Pleurotus Sapidus Research Articles

Elucidation of the regio- and chemoselectivity of enzymatic allylic oxidations with Pleurotus sapidus – conversion of selected spirocyclic terpenoids and computational analysis

Allylic oxidations of olefins to enones allow the efficient synthesis of value-added products from simple olefinic precursors like terpenes or terpenoids. Biocatalytic variants have a large potential for industrial applications, particularly in the pharmaceutical and food industry. Herein we report efficient biocatalytic allylic oxidations of spirocyclic terpenoids by a lyophilisate of the edible fungus Pleurotus sapidus. This ‘’mushroom catalysis’’ is operationally simple and allows the conversion of various unsaturated spirocyclic terpenoids. A number of new spirocyclic enones have thus been obtained with good regio- and chemoselectivity and chiral separation protocols for enantiomeric mixtures have been developed. The oxidations follow a radical mechanism and the regioselectivity of the reaction is mainly determined by bond-dissociation energies of the available allylic CH-bonds and steric accessibility of the oxidation site.

Orthologous lipoxygenases of Pleurotus spp. – A comparison of substrate specificity and sequence homology

A selection of Pleurotus spp. was screened for intracellular lipoxygenase activity, and strains with distinguished activities were chosen for a screening on the molecular level. Lipoxygenase genes from five different Pleurotus spp. were amplified from the corresponding cDNA, functionally expressed using a cold shock expression system in Escherichia coli BL21DE3 Star cells and characterized for specific activity and reaction optima. All lipoxygenase sequences coded for proteins of 643 amino acids, sharing similarities >95% among each other and to a previously characterized LOXPsa1 from Pleurotus sapidus. Lipoxygenase activities were quantified using linoleic acid as substrate and reached similar values ranging from 95 U/mg to 118 U/mg, with Km values between 58 and 106 μM. Optimum reaction conditions were pH 7 and 30–35 ̊C. However, the uncommon trait of accepting (+)-valencene, a sesquiterpene hydrocarbon substrate, differed strongly between two clusters of highly homologous sequences. No exchange of amino acids adjacent to the active site was found. Cloning and expression of a truncated LOXPsa1 sequence missing 144 amino acid residues of the N-terminal barrel domain yielded soluble protein but no measurable activity.

Induction, characterization, and heterologous expression of a carotenoid degrading versatile peroxidase from Pleurotus sapidus

Abstract A β-carotene degrading versatile peroxidase (VP) was successfully induced in submerged cultures of the white-rot basidiomycete Pleurotus sapidus by use of residues of a biogas plant as a carbon and nitrogen source. After three chromatographic steps, the VP was isolated with an overall yield of 12% and a purification factor of 130. The purified enzyme showed a molecular mass of 38 kDa and an isoelectric point of 3.6. Highest affinity to β-carotene ( K m = 50 ± 5 μM) was observed at 30 °C and pH 4.5. The purified VP was capable of degrading suspended lignin organosolv particles. N-terminal and internal peptide sequences were obtained from Edman degradation and mass spectrometric peptide sequencing. The VP encoding cDNA was identified by colony hybridization and amplified by PCR. Bioinformatic analyses revealed an open reading frame of 1083 bp and similarities of 90% to VPs from P. eryngii . The recombinant VP was produced successfully with an activity of 450 ± 20 mU mg −1 in cultures of H. polymorpha .

Rapid Differentiation of Pleurotus Ostreatus from Pleurotus Sapidus Using PCR Technique

Pleurotus ostreatus and Pleurotus sapidus are difficult to differentiate using standard morphologically based characteristics. This study, using two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F & ITS4, were intended to be specific for the higher fungi, respectively. RAPD-PCR technique was used to reveal DNA polymorphism in DNA of the two fungi Pleurotus ostreatus and Pleurotus sapidus in order to search for the sources of differences that could be used as a DNA marker represent the differentiation between these two species . In RAPD-PCR, eight different decamer primers chosen randomly were employed to detect the genetic polymorphisms among studied strains .

Molecular Characterization of a Lipoxygenase from the Basidiomycete MushroomPleurotus ostreatus

The full-length cDNA of the gene PoLOX1 encoding a lipoxygenase (LOX) and its corresponding genomic DNA were isolated from the basidiomycete mushroom Pleurotus ostreatus strain H1. The deduced amino acid sequence of PoLOX1 showed similarity to a valencene dioxygenase of Pleurotus sapidus, putative LOX-like proteins from ascomycete, basidiomycete, and deuteromycete fungi, and known LOXs from plants, animals, and bacteria. PoLOX1 also contained the LOX iron-binding catalytic domain in the C-terminal region, but not the polycystin-1, lipoxygenase, alpha-toxin (PLAT) domain, which is usually found in the N-terminal region of eukaryotic LOXs. Genomic sequence analysis revealed that PoLOX1 was interrupted by one intron, and that the promoter region included TATA and CAAT boxes. Southern blot analysis indicated that PoLOX1 is a member of a small gene family comprising highly similar genes. Northern blot analysis revealed that it is transcribed more abundantly in the stipes of the fruit bodies than in the caps.

An esterase from the basidiomycete Pleurotus sapidus hydrolyzes feruloylated saccharides

Investigating the secretion of esterases by the basidiomycetous fungus Pleurotus sapidus in a Tween 80-rich nutrient medium, an enzyme was discovered that hydrolyzed the ester bond of feruloylated saccharides. The enzyme was purified by ion exchange and size exclusion chromatography. Polyacrylamide gel electrophoresis analysis showed a monomeric protein of about 55kDa. The complete coding sequence with an open reading frame of 1,665bp encoded a protein (Est1) consisting of 554 amino acids. The enzyme showed no significant homology to any published feruloyl esterase sequences, but possessed putative conserved domains of the lipase/esterase superfamily. Substrate specificity studies classified the new enzyme as type-A feruloyl esterase, hydrolyzing methyl ferulate, methyl sinapate, and methyl p-coumarate but no methyl caffeate. The enzyme had a pH optimum of 6 and a temperature optimum at 50°C. Ferulic acid was efficiently released from ferulated saccharides, and the feruloyl esterase exhibited moderate stability in biphasic systems (50% toluene or tert-butylmethyl ether).

LOXPsa1, the first recombinant lipoxygenase from a basidiomycete fungus

Abstract A dioxygenase from the edible basidiomycete Pleurotus sapidus , originally researched because of its distinct ability to convert the sequiterpene (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was identified as a potent lipoxygenase (LOX Psa 1). Kinetic parameters, pH and temperature optima of the pure recombinant enzyme were determined using linoleic acid as the substrate. K m , v max , and k cat were 40.3 μM, 130.3 U mg −1 , and 157 s −1 , respectively. The maximal enzymatic activity was found at pH 7.0 and 35 °C. Showing high specificity toward free linoleic acid, the enzyme was classified as lipoxygenase type 1. Conversion of linoleic acid yielded mainly ( S )-13-hydroperoxy-9 Z ,11 E -octadecadienoic acid (94% ee), as was confirmed by chiral HPLC analysis of the hydroperoxides. The amino acid sequence showed homology to lipoxygenases catalyzing S stereospecific oxygenation, and thus the enzyme was characterized as a 13 S -lipoxygenase. This is the first lipoxygenase described to accept terpene hydrocarbons as substrates.

Membrane chromatography for the purification of laccase from the supernatant of Pleurotus sapidus

Extracellular laccase from the fermentation broth of Pleurotus sapidus was purified using membrane chromatography. Five laccase iso-enzymes produced in submerged cultures were purified; the enzymes had isoelectric points (pI) ranging from 3.3 to 4.7 and a calculated molecular weight of 57.4kDa. The fermentation broth had a low product titre of 80μg/l. Hydrophobic interaction (HIC) and ion exchange (IEX) membranes chromatography modules were used in an ÄKTApurifier 100 liquid chromatography system for the dynamic experiments. The pre-treatment of the fermentation broth was not required. The enzyme activity was retained, and enrichment up to 15-fold was observed via activity assays and SDS-Page analysis.

Bioreduction of chloroaurate ions to gold nanoparticles by culture filtrate of Pleurotus sapidus Quél

The present study demonstrates an eco-friendly, non-toxic and cost-effective protocol for synthesis of gold nanoparticles (AuNPs) in aqueous medium using Pleurotus sapidus culture filtrate as the reducing and stabilizing agent. On treating chloroauric acid solutions with fungal culture filtrate, rapid reduction of chloroaurate ions is observed leading to the formation of highly stable AuNPs in solution. The particles thereby obtained were characterized by different spectroscopic and X-ray analysis. EDX-spectrum revealed the presence of gold in the nanoparticles. FTIR spectroscopy confirmed the presence of a protein shell outside the nanoparticles which in turn also support their stabilization. DLS and TEM analysis of the AuNPs indicated that they ranged in size from 15 to 100nm with average size of 65±5nm. A probable mechanism behind the biosynthesis is discussed, which leads to the possibility of using the present protocol in future “nano-factories”.

Enzymatic allylic oxidations with a lyophilisate of the edible fungus Pleurotus sapidus

Allylic oxidations belong to the most attractive synthetic transformations because they convert readily available and cheap starting materials into value-added products. In this study, we describe oxidative conversions of terpenoids and a number of related cycloalkenes with a lyophilisate of the edible fungus Pleurotus sapidus. The biocatalytic protocol is simple and the biocatalyst is readily available. The conversions of various cycloalkenes proceed cleanly in most cases to the corresponding enones. The substrate scope is remarkable and includes a number of mono- and sequiterpenes, functionalized terpenoids as well as simple cyclohexenes and benzylic substrates. Enzymatic allylic oxidations by Pleurotus sapidus are thus an excellent non-toxic alternative to metal-mediated oxidation procedures in academic labs and for industrial application in food technology, cosmetics or pharmaceutical research.

Functional expression of a valencene dioxygenase from Pleurotus sapidus in E. coli

Valencene dioxygenase (ValOx) from the edible basidiomycete Pleurotus sapidus converted the sesquiterpene (+)-valencene to the valuable grapefruit flavour (+)-nootkatone and to nootkatols through intermediate hydroperoxides. Expression of the enzyme was carried out in the cytosol and periplasm of Escherichia coli. The heterologous production led to high yields of inclusion bodies. The poor yield of soluble recombinant protein was improved by various strategies including cold shock expression, chaperone co-expression, and employment of mutant E. coli strains. Up to 60mg of the biologically active, soluble ValOx was produced by cold shock under control of the cspA promoter at 8°C in the BL21(DE3)Star strain and co-expression of the E. coli trigger factor. The recombinant enzyme, purified using the N-terminal His tag, showed the catalytic properties of the wild-type enzyme, as was confirmed by the LC-MS analysis of hydroperoxide intermediates and GC–MS analysis of the volatile products.

Analysis of Car-3-en-5-hydroperoxide

HRGC-MS, using split/splitless injection (230 degrees C), showed that a dioxygenase from Pleurotus sapidus regio-selectively transformed (+)-car-3-ene to car-3-en-5-one as the major volatile product to minor amounts of the corresponding alcohol, and to some other volatiles. Thus, the reaction was assumed to be radical mediated and similar to the lipoxygenase catalyzed peroxidation of polyunsaturated fatty acids, but the expected car-3-ene-hydroperoxides were not detected. TLC of the reaction products, followed by hydroperoxide specific staining, visually indicated the presence of hydroperoxides. TLC spots were eluted and re-analyzed using cool on-column injection, but only tailing peaks showing a mixed mass spectrum of car-3-en-5-ol/one were obtained. An unequivocal identification of car-3-en-5-hydroperoxides was achieved only after using APCI(+)-LC-MS. Upon structural confirmation, the car-3-en-5-hydroperoxide was accumulated by preparative HPLC, re-injected cool on-column, and the continuing degradation of the hydroperoxide to monoterpene ketone and alcohol during chromatography was verified. It was concluded that terpene hydroperoxides may occur in essential oils more frequently than anticipated, but are not recognized due to the principal blindness of capillary gas chromatography techniques and UV/vis LC-detectors.

Bioconversion of car-3-ene by a dioxygenase of Pleurotus sapidus

Mycelium of the basidiomycete Pleurotus sapidus known to contain a novel dioxygenase was used for the bioconversion of car-3-ene [I]. After 4h of incubation 25.3mgL(-1) car-3-en-5-one [V], 5.4mgL(-1) car-3-en-2-one [VII], and 7.3mgL(-1) car-2-en-4-one [XV] accumulated as major oxidation products. The identity of the respective carenones and their corresponding alcohols was confirmed by comparison with MS and NMR spectral data obtained for synthesized authentic compounds. The peak areas of oxidation products were at least five times higher as compared with autoxidation. A radical mechanism similar to lipoxygenase catalysis was proposed and substantiated with detailed product analyses. The reduction of assumed car-3-ene hydroperoxides to the corresponding alcohols evidenced the radical initiated formation of hydroperoxides and confirmed the regio- and stereo-selectivity of the dioxygenase. The introduction of molecular oxygen into the bicyclic car-3-ene [I] molecule occurred at allylic positions of a cyclic isopentenyl moiety with a pronounced preference for the position adjacent to the non-substituted carbon atom of the C-C-double bond. This co-factor independent selective oxygenation presents an alternative to P450 mono-oxygenase based approaches for the production of terpene derived flavor compounds, pharmaceuticals and other fine chemicals.

Performance of a Novel dioxygenase of pleurotus sapidus to generate terpene-derived flavours

Performance of a Novel dioxygenase of pleurotus sapidus to generate terpene-derived flavours

Autoxidation versus Biotransformation of α-Pinene to Flavors with Pleurotus sapidus: Regioselective Hydroperoxidation of α-Pinene and Stereoselective Dehydrogenation of Verbenol

The enzymatic conversion of alpha-pinene to verbenols, verbenone, and minor volatile flavors was studied using submerged cultured cells, lyophilisate, and microsomal fractions of the edible basidiomycete Pleurotus sapidus . The similarity of the product range obtained by the bioconversions with the range of products found after autoxidation of alpha-pinene at 100 degrees C suggested similar initial pinene radicals. Extracts of the bioconversions were analyzed using thin layer chromatography with hydroperoxide staining and cool on-column capillary gas chromatography-mass spectrometry. Two isomer alpha-pinene hydroperoxides were identified as the key intermediates and their structures confirmed by comparison with synthesized reference samples and by microchemical reduction to (Z)- and (E)-verbenol. When the biocatalysts were supplemented with one of the verbenols, only the (Z)-isomer was oxidized, indicating the activity of a highly stereospecific monoterpenol dehydrogenase. The structural comparison of subunits shows that fungal oxifunctionalization reactions of some common terpene substrates, such as (+)-limonene or (+)-valencene, might likewise be catalyzed by dioxygenases rather than by CYP450 enzymes, as previously assumed.

A dioxygenase of Pleurotus sapidus transforms (+)-valencene regio-specifically to (+)-nootkatone via a stereo-specific allylic hydroperoxidation

A selective and highly efficient allylic oxidation of the sesquiterpene (+)-valencene to the grapefruit flavour compound (+)-nootkatone was achieved with lyophilisate of the edible mushroom Pleurotus sapidus. The catalytic reaction sequence was elucidated through the identification of intermediate, (+)-valencene derived hydroperoxides. A specific staining of hydroperoxides allowed the semi-preparative isolation of two secondary (+)-valencene hydroperoxides, 6(R)-Isopropenyl-4(R),4a(S)-dimethyl-2,3,4,4a,5,6,7,8-octahydro-naphthalen-4(S)-yl-hydroperoxide and 6(R)-Isopropenyl-4(R),4a(S)-dimethyl-2,3,4,4a,5,6,7,8-octahydro-naphthalen-2(R)-yl-hydroperoxide. Chemical reduction of the biotransformation products yielded a tertiary alcohol identified as 2(R)-Isopropenyl-8(R),8a(S)-dimethyl-1,3,4,7,8,8a-hexahydro-2H-naphthalen-4a(R)-ol. This suggested a lipoxygenase-type oxidation of (+)-valencene via secondary and tertiary hydroperoxides and confirmed homology data of the key enzyme obtained previously from amino acid sequencing.

A novel oxygenase from Pleurotus sapidus transforms valencene to nootkatone

A selective and highly efficient allylic oxidation of the sesquiterpene (+)-valencene to (+)-nootkatone was achieved with lyophilisate of the basidiomycete Pleurotus sapidus. The responsible enzymatic activity was biochemically characterised and purified by chromatographic and electrophoretic methods. Peptide sequences obtained by mass spectrometry showed homologies to oxygenases from various ascomycetes. Based on the peptide sequences, the encoding cDNA was amplified from a cDNA library of P. sapidus by PCR. The cloned sequence consisted of 1309bp with an open reading frame of 1191bp. Based on database research, the translated amino acid sequence of 396 amino acids showed on the protein level homologies of ∼50% to putative lipoxygenases from Aspergillus fumigatus and Laccaria bicolor as well as 26% homology to the sequence of lipoxygenase-1 from soy bean (Glycine max). A lipoxygenase from a basidiomycetous fungus has not yet been characterised on a molecular level.

Separation of Extracellular Esterases from Pellet Cultures of the Basidiomycete Pleurotus sapidus by Foam Fractionation

AbstractTwo extracellular esterases were produced in submerged cultures of the basidiomycete Pleurotus sapidus. A foam fractionation device was designed and employed for the isolation of the esterolytic enzymes. The recovery of enzyme activity in the liquefied foam strongly depended on the superficial gas velocity. High purification and enrichment factors (Ea = 62.0, P = 15.5) were achieved using nitrogen at 1.87 cm min−1 within 100 min. Increasing the superficial gas velocity to 2.49 cm min−1 improved the recovery of total esterase activity in the foam to >95% at the expense of reduced enrichment and purification factors. Differences in their physicochemical characteristics resulted in differing foaming properties of the two esterases secreted by P. sapidus. By variation of the pH value of the culture medium and addition of Triton X‐100, both esterases were successively and quantitatively transferred into the foam in a two‐step fractionation process.

Removal of Furanocoumarins in Grapefruit Juice by Edible Fungi

Furanocoumarins (FCs) in the human diet irreversibly inhibit human cytochrome P450 3A4 (CYP 3A4) and are responsible for the "grapefruit/drug""interaction phenomenon. Previously, we reported that FCs in grapefruit juice (GFJ) bind to autoclaved Aspergillus niger, and this binding reduced the GFJ inhibition of CYP 3A4. However, A. niger is not an edible fungus, and thus, potentially similar binding by edible fungi was also characterized. In this study, autoclaved Morchella esculenta, an edible ascomycete, removed much of the FC content in GFJ, resulting in decreased inhibition of CYP 3A4 activity by the GFJ. Three other edible fungi, Monascus purpureus, Pleurotus sapidus, and Agaricus bisporus, were evaluated for their binding with two of the major FCs in GFJ, 6',7'-dihydroxybergamottin (DHB) and bergamottin (BM). These autoclaved edible fungi removed these FCs from GFJ, similar to M. esculenta, indicating that binding is a general, passive interaction between FCs and fungal hyphae. The removal of FCs was independent of pH in GFJ. Dried fungal material of M. esculenta was also effective in removing FCs from GFJ and occurred with GFJ samples prepared from both fresh grapefruit and GFJ concentrate.

Functional expression of the lipase gene Lip2 of Pleurotus sapidus in Escherichia coli

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.