Comparative Untargeted Metabolic Profiling of Different Parts of Citrus sinensis Fruits via Liquid Chromatography-Mass Spectrometry Coupled with Multivariate Data Analyses to Unravel Authenticity.

Differences between seven authentic samples of Citrus sinensis var. Valencia peel (albedo and flavedo) and juices from Spain and Uruguay, in addition to a concentrate obtained from Brazil, were investigated by untargeted metabolic profiling. Sixty-six metabolites were detected by nano-liquid chromatography coupled to a high-resolution electrospray-ionization quadrupole time-of-flight mass spectrometer (nLC-ESI-qTOF-MS) belonging to phenolic acids, coumarins, flavonoid glycosides, limonoids, terpenes, and fatty acids. Eleven metabolites were detected for the first time in Citrus sinensis and identified as citroside A, sinapic acid pentoside, apigenin-C-hexosyl-O-pentoside, chrysoeriol-C-hexoside, di-hexosyl-diosmetin, perilloside A, gingerol, ionone epoxide hydroxy-sphingenine, xanthomicrol, and coumaryl alcohol-O-hexoside. Some flavonoids were completely absent from the juice, while present most prominently in the Citrus peel, conveying more industrial and economic prospects to the latter. Multivariate data analyses clarified that the differences among orange parts overweighed the geographical source. PCA analysis of ESI-(-)-mode data revealed for hydroxylinoleic acid abundance in flavedo peel from Uruguay the most distant cluster from all others. The PCA analysis of ESI-(+)-mode data provided a clear segregation of the different Citrus sinensis parts primarily due to the large diversity of flavonoids and coumarins among the studied samples.

Impact of Agro-Industrial Side-Streams on Sesquiterpene Production by Submerged Cultured Cerrena unicolor.

The quality and harvest of essential oils depend on a large number of factors, most of which are hard to control in an open-field environment. Therefore, Basidiomycota have gained attention as a source for biotechnologically produced terpenoids. The basidiomycete Cerrena unicolor (Cun) was cultivated in submerged culture, and the production of sesquiterpenoids was analyzed via stir bar sorptive extraction (SBSE), followed by thermo-desorption gas chromatography coupled with mass spectrometry (TDS-GC-MS). Identification of aroma-active sesquiterpenoids was supported by GC, coupled with an olfactory detection port (ODP). Following the ideal of a circular bioeconomy, Cun was submerged (up-scalable) cultivated, and supplemented with a variety of food industrial side-streams. The effects of the different supplementations and of pure fatty acids were evaluated by liquid extraction and analysis of the terpenoids via GC-MS. As sesquiterpenoid production was enhanced by the most by lipid-rich side-streams, a cultivation with 13C-labeled acetate was conducted. Data confirmed that lipid-rich side-streams enhanced the sesquiterpene production through an increased acetyl-CoA pool.

Enzymatic hydrolysis of kaempferol 3-O-(2‴-O-sinapoyl-ß-sophoroside), the key bitter compound of rapeseed (Brassica napus L.) protein isolate.

BACKGROUND: The use of rapeseed protein for human nutrition is primarily limited by its strong bitterness, which is why the key bitter compound, kaempferol 3-O-(2‴-O-sinapoyl-ß-sophoroside), is enzymatically degraded. RESULTS: Mass spectrometry analyses of an extract from an untreated rapeseed protein isolate gave three signals for m/z 815 [M-H]. The predominant compound among the three compounds was confirmed as kaempferol-3-O-(2‴-O-sinapoyl-ß-sophoroside). Enzymatic hydrolysis of this key bitter compound was achieved using a sinapyl ester cleaving side activity of a ferulic acid esterase (FAE) from the basidiomycete Schizophyllum commune (ScoFAE). Recombinant ferulic acid esterases from Streptomyces werraensis (SwFAE) and from Pleurotus eryngii (PeFAE) possessed better cleavage activity towards methyl sinapate but did not hydrolyze the sinapyl ester linkage of the bitter kaempferol sophoroside. CONCLUSION: Kaempferol-3-O-(2‴-O-sinapoyl-ß-sophoroside) was successfully degraded by enzymatic treatment with ScoFAE, which may provide a means to move the status of rapeseed protein from feed additive to food ingredient. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Immunomodulating Effect of the Consumption of Watercress (Nasturtium officinale) on Exercise-Induced Inflammation in Humans.

The vegetable watercress (Nasturtium officinale R.Br.) is, besides being a generally nutritious food, a rich source of glucosinolates. Gluconasturtiin, the predominant glucosinolate in watercress, has been shown to have several health beneficial properties through its bioactive breakdown product phenethyl isothiocyanate. Little is known about the immunoregulatory effects of watercress. Moreover, anti-inflammatory effects have mostly been shown in in vitro or in animal models. Hence, we conducted a proof-of-concept study to investigate the effects of watercress on the human immune system. In a cross-over intervention study, 19 healthy subjects (26.5 ± 4.3 years; 14 males, 5 females) were given a single dose (85 g) of fresh self-grown watercress or a control meal. Two hours later, a 30 min high-intensity workout was conducted to promote exercise-induced inflammation. Blood samples were drawn before, 5 min after, and 3 h after the exercise unit. Inflammatory blood markers (IL-1ß, IL-6, IL-10, TNF-α, MCP-1, MMP-9) were analyzed in whole blood cultures after ex vivo immune cell stimulation via lipopolysaccharides. A mild pro-inflammatory reaction was observed after watercress consumption indicated by an increase in IL-1ß, IL-6, and TNF-α, whereas the immune response was more pronounced for both pro-inflammatory and anti-inflammatory markers (IL-1ß, IL-6, IL-10, TNF-α) after the exercise unit compared to the control meal. During the recovery phase, watercress consumption led to a stronger anti-inflammatory downregulation of the pro-inflammatory cytokines IL-6 and TNF-α. In conclusion, we propose that watercress causes a stronger pro-inflammatory response and anti-inflammatory counter-regulation during and after exercise. The clinical relevance of these changes should be verified in future studies.

Phenylacrylic acids addition to potato and sweet potato showed no impact on acrylamide concentration via oxa-Michael-addition during frying.

Three phenolic acids, p-coumaric, ferulic and caffeic acid as well as cinnamic acid were added to raw potatoes and sweet potatoes before frying. A distinct mitigation of acrylamide was not detected. Fried samples were analysed for postulated adducts of a direct reaction between acrylamide and these phenolic acids using LC-MS. In a model system with pure compounds (phenylacrylic acid and acrylamide) heated on 10% hydrated silica gel one specific adduct (respective m/z for M â€‹+ â€‹H+) was formed in each reaction. MS/MS-data suggested an oxa-Michael formation of 3-amino-3-oxopropyl-phenylacrylates, which was confirmed by de novo syntheses along an SN2 substitution of 3-chloropropanamide. Exemplarily, the structure of the ester was confirmed for p-coumaric acid by NMR-data. Standard addition revealed that 3-amino-(3-oxopropyl-phenyl)-acrylates occurred neither in fried potato nor in sweet potato, while a formation was shown in phenylacrylic acid plus acrylamide supplemented potatoes and sweet potatoes.

Formation of Decatrienones with a Pineapple-like Aroma from 1-13C-Acetate by Cell Cultures of the Birch Polypore, Fomitopsis betulina.

During submerged cultivation, the edible basidiomycete Fomitopsis betulina (previously Piptoporus betulinus) developed a fruity odor, strongly reminding of pineapple. Olfactometric analysis showed that this impression was mainly caused by the two (5E/Z,7E,9)-decatrien-2-ones. At the time of maximum concentration on the 5th day, the (5E/5Z)-ratio was 94:6. Three hypotheses were experimentally examined to shed light onto the genesis of the uncommon volatiles: first, an indirect effect of agro-industrial side-streams, such as cabbage cuttings, supporting good growth; second, an unsaturated odd-numbered fatty acid precursor; and third, a polyketide-like pathway. In the presence of 1-13C- or 2-13C-acetate up to five acetates were incorporated into the molecular ions of the C10-body. Addition of 1-13C-pyruvate or 1-13C-lactate did not confirm an odd-numbered starter of the polyketide chain. None of the methylketones was found in pineapple or any other food before.

(5E/Z,7E,9)-Decatrien-2-ones, Pineapple-like Flavors from Fomitopsis betulina-Structure Elucidation and Sensorial Properties.

During the cultivation of the edible mushroom Fomitopsis betulina on agro-industrial side streams, a pleasant flavor strongly reminiscent of pineapple was perceived. Aroma extract dilution analyses identified two flavor components with a distinct pineapple odor. On the basis of mass spectrometric data, a Wittig reaction of (E)-penta-2,4-dien-1-yltriphosphonium bromide with ethyl levulinate was conducted. The resulting (5E/Z,7E,9)-decatrien-2-ones were identical to the compounds isolated from the fungal culture. Some structurally related methyl ketones were synthesized, confirmed by nuclear magnetic resonance and mass spectrometry, and their odor was characterized. The lowest odor threshold and most characteristic pineapple-like odor was found for (5Z,7E,9)-decatrien-2-one. Global minimum energy calculation of the methyl ketones and the comparison to (1,3E,5Z)-undecatriene, a character impact compound of fresh pineapple, showed that a chain length of at least 10 carbon atoms and a terminal double bond embedded in a "L"-shaped conformation were common to compounds imparting an intense pineapple-like odor. Both (5E/Z,7E,9)-decatrien-2-ones have not been described as natural flavor compounds.

Highly Variable Pharmacokinetics of Tyramine in Humans and Polymorphisms in OCT1, CYP2D6, and MAO-A.

Tyramine, formed by the decarboxylation of tyrosine, is a natural constituent of numerous food products. As an indirect sympathomimetic, it can have potentially dangerous hypertensive effects. In vitro data indicated that the pharmacokinetics of tyramine possibly depend on the organic cation transporter OCT1 genotype and on the CYP2D6 genotype. Since tyramine is a prototypic substrate of monoamine oxidase A (MAO-A), genetic polymorphisms in MAO-A may also be relevant. The aims of this study were to identify to what extent the interindividual variation in pharmacokinetics and pharmacodynamics of tyramine is determined by genetic polymorphisms in OCT1, CYP2D6, and MAO-A. Beyond that, we wanted to evaluate tyramine as probe drug for the in vivo activity of MAO-A and OCT1. Therefore, the pharmacokinetics, pharmacodynamics, and pharmacogenetics of tyramine were studied in 88 healthy volunteers after oral administration of a 400 mg dose. We observed a strong interindividual variation in systemic tyramine exposure, with a mean AUC of 3.74 min*µg/ml and a high mean CL/F ratio of 107 l/min. On average, as much as 76.8% of the dose was recovered in urine in form of the MAO-catalysed metabolite 4-hydroxyphenylacetic acid (4-HPAA), confirming that oxidative deamination by MAO-A is the quantitatively most relevant metabolic pathway. Systemic exposure of 4-HPAA varied only up to 3-fold, indicating no strong heritable variation in peripheral MAO-A activity. Systolic blood pressure increased by more than 10 mmHg in 71% of the volunteers and correlated strongly with systemic tyramine concentration. In less than 10% of participants, individually variable blood pressure peaks by >40 mmHg above baseline were observed at tyramine concentrations of >60 µg/l. Unexpectedly, the functionally relevant polymorphisms in OCT1 and CYP2D6, including the CYP2D6 poor and ultra-rapid metaboliser genotypes, did not significantly affect tyramine pharmacokinetics or pharmacodynamics. Also, the MOA-A genotypes, which had been associated in several earlier studies with neuropsychiatric phenotypes, had no significant effects on tyramine pharmacokinetics or its metabolism to 4-HPAA. Thus, variation in tyramine pharmacokinetics and pharmacodynamics is not explained by obvious genomic variation, and human tyramine metabolism did not indicate the existence of ultra-low or -high MAO-A activity.

Response of the sesquiterpene synthesis in submerged cultures of the Basidiomycete Tyromyces floriformis to the medium composition.

Tyromyces floriformis, a potent fungal sesquiterpene producer, was grown Cerrena unicolor, as a model organism in submerged culture to search for chemicals affecting sesquiterpene biosynthesis in vitro. Thirty-one sesquiterpenes and sesquiterpenoids were identified in the supernatant, among them the fruity α-ylangene as the main volatile. Additives, such as some polysaccharides or lipids, did not affect the qualitative product spectrum but strongly affected the quantitative synthesis. Rye arabinoxylan and other polysaccharides, such as chitin, starch, and agarose, almost blocked the synthesis of α-ylangene. Single addition of the building blocks of arabinoxylan, arabinose, xylose, or ferulic acid showed no inhibitory effect, whereas 0.05% (w/v) 32-α-l-arabinofuranosyl-xylobiose and larger oligosaccharides resulted in a significant suppression. In contrast, addition of acetyl donors boosted the α-ylangene concentration by 1 order of magnitude up to >40 mg L-1. Both increased as well as decreased α-ylangene concentrations correlated with the intracellular sesquiterpene cyclase activity. Similar experiments using submerged cultured Cerrena unicolor, Postia placenta, and Coprinopsis cinerea showed that the additives affected fungal sesquiterpenoid synthesis differently. Whereas the addition of acetyl donors boosted the synthesis in all biphasic cultures, it was inhibited by polysaccharides in fungi preferably interacting with lignified plants. In contrast, Cerrena unicolor, known for a symbiotic lifestyle with wasps, responded by forming higher concentrations of the possibly insect-attracting sesquiterpenes.

A Portable Biosensor for 2,4-Dinitrotoluene Vapors.

Buried explosive material, e.g., landmines, represent a severe issue for human safety all over the world. Most explosives consist of environmentally hazardous chemicals like 2,4,6-trinitrotoluene (TNT), carcinogenic 2,4-dinitrotoluene (2,4-DNT) and related compounds. Vapors leaking from buried landmines offer a detection marker for landmines, presenting an option to detect landmines without relying on metal detection. 2,4-Dinitrotoluene (DNT), an impurity and byproduct of common TNT synthesis, is a feasible detection marker since it is extremely volatile. We report on the construction of a wireless, handy and cost effective 2,4-dinitrotoluene biosensor combining recombinant bioluminescent bacterial cells and a compact, portable optical detection device. This biosensor could serve as a potential alternative to the current detection technique. The influence of temperature, oxygen and different immobilization procedures on bioluminescence were tested. Oxygen penetration depth in agarose gels was investigated, and showed that aeration with molecular oxygen is necessary to maintain bioluminescence activity at higher cell densities. Bioluminescence was low even at high cell densities and 2,4-DNT concentrations, hence optimization of different prototypes was carried out regarding radiation surface of the gels used for immobilization. These findings were applied to sensor construction, and 50 ppb gaseous 2,4-DNT was successfully detected.

Cell-free one-pot conversion of (+)-valencene to (+)-nootkatone by a unique dye-decolorizing peroxidase combined with a laccase from Funalia trogii.

A combined system of a unique dye-decolorizing peroxidase (Ftr-DyP) and a laccase obtained from the basidiomycete Funalia trogii converted the precursor (+)-valencene completely to the high-value grapefruit flavour constituent (+)-nootkatone, reaching a concentration maximum of 1100 mg/L. In the presence of 1 mM Mn2+ and 2.5 mM p-coumaric acid, (+)-nootkatone was the predominating volatile product, and only traces of substrate and the nootkatols were detectable after 24 h. Hence, the two-enzyme-system reproduced the oxidizing activity observed before for the crude culture supernatant. The newly discovered Ftr-DyP was purified, sequenced and further characterized as a thermostable, non-glycosylated protein with a pH-optimum in the acidic range and a calculated mass of 52.3 kDa. Besides the typical activity of DyPs towards anthraquinone dyes, Ftr-DyP also oxidized Mn2+ and showed activity in the absence of hydrogen peroxide. Neither the DyP from Mycetinis scorodonius nor the manganese peroxidase from Nematoloma frowardii were able to replace Ftr-DyP in this reaction. A hypothetical reaction mechanism is presented.

Volatiles from Cinnamomum cassia buds.

While the chemical composition of leaf and stem bark essential oils of the Chinese cinnamon, Cinnamomum cassia (L.) J. Presl, has been well investigated, little is known about the volatilom of its buds, which appeared recently on German markets. Soxhlet extracts of the commercial samples were prepared, fractionated using silica gel and characterised by gas chromatography-flame ionisation detector (GC-FID) for semi-quantification, by gas chromatography-mass spectrometry (GC-MS) for identification and by GC-FID/olfactometry for sensory evaluation. Cinnamaldehyde was the most abundant compound with concentrations up to 40 mg/g sample. In total, 36 compounds were identified and 30 were semi-quantified. The extracts contained mostly phenylpropanoids, mono- and sesquiterpene hydrocarbons and oxygenated derivatives. Because of the high abundance of cinnamaldehyde, the aldehyde fraction was removed from the extracts by adding hydrogen sulphite to improve both the detection of trace compounds and column chromatography. The aldehyde fraction was analysed by GC-MS separately. The highest flavour dilution factor of 316 was calculated for cinnamaldehyde. Other main sensory contributors were 2-phenylethanol and cinnamyl alcohol. This report provides the first GC-olfactometry data of a plant part of a Cinnamomum species. The strongly lignified C. cassia buds combine a high abundance of cinnamaldehyde with comparably low coumarin concentrations (<0.48 mg/g), and provide a large cinnamaldehyde depot for slow release applications.

Cold generation of smoke flavour by the first phenolic acid decarboxylase from a filamentous ascomycete - Isaria farinosa.

A decarboxylase (IfPAD) from the ascomycete Isaria farinosa converted ferulic acid to 4-vinylguaiacol (4-VG), a volatile which imparts the distinct "smoke flavor" of pyrolized wood. The activity was enhanced by adding (E)-ferulic acid to the culture medium and peaked with 3.6 U g-1 mycelium (1 µmol 4-VG min-1). The coding sequence of 543 bp was translated into a 25 kDa protein with a homology of 91 % to putative phenolic acid decarboxylases of its teleomorph, Cordyceps militaris, and Beauveria bassiana, the anamorph of Cordyceps bassiana. Cold shock expression in Escherichia coli yielded 411 U g-1 wet mass. Substrate conversion required a hydroxyl substituent para to a trans-unsaturated C3-side chain of the aromatic ring. Km and kcat/Km values were determined to 0.3 mM and 78.4 mM-1s-1 for p-coumaric acid and 1.9 mM and 45.1 mM-1s-1 for (E)-ferulic acid, respectively. The native enzyme and its recombinant counterpart showed pH-optima at pH 6.0 and pH 5.5, and low temperature optima of 19 °C and 14 °C, respectively. IfPAD produced 4-VG from destarched wheat bran and sugar beet fiber, confirming activity on complex plant biomass. This is the first report on the biochemical characterization of a phenolic acid decarboxylase from a filamentous ascomycete.

Heterologous production of the stain solving peptidase PPP1 from Pleurotus pulmonarius.

A novel stain solving subtilisin-like peptidase (PPP1) was identified from the culture supernatant of the agaricomycete Pleurotus pulmonarius. It was purified to homogeneity using a sequence of preparative isoelectric focusing, anion exchange and size exclusion chromatography. Peptides were identified by ab initio sequencing (nLC-ESI-QTOF-MS/MS), characterizing the enzyme as a member of the subtilase family (EC 3.4.21.X). An expression system was established featuring the pPIC9K vector, an alternative Kozak sequence, the codon optimized gene ppp1 gene without the native signal sequence with C-terminal hexa-histidine tag, and Pichia pastoris GS115 as expression host. Intracellular active enzyme was obtained from cultivations in shake flasks and in a five liter bioreactor. With reaction optima of 40 °C and a pH > 8.5, considerable bleaching of pre-stained fabrics (blood, milk and India ink), and the possibility of larger-scale production, the heterologous enzyme is well suitable for detergent applications, especially at lower temperatures as part of a more energy- and cost-efficient washing process. Showing little sequence similarity to other subtilases, this unique peptidase is the first subtilisin-like peptidase from Basidiomycota, which has been functionally produced in Pichia pastoris.

Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl-saccharide esters.

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.

A three-enzyme-system to degrade curcumin to natural vanillin.

The symmetrical structure of curcumin includes two 4-hydroxy-3-methoxyphenyl substructures. Laccase catalyzed formation of a phenol radical, radical migration and oxygen insertion at the benzylic positions can result in the formation of vanillin. As vanillin itself is a preferred phenolic substrate of laccases, the formation of vanillin oligomers and polymers is inevitable, once vanillin becomes liberated. To decelerate the oligomerization, one of the phenolic hydroxyl groups was protected via acetylation. Monoacetyl curcumin with an approximate molar yield of 49% was the major acetylation product, when a lipase from Candida antarctica (CAL) was used. In the second step, monoacetyl curcumin was incubated with purified laccases of various basidiomycete fungi in a biphasic system (diethyl ether/aqueous buffer). A laccase from Funalia trogii (LccFtr) resulted in a high conversion (46% molar yield of curcumin monoacetate) to vanillin acetate. The non-protected vanillin moiety reacted to a mixture of higher molecular products. In the third step, the protecting group was removed from vanillin acetate using a feruloyl esterase from Pleurotus eryngii (PeFaeA) (68% molar yield). Alignment of the amino acid sequences indicated that high potential laccases performed better in this mediator and cofactor-free reaction.

A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 µM, 64.1 µM, 72.5 µM, and 101.8 µM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.

Crosses between monokaryons of Pleurotus sapidus or Pleurotus florida show an improved biotransformation of (+)-valencene to (+)-nootkatone.

Several hundred monokaryotic and new dikaryotic strains derived thereof were established from (+)-valencene tolerant Pleurotus species. When grouped according to their growth rate on agar plates and compared to the parental of Pleurotus sapidus 69, the slowly growing monokaryons converted (+)-valencene more efficiently to the grapefruit flavour compound (+)-nootkatone. The fast growing monokaryons and the slow×slow and the fast×fast dikaryotic crosses showed similar or inferior yields. Some slow×fast dikaryons, however, exceeded the biotransformation capability of the parental dikaryon significantly. The activity of the responsible enzyme, lipoxygenase, showed a weak correlation with the yields of (+)-nootkatone indicating that the determination of enzyme activity using the primary substrate linoleic acid may be misleading in predicting the biotransformation efficiency. This exploratory study indicated that a classical genetics approach resulted in altered and partly improved terpene transformation capability (plus 60%) and lipoxygenase activity of the strains.

Isolation and characterization of wild-type lipoxygenase LOX(Psa)1 from Pleurotus sapidus.

The lipoxygenase LOX(Psa) 1 of Pleurotus sapidus, originally investigated because of its ability to oxidize (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was isolated from the peptidase-rich lyophilisate using a three-step purification scheme including preparative isoelectric focusing and chromatographic techniques. Nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS) of the purified enzyme and peptide mass fingerprint analysis gave 38 peptides of the lipoxygenase from P. sapidus. Nearly 50% of the 643 amino acids long sequence encoded by the cDNA was covered. Both terminal peptides of the native LOX(Psa) 1 were identified by de novo sequencing, and the postulated molecular mass of 72.5 kDa was confirmed. With linoleic acid as the substrate, the LOX(Psa)1 showed a specific activity of 113 U mg(-1) and maximal activity at pH 7.0 and 30 degrees C, respectively.

Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris.

The first heterologous expression of an iron-containing lipoxygenase from a basidiomycete in Pichia pastoris is reported. Five different expression constructs of the lipoxygenase gene LOX1 from Pleurotus sapidus were cloned and successfully transferred into P. pastoris SMD1168, but only one pPIC9K vector construct was functionally expressed. In this construct the vector-provided α-factor signal sequence was replaced by insertion of a second Kozak sequence between the signal sequence and the LOX1 gene. His(+) transformants were screened for their level of resistance to geneticin (G418). Lox1 was expressed under different culture conditions and purified using the N-terminal His-tag. Relative enzyme activity increased significantly 48h after methanol induction and was highest with 2mll(-1) inducer. The recombinant enzyme showed an optimal lipoxygenase activity at pH 7 and 30-35°C and a vmax like the wild-type enzyme.