Role and structure of the small subunit forming heterodimers with laccase-like enzymes.

Unlike laccases sensu stricto, which are usually monomeric enzymes, laccase-like enzymes recently re-classified as Novel Laccases (NLACs) are characterized by the formation of heterodimers with small proteins (subunits) of unknown function. Here the NLAC from Pleurotus eryngii (PeNL) and a small protein selected from the fungal genome, that is homologous to reported POXA3 from Pleurotus ostreatus, were produced in Aspergillus oryzae separately or together. The two proteins interacted regardless of whether the small subunit was co-expressed or exogenously added to the enzyme. The stability and catalytic activity of PeNL was significantly enhanced in the presence of the small subunit. Size exclusion chromatography-multi angle light scattering (SEC-MALS) analysis confirmed that the complex PeNL-ss is a heterodimer of 77.4 kDa. The crystallographic structure of the small protein expressed in Escherichia coli was solved at 1.6 Å resolution. This is the first structure elucidated of a small subunit of a NLAC. The helix bundle structure of the small subunit accommodates well with the enzyme model structure, including interactions with specific regions of NLACs and some amino acid residues of the substrate-binding loops.

Efficient degradation of various emerging pollutants by wild type and evolved fungal DyP4 peroxidases.

The accumulation of emerging pollutants in the environment remains a major concern as evidenced by the increasing number of reports citing their potential risk on environment and health. Hence, removal strategies of such pollutants remain an active area of investigation. One way through which emerging pollutants can be eliminated from the environment is by enzyme-mediated bioremediation. Enzyme-based degradation can be further enhanced via advanced protein engineering approaches. In the present study a sensitive and robust bioanalytical liquid chromatography-tandem mass spectrometry (LCMSMS)-based approach was used to investigate the ability of a fungal dye decolorizing peroxidase 4 (DyP4) and two of its evolved variants-that were previously shown to be H2O2 tolerant-to degrade a panel of 15 different emerging pollutants. Additionally, the role of a redox mediator was examined in these enzymatic degradation reactions. Our results show that three emerging pollutants (2-mercaptobenzothiazole (MBT), paracetamol, and furosemide) were efficiently degraded by DyP4. Addition of the redox mediator had a synergistic effect as it enabled complete degradation of three more emerging pollutants (methyl paraben, sulfamethoxazole and salicylic acid) and dramatically reduced the time needed for the complete degradation of MBT, paracetamol, and furosemide. Further investigation was carried out using pure MBT to study its degradation by DyP4. Five potential transformation products were generated during the enzymatic degradation of MBT, which were previously reported to be produced during different bioremediation approaches. The current study provides the first instance of the application of fungal DyP4 peroxidases in bioremediation of emerging pollutants.

Two adjacent C-terminal mutations enable expression of aryl-alcohol oxidase from Pleurotus eryngii in Pichia pastoris.

Fungal aryl-alcohol oxidases (AAOs) are attractive biocatalysts because they selectively oxidize a broad range of aromatic and aliphatic allylic primary alcohols while yielding hydrogen peroxide as the only by-product. However, their use is hampered by challenging and often unsuccessful heterologous expression. Production of PeAAO1 from Pleurotus eryngii ATCC 90787 in Pichia pastoris failed, while PeAAO2 from P. eryngii P34 with an amino acid identity of 99% was expressed at high yields. By successively introducing mutations in PeAAO1 to mimic the sequence of PeAAO2, the double mutant PeAAO1 ER with mutations K583E and Q584R was constructed, that was successfully expressed in P. pastoris. Functional expression was enhanced up to 155 U/l via further replacements D361N (variant NER) or V367A (variant AER). Fed-batch cultivation of recombinant P. pastoris yielded up to 116 mg/l of active variants. Glycosylated PeAAO1 variants demonstrated high stability and catalytic efficiencies similar to PeAAO2. Interestingly, P. pastoris expressing PeAAO1 variant ER contained roughly 13 gene copies but showed similar volumetric activity as NER and AER with one to two gene copies and four times lower mRNA levels. Additional H-bonds and salt bridges introduced by mutations K583E and Q584R might facilitate heterologous expression by enhanced protein folding.Key points• PeAAO1 not expressed in P. pastoris and PeAAO2 well-expressed in Pichia differ at 7 positions.• Expression of PeAAO1 in P. pastoris achieved through mutagenesis based on PeAAO2 sequence.• Combination of K583E and Q584R is essential for expression of PeAAO1 in P. pastoris.

Mycoremediation of oil contaminant by Pleurotus florida (P.Kumm) in liquid culture.

This study investigated the potential functions of Pleurotus florida (an edible mushroom) in the biodegradation of gas oil at concentrations of 0 (control), 2.5, 5, and 10% (V: V) for 30 days. The gas oil increased dry weight and protein concentration in all treatments (by an average of 19.5 and 108%, respectively). Moreover, the pH, surface tension (ST), and interfacial tension (IFT) were reduced by the mushroom supplementation. The lowest surface tension (31.9 mN m-1) and the highest biosurfactant production belonged to the 10% gas oil treatment (0.845 ± 0.03 mg mL-1). The results demonstrated that the adsorption isotherm agreed well with the Langmuir isotherm. The maximum Langmuir adsorption capacity was calculated at 0.743 mg g-1 wet biomass of P. florida. The fungal supplementation efficiently remedied the total petroleum hydrocarbons (TPHs) by an average of 55% after 30 days. Gas chromatography (GC) analysis revealed that P. florida effectively detoxified C13-C28 hydrocarbons, Pristane, and Phytane, implying its high mycoremediation function. The toxicity test showed that mycoremediation increased the germination by an average of 35.82% ± 8.89 after 30 days. Laccase activity increased significantly with increasing gas oil concentration in the treatments. The maximum laccase activity was obtained in the 10% gas oil treatment (142.25 ± 0.72 U L-1). The presence of pollutants was also associated with induction in the tyrosinase activity when compared to the control. These results underline the high mycoremediation capacity of P. florida through the involvement of biosurfactants, laccase, and tyrosinase.

Alternative oxidase gene induced by nitric oxide is involved in the regulation of ROS and enhances the resistance of Pleurotus ostreatus to heat stress.

BACKGROUND: In China, during the cultivation process of Pleurotus ostreatus, the yield and quality of fruiting bodies are easily affected by high temperatures in summer. Nitric oxide (NO) plays an important regulatory role in the response to abiotic stress, and previous studies have found that NO can induce alternative oxidase (aox) experssion in response to heat stress (HS) by regulating aconitase. However, the regulatory pathway of NO is complex, and the function and regulation of the aox gene in the response to HS remain unclear. RESULTS: In this study, we found that NO affected nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) levels, reduced hydrogen peroxide (H2O2) and superoxide anion (O2-) contents, and slowed O2- production. Further RNA-Seq results showed that NO regulated the oxidation-reduction process and oxidoreductase activity, affected the cellular respiration pathway and activated aox gene expression. The function of aox was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the OE-aox strains exhibited obviously improved growth recovery after exposure to HS. During exposure to HS, the OE-aox strains exhibited reduced levels of NADH, the product of the tricarboxylic acid (TCA) cycle, and decreased synthesis of ATP, which reduced the production and accumulation of reactive oxygen species (ROS), whereas the RNAi-aox strains exhibited the opposite result. In addition, aox mediated the expression of antioxidant enzyme genes in the mycelia of P. ostreatus under HS through the retrograde signaling pathway. CONCLUSIONS: This study shows that the expression of the aox gene in P. ostreatus mycelia can be induced by NO under HS, that it regulates the TCA cycle and cell respiration to reduce the production of ROS, and that it can mediate the retrograde signaling pathway involved in the mycelial response to HS.

Co-Oxidative Transformation of Piperine to Piperonal and 3,4-Methylenedioxycinnamaldehyde by a Lipoxygenase from Pleurotus sapidus.

The valuable aroma compound piperonal with its vanilla-like olfactory properties is of high interest for the fragrance and flavor industry. A lipoxygenase (LOXPsa 1) of the basidiomycete Pleurotus sapidus was identified to convert piperine, the abundant pungent principle of black pepper (Piper nigrum), to piperonal and a second volatile product, 3,4-methylenedioxycinnamaldehyde, with a vanilla-like odor through an alkene cleavage. The reaction principle was co-oxidation, as proven by its dependence on the presence of linoleic or α-linolenic acid, common substrates of lipoxygenases. Optimization of the reaction conditions (substrate concentrations, reaction temperature and time) led to a 24-fold and 15-fold increase of the piperonal and 3,4-methylenedioxycinnamaldehyde concentration using the recombinant enzyme. Monokaryotic strains showed different concentrations of and ratios between the two reaction products.

Upcycling the Spent Mushroom Substrate of the Grey Oyster Mushroom Pleurotus pulmonarius as a Source of Lignocellulolytic Enzymes for Palm Oil Mill Effluent Hydrolysis.

Mushroom cultivation along with the palm oil industry in Malaysia have contributed to large volumes of accumulated lignocellulosic residues that cause serious environmental pollution when these agroresidues are burned. In this study, we illustrated the utilization of lignocellulolytic enzymes from the spent mushroom substrate of Pleurotus pulmonarius for the hydrolysis of palm oil mill effluent (POME). The hydrolysate was used for the production of biohydrogen gas and enzyme assays were carried out to determine the productivities/activities of lignin peroxidase, laccase, xylanase, endoglucanase and ß-glucosidase in spent mushroom substrate. Further, the enzyme cocktails were concentrated for the hydrolysis of POME. Central composite design of response surface methodology was performed to examine the effects of enzyme loading, incubation time and pH on the reducing sugar yield. Productivities of the enzymes for xylanase, laccase, endoglucanase, lignin peroxidase and ß-glucosidase were 2.3, 4.1, 14.6, 214.1, and 915.4 U g-1, respectively. A maximum of 3.75 g/l of reducing sugar was obtained under optimized conditions of 15 h incubation time with 10% enzyme loading (v/v) at a pH of 4.8, which was consistent with the predicted reducing sugar concentration (3.76 g/l). The biohydrogen cumulative volume (302.78 ml H2.L-1 POME) and 83.52% biohydrogen gas were recorded using batch fermentation which indicated that the enzymes of spent mushroom substrate can be utilized for hydrolysis of POME.

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.

Self-assembling thermostable chimeras as new platform for arsenic biosensing.

The correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either for the realization of biosensors, as well as to guarantee the efficacy of the developed biomaterials. In this work we produced two versions of a chimeric protein, namely ArsC-Vmh2 and Vmh2-ArsC, which combined the self-assembling properties of Vmh2, a hydrophobin from Pleurotus ostreatus, with that of TtArsC, a thermophilic arsenate reductase from Thermus thermophilus; both chimeras were heterologously expressed in Escherichia coli and purified from inclusion bodies. They were characterized for their enzymatic capability to reduce As(V) into As(III), as well as for their immobilization properties on polystyrene and gold in comparison to the native TtArsC. The chimeric proteins immobilized on polystyrene can be reused up to three times and stored for 15 days with 50% of activity loss. Immobilization on gold electrodes showed that both chimeras follow a classic Langmuir isotherm model towards As(III) recognition, with an association constant (KAsIII) between As(III) and the immobilized enzyme, equal to 650 (± 100) L mol-1 for ArsC-Vmh2 and to 1200 (± 300) L mol-1 for Vmh2-ArsC. The results demonstrate that gold-immobilized ArsC-Vmh2 and Vmh2-ArsC can be exploited as electrochemical biosensors to detect As(III).

Characterization of a Novel Laccase LAC-Yang1 from White-Rot Fungus Pleurotus ostreatus Strain Yang1 with a Strong Ability to Degrade and Detoxify Chlorophenols.

In this study, a laccase LAC-Yang1 was successfully purified from a white-rot fungus strain Pleurotus ostreatus strain yang1 with high laccase activity. The enzymatic properties of LAC-Yang1 and its ability to degrade and detoxify chlorophenols such as 2,6-dichlorophenol and 2,3,6-trichlorophenol were systematically studied. LAC-Yang1 showed a strong tolerance to extremely acidic conditions and strong stability under strong alkaline conditions (pH 9-12). LAC-Yang1 also exhibited a strong tolerance to different inhibitors (EDTA, SDS), metal ions (Mn2+, Cu2+, Mg2+, Na+, K+, Zn2+, Al3+, Co2+, and metal ion mixtures), and organic solvents (glycerol, propylene glycol). LAC-Yang1 showed good stability in the presence of Mg2+, Mn2+, glycerol, and ethylene glycol. Our results reveal the strong degradation ability of this laccase for high concentrations of chlorophenols (especially 2,6-dichlorophenol) and chlorophenol mixtures (2,6-dichlorophenol + 2,3,6-trichlorophenol). LAC-Yang1 displayed a strong tolerance toward a variety of metal ions (Na2+, Zn2+, Mn2+, Mg2+, K+ and metal ion mixtures) and organic solvents (glycerol, ethylene glycol) in its degradation of 2,6-dichlorophenol and 2,3,6-trichlorophenol. The phytotoxicity of 2,6-dichlorophenol treated by LAC-Yang1 was significantly reduced or eliminated. LAC-Yang1 demonstrated a good detoxification effect on 2,6-dichlorophenol while degrading this compound. In conclusion, LAC-Yang1 purified from Pleurotus ostreatus has great application value and potential in environmental biotechnology, especially the efficient degradation and detoxification of chlorophenols.

Enzyme Cascade Reaction Involving Lytic Polysaccharide Monooxygenase and Dye-Decolorizing Peroxidase for Chitosan Functionalization.

In situ H2O2 generation systems are efficient for H2O2-dependent biocatalytic oxidation reactions. Here, we report that lytic polysaccharide monooxygenases (LPMOs), copper-dependent polysaccharide monooxygenases, can efficiently supply H2O2 in situ to dye-decolorizing peroxidases (DyPs) using substrate gallic acid (GA) for chitosan functionalization. The maximum grafting ratio induced by the cascade reaction was significantly higher than that achieved by a reaction with initial exogenous H2O2. The maximum grafting ratio was obtained with 12 g/L GA, 5.6 mg/L DyP, 20-30 mg/L LPMO, and pH 4.5-5.0. UV-vis, Fourier transform infrared (FT-IR), and nuclear magnetic resonance (1H NMR) spectroscopy confirmed GA grafting onto chitosan. X-ray diffraction (XRD) analysis and thermogravimetric analysis (TGA) indicated that GA-chitosan conjugates had lower thermal stability and crystallinity than chitosan. The GA-chitosan conjugates had significantly higher antioxidant activity than chitosan. This study supplies a green and high-efficiency approach to achieve an enzymatic cascade reaction for chitosan functionalization and has potential applications in H2O2-dependent biocatalytic oxidation reactions.

TMT-MS/MS proteomic analysis of the carbohydrate-active enzymes in the fruiting body of Pleurotus tuoliensis during storage.

BACKGROUND: The fruiting body of Pleurotus tuoliensis deteriorates rapidly after harvest, causing a decline in its commercial value and a great reduction in its shelf life. According to the present research, carbohydrate-active enzymes (CAZymes) may cause the softening, liquefaction and autolysis of mature mushrooms after harvest. To further understand the in vivo molecular mechanism of CAZymes affecting the postharvest quality of P. tuoliensis fruiting bodies, a tandem mass tags labelling combined liquid chromatography-tandem mass spectrometry (TMT-MS/MS) proteomic analysis was performed on P. tuoliensis fruiting bodies during storage at 25 °C. RESULTS: A total of 4737 proteins were identified, which had at least one unique peptide and had a confidence level above 95%. Consequently, 1307 differentially expressed proteins (DEPs) were recruited using the criteria of abundance fold change (FC) >1.5 or < 0.67 and P < 0.05. The identified proteins were annotated by dbCAN2, a meta server for automated CAZymes annotation. Subsequently, 222 CAZymes were obtained. Several CAZymes participating in the cell wall degradation process, including ß-glucosidase, glucan 1,3-ß-glucosidase, endo-1,3(4)-ß-glucanase and chitinases, were significantly upregulated during storage. The protein expression level of CAZymes, such as xylanase, amylase and glucoamylase, were upregulated significantly, which may participate in the P. tuoliensis polysaccharide degradation. CONCLUSIONS: The identified CAZymes degraded the polysaccharides and lignin, destroying the cell wall structure, preventing cell wall remodeling, causing a loss of nutrients and the browning phenomenon, accelerating the deterioration of P. tuoliensis fruiting body. © 2020 Society of Chemical Industry.

Enhanced konjac glucomannan hydrolysis by lytic polysaccharide monooxygenases and generating prebiotic oligosaccharides.

Konjac glucomannan (KGM) hydrolysate is a potentially valuable prebiotic that could improve gastrointestinal health by modulating the growth of probiotic bacteria and by promoting the production of short-chain fatty acids (SCFAs). In this study, we used lytic polysaccharide monooxygenases (LPMOs) to produce oligosaccharides from KGM and studied their prebiotic functions. The LPMO from Pleurotus ostreatus (PoLPMO9D) was shown to efficiently depolymerize KGM and produce a broad range of small oligomers. PoLPMO9D showed maximal activities at 50-60 °C and pH 4.0. When KGM-depolymerizing products produced by PoLPMO9D were employed as the carbon source instead of untreated KGM polymers, the growth of faecal microbiota was 2.76 times higher, a significant increase in the genus Lactococcus was observed, and the production of SCFAs increased by 14.6-fold with a significant pH decrease. This study shows that LPMOs may be a promising alternative enzyme for depolymerizing polysaccharide to prepare prebiotics from KGM.

A water-soluble selenium-enriched polysaccharide produced by Pleurotus ostreatus: Purification, characterization, antioxidant and antitumor activities in vitro.

The development and application of new selenium-enriched polysaccharides has become a critical topic in recent years. In this study, a natural selenium-enriched polysaccharide fraction (Se-POP-21) produced by Pleurotus ostreatus was purified, characterized, and investigated the antioxidant and antitumor activities in vitro. The Se-POP-21 was mainly composed of mannose, glucose, galactose and arabinose, with a molar ratio of 18.01:2.40:26.15:7.34, of which molecular weight was 15,888 Da and the selenium content was 5.31 µg/g. Spectral analysis demonstrated that Se-POP-21 represented a non-triple helix pyranopolysaccharide and selenium occurred in the form of C-O-Se and SeO. Molecular size and morphology studies showed that Se-POP-21 exhibited a spherical shape with a particle size distribution between 100 and 200 nm, even though Se-POP-21 aggregates were also found with a size between 500 and 600 nm. In addition, Se-POP-21 showed strong scavenging capacity to DPPH and hydroxyl radical. More, cell experiments showed that Se-POP-21 could reduce viability of A549, SKOV3, HepG2 and MCF-7 cells, induce apoptosis and inhibit metastasis of A549 cells. A potential mechanism was that Se-POP-21 inhibited the epithelial-to-mesenchymal transition of cancer cells. Se-POP-21 featured no significant effect on normal cells. Se-POP-21 showed great potential to develop into a natural antioxidant or low-toxic antitumor drug.

Preparation of Antioxidant Protein Hydrolysates from Pleurotus geesteranus and Their Protective Effects on H2O2 Oxidative Damaged PC12 Cells.

Pleurotus geesteranus is a promising source of bioactive compounds. However, knowledge of the antioxidant behaviors of P. geesteranus protein hydrolysates (PGPHs) is limited. In this study, PGPHs were prepared with papain, alcalase, flavourzyme, pepsin, and pancreatin, respectively. The antioxidant properties and cytoprotective effects against oxidative stress of PGPHs were investigated using different chemical assays and H2O2 damaged PC12 cells, respectively. The results showed that PGPHs exhibited superior antioxidant activity. Especially, hydrolysate generated by alcalase displayed the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (91.62%), 2,2-azino-bis (3-ethylbenzothia zoline-6-sulfonic acid) (ABTS) radical scavenging activity (90.53%), ferric reducing antioxidant power, and metal ion-chelating activity (82.16%). Analysis of amino acid composition revealed that this hydrolysate was rich in hydrophobic, negatively charged, and aromatic amino acids, contributing to its superior antioxidant properties. Additionally, alcalase hydrolysate showed cytoprotective effects on H2O2-induced oxidative stress in PC12 cells via diminishing intracellular reactive oxygen species (ROS) accumulation by stimulating antioxidant enzyme activities. Taken together, alcalase hydrolysate of P. geesteranus protein can be used as beneficial ingredients with antioxidant properties and protective effects against ROS-mediated oxidative stress.

The ribotoxin-like protein Ostreatin from Pleurotus ostreatus fruiting bodies: Confirmation of a novel ribonuclease family expressed in basidiomycetes.

Fungi produce several toxins active against plants, animal or humans. Among them, ribotoxins are enzymes that specifically attack ribosomes irreparably compromising protein synthesis, useful as insecticides or as anticancer agents. Here, a novel ribotoxin from the edible mushroom Pleurotus ostreatus has been purified and characterized. This ribotoxin, named Ostreatin, is a specific ribonuclease releasing α-fragment when incubated with yeast or rabbit ribosomes. Ostreatin shows IC50 of 234 pM in rabbit reticulocyte lysate, and metal dependent endonuclease activity. Following the completion of Ostreatin primary structure, we ascertained that this toxin is homologous to Ageritin, the first ribotoxin-like protein from the basidiomycete Agrocybe aegerita, with which it shares 38.8% amino acid sequence identity. Ostreatin consists of 131 amino acid residues with an experimental molecular mass of 14,263.51 Da ([M+H+]+). Homology modeling revealed that Ostreatin and Ageritin share a similar fold in which the common catalytic triad is conserved. Purified Ostreatin lacks N-terminal and C-terminal peptides, which instead are present in the Ostreatin coding sequence. Such peptides are probably involved in protein sorting and for this they could be removed. Our findings confirm the presence of ribotoxin-like proteins in basidiomycetes edible mushrooms, that we propose as novel tool for biotechnological applications.

Glucose counteracts wood-dependent induction of lignocellulolytic enzyme secretion in monokaryon and dikaryon submerged cultures of the white-rot basidiomycete Pleurotus ostreatus.

The secretome complexity and lignocellulose degrading capacity of Pleurotus ostreatus monokaryons mkPC9 and mkPC15 and mated dikaryon dkN001 were studied in submerged liquid cultures containing wood, glucose, and wood plus glucose as carbon sources. The study revealed that this white-rot basidiomycete attacks all the components of the plant cell wall. P. ostreatus secretes a variety of glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases, especially when wood is the only carbon source. The presence of wood increased the secretome complexity, whereas glucose diminished the secretion of enzymes involved in cellulose, hemicellulose and pectin degradation. In contrast, the presence of glucose did not influence the secretion of redox enzymes or proteases, which shows the specificity of glucose on the secretion of cellulolytic enzymes. The comparison of the secretomes of monokaryons and dikaryons reveals that secretome complexity is unrelated to the nuclear composition of the strain.

Transcriptional shifts in delignification-defective mutants of the white-rot fungus Pleurotus ostreatus.

White-rot fungi efficiently degrade lignin and, thus, play a pivotal role in the global carbon cycle. However, the mechanisms of lignin degradation are largely unknown. Recently, mutations in four genes, namely wtr1, chd1, pex1, and gat1, were shown to abrogate the wood lignin-degrading ability of Pleurotus ostreatus. In this study, we conducted a comparative transcriptome analysis to identify genes that are differentially expressed in ligninolysis-deficient mutant strains. Putative ligninolytic genes that are highly expressed in parental strains are significantly downregulated in the mutant strains. On the contrary, many putative cellulolytic and xylanolytic genes are upregulated in the chd1-1, Δpex1, and Δgat1 strains. Identifying transcriptional alterations in mutant strains could provide new insights into the regulatory mechanisms of lignocellulolytic genes in P. ostreatus.

Crosslinked Enzyme Aggregates (CLEAs) of Laccases from Pleurotus citrinopileatus Induced in Olive Oil Mill Wastewater (OOMW).

The enzymatic factory of ligninolytic fungi has proven to be a powerful tool in applications regarding the degradation of various types of pollutants. The degradative potential of fungi is mainly due to the production of different types of oxidases, of which laccases is one of the most prominent enzymatic activities. In the present work, crude laccases from the supernatant of Pleurotus citrinopileatus cultures grown in olive oil mill wastewater (OOMW) were immobilized in crosslinked enzyme aggregates (CLEAs), aiming at the development of biocatalysts suitable for the enzymatic treatment of OOMW. The preparation of laccase CLEAs was optimized, resulting in a maximum of 72% residual activity. The resulting CLEAs were shown to be more stable in the presence of solvents and at elevated temperatures compared to the soluble laccase preparation. The removal of the phenolic component of OOMW catalyzed by laccase-CLEAs exceeded 35%, while they were found to retain their activity for at least three cycles of repetitive use. The described CLEAs can be applied for the pretreatment of OOMW, prior to its use for valorization processes, and thus, facilitate its complete biodegradation towards a consolidated process in the context of 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.