Characterization of a GDS(L)-like hydrolase from Pleurotus sapidus with an unusual SGNH motif.

The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L-1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8-25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes.

Optimization of polysaccharide conditions and analysis of antioxidant capacity in the co-culture of Sanghuangporus vaninii and Pleurotus sapidus.

Fungal polysaccharides are commonly utilized in the food industry and biomedical fields as a natural and safe immune modulator. Co-culturing is a valuable method for enhancing the production of secondary metabolites. This study used intracellular polysaccharide (IPS) content as a screening index, co-culturing seven different fungi with Sanghuangporus vaninii. The seed pre-culture liquid culture time was selected through screening, and conditions were assessed using single factor experimentation, a Plackett-Burman (PB) design, and response surface methodology (RSM) optimization. RSM optimization was conducted, leading to the measurement of antioxidant capacity. Results indicated that the co-culture of S. vaninii and Pleurotus sapidus exhibited the most effective outcome. Specifically, pre-culturing S. vaninii and P. sapidus seed cultures for 2 days and 0 days, respectively, followed by co-culturing, significantly increased IPS content compared to single-strain culturing. Further optimization of co-culture conditions revealed that yeast extract concentration, liquid volume, and S. vaninii inoculum ratio notably influenced IPS content in the order of yeast extract concentration > liquid volume > S. vaninii inoculum ratio. Under the optimal conditions, IPS content reached 69.9626 mg/g, a 17.04% increase from pre-optimization co-culture conditions. Antioxidant capacity testing demonstrated that co-cultured IPS exhibited greater scavenging abilities for DPPH and ABTS free radicals compared to single strain cultures. These findings highlight the potential of co-culturing S. vaninii and P. sapidus to enhance IPS content and improve antioxidant capacity, presenting an effective strategy for increasing fungal polysaccharide production.

Improvement in the Stability and Enzymatic Activity of Pleurotus sapidus Lipoxygenase Dissolved in Natural Deep Eutectic Solvents (NADESs).

Natural deep eutectic solvents (NADESs) can serve as solvents for enzymes, are biodegradable, and have low toxicities. Eight NADESs with different hydrogen bond acceptors and donors were tested to improve the stability and activity of a lipoxygenase from Basidiomycete Pleurotus sapidus (LOXPSA). Betaine:sorbitol:water (1:1:3, BSorbW) and betaine:ethylene glycol (1:3, BEtGly) had the best impact on the peroxidation of linoleic acid and the side reaction of piperine to the vanilla-like scented compound piperonal. The yield of piperonal in NADESs increased by 43% in BSorbW and 40% in BEtGly compared to the control. The addition of BSorbW also enhanced the enzyme's stability at various temperatures and increased its activity during incubation at 60 °C. The demonstrated improvement in lipoxygenase activity and stability indicates versatile applications in industry, expanding the potential uses of the enzyme.

Production of an Anise- and Woodruff-like Aroma by Monokaryotic Strains of Pleurotus sapidus Grown on Citrus Side Streams.

The production of natural flavors by means of microorganisms is of great interest for the food and flavor industry, and by-products of the agro-industry are particularly suitable as substrates. In the present study, Citrus side streams were fermented using monokaryotic strains of the fungus Pleurotus sapidus. Some of the cultures exhibited a pleasant smell, reminiscent of woodruff and anise, as well as herbaceous notes. To evaluate the composition of the overall aroma, liquid/liquid extracts of submerged cultures of a selected monokaryon were prepared, and the volatiles were isolated via solvent-assisted flavor evaporation. Aroma extract dilution analyses revealed p-anisaldehyde (sweetish, anisic- and woodruff-like) with a flavor dilution factor of 218 as a character impact compound. The coconut-like, herbaceous, and sweetish smelling acyloin identified as (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone also contributed to the overall aroma and was described as an aroma-active substance with an odor threshold in air of 0.2 ng L-1 to 2.4 ng L-1 for the first time. Supplementation of the culture medium with isotopically substituted l-tyrosine elucidated this phenolic amino acid as precursor of p-anisaldehyde as well as of (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone. Chiral analysis via HPLC revealed an enantiomeric excess of 97% for the isolated product produced by P. sapidus.

Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression.

The basidiomycete Pleurotus sapidus produced a dye-decolorizing peroxidase (PsaPOX) with alkene cleavage activity, implying potential as a biocatalyst for the fragrance and flavor industry. To increase the activity, a daughter-generation of 101 basidiospore-derived monokaryons (MK) was used. After a pre-selection according to the growth rate, the activity analysis revealed a stable intraspecific variability of the strains regarding peroxidase and alkene cleavage activity of PsaPOX. Ten monokaryons reached activities up to 2.6-fold higher than the dikaryon, with MK16 showing the highest activity. Analysis of the PsaPOX gene identified three different enzyme variants. These were co-responsible for the observed differences in activities between strains as verified by heterologous expression in Komagataella phaffii. The mutation S371H in enzyme variant PsaPOX_high caused an activity increase alongside a higher protein stability, while the eleven mutations in variant PsaPOX_low resulted in an activity decrease, which was partially based on a shift of the pH optimum from 3.5 to 3.0. Transcriptional analysis revealed the increased expression of PsaPOX in MK16 as reason for the higher PsaPOX activity in comparison to other strains producing the same PsaPOX variant. Thus, different expression profiles, as well as enzyme variants, were identified as crucial factors for the intraspecific variability of the PsaPOX activity in the monokaryons.

Biotransformation of rice and sunflower side-streams by dikaryotic and monokaryotic strains of Pleurotus sapidus: Impact on phenolic profiles and bioactive properties.

Fungi are known to modify the properties of lignocellulosic materials during solid-state fermentation (SSF). In this study, agricultural side-streams (sunflower seed hulls, rice husks and rice straw) were used as substrates for SSF with dikaryotic and monokaryotic strains of Pleurotus sapidus. The phenolic profiles of the mentioned substrates were characterized by LC-DAD/ESI-MSn pre- and post- fermentation. Moreover, antioxidant, cytotoxic and antimicrobial activities were screened against oxidizable cellular substrates, tumour and primary cell lines, and different bacteria and fungi, respectively. The concentration of phenolic compounds in the crop side-streams was reduced after fermentation with both strains of the fungus. The fermented extracts also displayed lower antioxidant and cytotoxic activities and had no hepatotoxicity. The antimicrobial activity depended upon the crop side-stream and/or SSF conditions. These results indicate that P. sapidus represent a good candidate to modify the phenolic fraction presents in crop side-streams with a consequent decrease in its bioactivities. However, the SSF with P. sapidus strains play an interesting role in the detoxification of plant materials which can be used for different applications according to the "reduce - reuse - recycle" concept contributing with the sustainable land use and circular economy.

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity.

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of ß-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

Biorefining via solid-state fermentation of rice and sunflower by-products employing novel monosporic strains from Pleurotus sapidus.

Pleurotus sapidus monokaryotic strains (Mk) were screened as a novel source of mycelia to valorize rice straw (RS), rice husks (RH) and sunflower seed hulls (SSH) into value-added products through solid-state fermentation (SSF). P. sapidus Dk3174 basidiospores were cultured in the presence of Remazol Brillant Blue R for strain selection, revealing the ligninolytic ability of emerging colonies. Further screening demonstrated the intraspecific variability in dye degradation and enzyme production of 63 strains. Growth rate, biomass and enzyme production in plates containing RS, RH or SSH pointed at MkP6 as a suitable strain for pilot-scale SSF. MkP6 presented a similar laccase profile as the parental Dk3174, being greater in pasteurized substrates (300-1200 U/Kg) than in sterilized substrates (30-250 U/Kg). Peroxidase represented 25% of the total ligninolytic activity measured. The SSH fermented biomass with MkP6 obtained good yields of nanocellulose (67%) and the saccharide release for ethanol production increased by 3-4 times.

Biosynthesis of Stereoisomers of Dill Ether and Wine Lactone by Pleurotus sapidus.

The white-rot fungus Pleurotus sapidus (PSA) biosynthesizes the bicyclic monoterpenoids 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran (dill ether) (1) and 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-one (wine lactone) (2). Submerged cultures grown in different media were analyzed by gas chromatography-mass spectrometry. The stereochemistry of the formed isomers was elucidated by comparing their retention indices to those of reference compounds by enantioselective multidimensional gas chromatography. The basidiomycete produced the rare (3R,3aR,7aS) and (3S,3aR,7aS) stereoisomers of dill ether and wine lactone. Kinetic analyses of the volatilome and bioprocess parameters revealed that the biosynthesis of the bicyclic monoterpenoids correlated with the availability of the primary carbon source glucose. Spiking the media with 13C-labeled glucose demonstrated that the compounds were produced de novo. Supplementation studies i.a. with isotopically labeled substrates further identified limonene and p-menth-1-en-9-ol as intermediate compounds in the fungal pathways. PSA was able to biotransform all enantiomeric forms of the latter compounds to the respective isomers of dill ether and wine lactone.

Submerged Cultivation of Pleurotus sapidus with Molasses: Aroma Dilution Analyses by Means of Solid Phase Microextraction and Stir Bar Sorptive Extraction.

The basidiomycete Pleurotus sapidus (PSA) was grown in submerged cultures with molasses as substrate for the production of mycelium as a protein source for food applications. The volatilomes of the substrate, the submerged culture, and the mycelia were analyzed by gas chromatography-tandem mass spectrometry-olfactometry. For compound identification, aroma dilution analyses by means of headspace solid phase microextraction and stir bar sorptive extraction were performed via variation of the split vent flow rate. Among the most potent odorants formed by PSA were arylic compounds (e.g., p-anisaldehyde), unsaturated carbonyls (e.g., 1-octen-3-one, ( E)-2-octenal, ( E, E)-2,4-decadienal), and cyclic monoterpenoids (e.g., 3,9-epoxy- p-menth-1-ene, 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3 H)-one). Several compounds from the latter group were described for the first time in Pleurotus spp. After separation of the mycelia from the medium, the aroma compounds were mainly enriched in the culture supernatant. The sensory analysis of the mycelium correlated well with the instrumental results.

Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus.

The coding sequence of a peroxidase from the secretome of Pleurotus sapidus was cloned from a cDNA library. Bioinformatic analyses revealed an open reading frame of 1551 bp corresponding to a primary translation product of 516 amino acids. The DyP-type peroxidase was heterologously produced in Trichoderma reesei with an activity of 55,000 U L-1. The enzyme was purified from the culture supernatant, biochemically characterized and the kinetic parameters were determined. The enzyme has an N-terminal signal peptide composed of 62 amino acids. Analysis by Blue Native PAGE and activity staining with ABTS, as well as gel filtration chromatography showed the native dimeric state of the enzyme (115 kDa). Analysis of the substrate range revealed that the recombinant enzyme catalyzes, in addition to the conversion of some classic peroxidase substrates such as 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) and substituted phenols like 2,6-dimethoxyphenol, also the decolorization of the anthraquinonic dye Reactive Blue 5. The enzyme also catalyzes bleaching of natural colorants such as ß-carotene and annatto. Surprisingly, ß-carotene was transformed in the presence and absence of H2O2 by rPsaDyP, however enzyme activity was increased by the addition of H2O2. This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family.

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.

Separation of active laccases from Pleurotus sapidus culture supernatant using aqueous two-phase systems in centrifugal partition chromatography.

For the production of bio active compounds, e.g., active enzymes or antibodies, a conserved purification process with a minimum loss of active compounds is necessary. In centrifugal partition chromatography (CPC), the separation effect is based on the different distribution of the components to be separated between two immiscible liquid phases. Thereby, one liquid phase is kept stationary in chambers by a centrifugal field and the mobile phase is pumped through via connecting ducts. Aqueous two phase systems (ATPS) are known to provide benign conditions for biochemical products and seem to be promising when used in CPC for purification tasks. However, it is not known if active biochemical compounds can "survive" the conditions in a CPC where strong shear forces can occur due to the two-phasic flow under centrifugal forces. Therefore, this aspect has been faced within this study by the separation of active laccases from a fermentation broth of Pleurotus sapidus. After selecting a suitable ATPS and operating conditions, the activity yield was calculated and the preservation of the active enzymes could be observed. Therefore, CPC could be shown as potentially suitable for the purification of bio-active compounds.

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.