOX6 showed that it is closely related to chloroplast-targeted LOX from potato (H1) and tomato (TomLOXC); both of them are implicated in the biosynthesis of C6 volatiles. Induction of PvLOX6 mRNA by wounding ethylene and jasmonic acid on the one side, and non-host pathogen, salicylic acid on the other indicates that common bean uses the same LOX to synthesize oxylipins in response to different stresses.

Interaction with and Effects on the Profile of Proteins of Botrytis cinerea by C6 Aldehydes

The natural volatile compounds cis-3-hexenal (c-3-H) and trans-2-hexenal (t-2-H) have significant antifungal activity with potential for use as postharvest fumigants of fruits and vegetables. However, the nature of their interaction with fungi and impact on fungal growth at the molecular level are largely unknown. The sites of interaction of these six carbon (C6) aldehydes with Botrytis cinerea, a common pathogen of many plant species, was characterized using 3H-labeled c-3-H and t-2-H. Radiolabeled C6 aldehydes were produced with lipoxygenase and hydroperoxide lyase extracts using [9,10,12,13,15,16-3H6]linolenic acid as a substrate. Following exposure of B. cinerea cultures to radiolabeled C6 aldehydes, radiolabel was recovered in protein-enriched but not lipid-enriched fractions. Radiolabel was incorporated at higher levels (6-fold per milligram of fresh weight and 4-fold per microgram of protein) into conidia than mycelia. About 95% of the radiolabeled aldehyde recovered in the protein fraction was from the surface of the fungal tissue, while 5% was from protein in internal tissue (cell wall, membrane, and cytosol). Exposure to t-2-H at both 5.4 and 85.6 micromol affected the protein profile of B. cinerea, changing the intensity of over one-third of all proteins. Both up-regulation and down-regulation of specific proteins were observed by two-dimensional gel electrophoresis, indicating a clear effect of t-2-H on changes in the protein profile of B. cinerea. This is the first evidence that fungal proteins are targets of the volatile C6 aldehydes and that sublethal levels of the aldehydes cause changes in the protein profile of a fungus.

Effect of the morphology of polyethylene glycol esters on the distribution of C1–C4 carboxylic acids in the gas phase-sorbent system

The effect of the morphology of polymer films on the distribution of volatile C1–C4 carboxylic acids in the sorbent-gas system is discussed. The efficiency of sorption of vapors of formic, acetic, propionic, and butyric acids on films of polyethylene glycol esters of various structures formed on the electrodes of a piezoquartz resonator by means of static evaporation of a drop and immersion of the resonator into a sorbent solution is evaluated.

Lipoxygenase Involvement in Ripening Strawberry

The enzymatic activity, subcellular localization, and immunolocalization of plant lipoxygenase (LOX) in strawberry fruits (Fragaria x ananassa, Duch) were investigated. Chemical and enzymatic properties of LOX have been characterized, and the LOX capability of oxygenating free and esterified unsaturated fatty acids into C6 volatile aldehydes has been confirmed. Fruits at unripe, turning, and ripe stages were analyzed for LOX activity and protein localization by Western blots, two-dimensional electrophoresis, and immunolocalization analyses. The ability of strawberry tissues to in vivo metabolize linolenic acid or linoleic acid into C6 volatile aldehydes and the LOX products was also analyzed. Analysis of strawberry proteins showed that a number of LOX forms, corresponding to at least two mobility groups of approximately 100 and 98 kDa and pI values ranging between 4.4 and 6.5, were present. Confocal and electron microscopy analyses support the idea that LOX proteins are associated to lipid-protein aggregates. Both exogenously supplied linoleate and linolenate were converted into hexanal and trans-2-hexenal at the three fruit-ripening stages. Our experiments suggest the presence of different LOX isoforms in strawberry fruits and that the lipoxygenase-hydroperoxide lyase pathway plays a role in converting lipids to C6 volatiles during ripening.

Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affect hexenal signaling in the Columbia and Landsberg erecta ecotypes of Arabidopsis.

The CYP74B2 gene in Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col) contains a 10-nucleotide deletion in its first exon that causes it to code for a truncated protein not containing the P450 signature typical of other CYP74B subfamily members. Compared to CYP74B2 transcripts in the Landsberg erecta (Ler) ecotype that code for full-length hydroperoxide lyase (HPL) protein, CYP74B2 transcripts in the Col ecotype accumulate at substantially reduced levels. Consistent with the nonfunctional HPL open reading frame in the Col ecotype, in vitro HPL activity analyses using either linoleic acid hydroperoxide or linolenic acid hydroperoxide as substrates show undetectable HPL activity in the Col ecotype and C6 volatile analyses using leaf homogenates show substantially reduced amounts of hexanal and no detectable trans-2-hexenal generated in the Col ecotype. P450-specific microarrays and full-genome oligoarrays have been used to identify the range of other transcripts expressed at different levels in these two ecotypes potentially as a result of these variations in HPL activity. Among the transcripts expressed at significantly lower levels in Col leaves are those coding for enzymes involved in the synthesis of C6 volatiles (LOX2, LOX3), jasmonates (OPR3, AOC), and aliphatic glucosinolates (CYP83A1, CYP79F1, AOP3). Two of the three transcripts coding for aliphatic glucosinolates (CYP83A1, AOP3) are also expressed at significantly lower levels in Col flowers.

Reduced levels of volatile emissions in jasmonate‐deficientspr2tomato mutants favour oviposition by insect herbivores

Tomato plants release volatile organic compounds (VOCs) following insect or mechanical damage. In this study, the constitutive and wound-induced emission levels of VOCs in suppressor of prosystemin-mediated responses2 (spr2) mutant plants, compromised in linolenic acid (LA) and jasmonic acid (JA) synthesis, and in 35S::prosystemin (35S::prosys) plants, having upregulated direct defence responses, were compared. The spr2 mutants produced constitutively lower levels of VOCs, which were nonetheless increased in response to (a)biotic damage, although at lower levels than wild-type (Wt) and 35S::prosys plants. No significant differences in VOC emissions were detected between the latter two genotypes, thereby suggesting that systemin does not regulate indirect defence responses, whereas differences in fatty acid composition in spr2 plants led to the predominant emission of saturated C6 volatiles in response to wounding. The expression of 1-deoxy-D-xylulose 5-phosphate synthase (DXS2), a key gene involved in VOC synthesis in the chloroplast, was only upregulated in Manduca sexta L.-damaged Wt and 35S::prosys plants. However, its expression was restored in spr2 plants by exogenous LA or JA, suggesting that abated VOC emissions in spr2 plants are correlated with lowered DXS2 expression. Bioassays with two different insects showed that adult females significantly preferred spr2 plants, indicating that lowered levels of VOCs in tomato influence plant selection by insects during oviposition.

Ozone induced emissions of biogenic VOC from tobacco: relationships between ozone uptake and emission of LOX products

ABSTRACTVolatile organic compound (VOC) emissions from tobacco (Nicotiana tabacum L. var. Bel W3) plants exposed to ozone (O3) were investigated using proton‐transfer‐reaction mass‐spectrometry (PTR‐MS) and gas chromatography mass‐spectrometry (GC‐MS) to find a quantitative reference for plants’ responses to O3 stress. O3 exposures to illuminated plants induced post‐exposure VOC emission bursts. The lag time for the onset of volatile C6 emissions produced within the octadecanoid pathway was found to be inversely proportional to O3 uptake, or more precisely, to the O3 flux density into the plants. In cases of short O3 pulses of identical duration the total amount of these emitted C6 VOC was related to the O3 flux density into the plants, and not to ozone concentrations or dose–response relationships such as AOT 40 values. Approximately one C6 product was emitted per five O3 molecules taken up by the plant. A threshold flux density of O3 inducing emissions of C6 products was found to be (1.6 ± 0.7) × 10−8 mol m−2 s−1.

Identification of a specific isoform of tomato lipoxygenase (TomloxC) involved in the generation of fatty acid-derived flavor compounds.

There are at least five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE) present in tomato (Lycopersicon esculentum Mill.) fruit, but their role in generation of fruit flavor volatiles has been unclear. To assess the physiological role of TomloxC in the generation of volatile C6 aldehyde and alcohol flavor compounds, we produced transgenic tomato plants with greatly reduced TomloxC using sense and antisense constructs under control of the cauliflower mosaic virus 35S promoter. The expression level of the TomloxC mRNA in some transgenic plants was selectively reduced by gene silencing or antisense inhibition to between 1% and 5% of the wild-type controls, but the expression levels of mRNAs for the four other isoforms were unaffected. The specific depletion of TomloxC in transgenic tomatoes led to a marked reduction in the levels of known flavor volatiles, including hexanal, hexenal, and hexenol, to as little as 1.5% of those of wild-type controls following maceration of ripening fruit. Addition of linoleic or linolenic acid to fruit homogenates significantly increased the levels of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than with the wild-type control. Confocal imaging of tobacco (Nicotiana tabacum) leaf cells expressing a TomloxC-GFP fusion confirmed a chloroplast localization of the protein. Together, these results suggest that TomloxC is a chloroplast-targeted lipoxygenase isoform that can use both linoleic and linolenic acids as substrates to generate volatile C6 flavor compounds. The roles of the other lipoxygenase isoforms are discussed.

Epoxide hydrolase: a mRNA induced by the fungal pathogen Alternaria alternata on rough lemon (Citrus jambhiri Lush).

An expression profile of genes induced by non-pathogenic Alternaria alternata on rough lemon leaves was obtained by sequencing 500 subtractive PCR clones generated from mRNA of leaves inoculated with the fungus after subtraction with that of non-inoculated leaves. About 6% of the cDNA sequences had homology to known putative defense-related genes including epoxide hydrolase. A full-length cDNA (951 bp) from rough lemon that encoded epoxy hydrolase was isolated by random amplification of cDNA ends (RACEs), based on sequence information from subtractive PCR, and designated as RlemEH. The product of this gene expressed with an in vitro translation system with Escherichia coli also had activity of a soluble type of epoxide hydrolase. The transcript of rough lemon RlemEH was not detected in flowers, fruits, stems or leaves, but was induced after inoculation of leaves with conidia of Alternaria alternata, wounding, or treatment with C6 volatiles, including trans-2-hexenol and cis-3-hexenol, and methyl jasmonate. The response of the epoxide hydrolase gene correlated well with the activation of defense mechanisms induced in plant-fungus interactions.

Development of a biocatalytic process for the production of c6-aldehydes from vegetable oils by soybean lipoxygenase and recombinant hydroperoxide lyase.

Volatile C6- and C9-aldehydes and alcohols are widely used as food flavors to reconstitute the "fresh green" odor of fruits and vegetables lost during processing. To meet the high demand for natural flavors, an efficient, cheap, and versatile biocatalytic process was developed to produce C6-aldehydes on a large scale. Vegetable oils were converted by soybean lipoxygenase and recombinant hydroperoxide lyase into hexanal and (2E)- or (3Z)-hexenal. In contrast to plant extracts, generally used as enzyme sources, high molar conversions were obtained with recombinant hydroperoxide lyase (50% for hexanal and 26% for hexenal formation), and no side products were formed. Furthermore, recombinant hydroperoxide lyase lacks isomerase activity, allowing production of (3Z)-hexenal, which could not be obtained in previously described processes. Recombinant hydroperoxide lyase is stable and can be stored at 4 degrees C for 1 month without significant loss of activity.

Fatty acid hydroperoxide lyase: a plant cytochrome p450 enzyme involved in wound healing and pest resistance.

Plants continuously have to defend themselves against life-threatening events such as drought, mechanical damage, temperature stress, and potential pathogens. Nowadays, more and more similarities between the defense mechanism of plants and that of animals are being discovered. In both cases, the lipoxygenase pathway plays an important role. In plants, products of this pathway are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity. The first step in the lipoxygenase pathway is the reaction of linoleic or linolenic acids with molecular oxygen, catalyzed by the enzyme lipoxygenase. The hydroperoxy fatty acids thus formed are highly reactive and dangerous for the plant and therefore further metabolized by other enzymes such as allene oxide synthase, hydroperoxide lyase, peroxygenase, or divinyl ether synthase. Recently, these enzymes have been characterized as a special class of cytochrome P450 enzymes. Hydroperoxide lyases cleave the lipoxygenase products, resulting in the formation of omega-oxo acids and volatile C6- and C9-aldehydes and -alcohols. These compounds are major contributors to the characteristic "fresh green" odor of fruit and vegetables. They are widely used as food flavors, for example, to restore the freshness of food after sterilization processes. The low abundance of these compounds in nature and the high demand make it necessary to synthesize them on a large scale. Lipoxygenase and hydroperoxide lyase are suitable biocatalysts for the production of "natural" food flavors. In contrast to lipoxygenase, which has been extensively studied, little is yet known about hydroperoxide lyase. Hydroperoxide lyases from different organisms have been isolated, and a few genes have been published lately. However, the structure and reaction mechanism of this enzyme are still unclear. The identification of this enzyme as a cytochrome P450 sheds new light on its structure and possible reaction mechanism, whereas recombinant expression brings a biocatalytic application into sight.