Novel Catechol O-methyltransferases from Lentinula edodes Catalyze the Generation of Taste-Active Flavonoids.

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O-methyltransferase activity was identified in the mycelium of Lentinula edodes, which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O-methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli. The purified enzymes confirmed the biocatalytic O-methylation activity against targeted flavonoids containing catechol motifs.

The Health-Promoting Properties and Clinical Applications of Rice Bran Arabinoxylan Modified with Shiitake Mushroom Enzyme-A Narrative Review.

Rice bran arabinoxylan compound (RBAC) is derived from defatted rice bran hydrolyzed with Lentinus edodes mycelial enzyme. It has been marketed as a functional food and a nutraceutical with health-promoting properties. Some research has demonstrated this rice bran derivative to be a potent immunomodulator, which also possesses anti-inflammatory, antioxidant, and anti-angiogenic properties. To date, research on RBAC has predominantly focused on its immunomodulatory action and application as a complementary therapy for cancer. Nonetheless, the clinical applications of RBAC can extend beyond cancer therapy. This article is a narrative review of the research on the potential benefits of RBAC for cancer and other health conditions based on the available literature. RBAC research has shown it to be useful as a complementary treatment for cancer and human immunodeficiency virus infection. It can positively modulate serum glucose, lipid and protein metabolism in diabetic patients. Additionally, RBAC has been shown to ameliorate irritable bowel syndrome and protect against liver injury caused by hepatitis or nonalcoholic fatty liver disease. It can potentially ease symptoms in chronic fatigue syndrome and prevent the common cold. RBAC is safe to consume and has no known side effects at the typical dosage of 2-3 g/day. Nevertheless, further research in both basic studies and human clinical trials are required to investigate the clinical applications, mechanisms, and effects of RBAC.

Recombinant Lentinula edodes xylanase improved the hydrolysis and in vitro ruminal fermentation of soybean straw by changing its fiber structure.

Soybean straw cannot be efficiently degraded and utilized by ruminants due to the complex cross-linked structure among cellulose, hemicellulose, and lignin in its cell wall. Xylanase can degrade the xylan component of hemicellulose, destroy the xylan-lignin matrix and, consequently, would theoretically improve the hydrolysis effectiveness of cellulose. Therefore, this study was performed to investigate the effects of recombinant Lentinula edodes xylanase (rLeXyn11A) on fiber structure, hydrolysis, and in vitro ruminal fermentation of soybean straw. Treatment with rLeXyn11A enhanced the hydrolysis of soybean straw with an evident increase in productions of ribose, rhamnose, and xylose. Soybean straw treated by rLeXyn11A had lower hemicellulose content and greater cellulose and lignin contents. The rLeXyn11A could remove xylan, loosen unordered fibrous networks, enhance substrate porosity, and rearrange lignin, consequently increasing the exposure of cellulose and improving the cellulase hydrolysis of soybean straw. Supplemental rLeXyn11A stimulated the dry matter digestion, volatile fatty acids production, and microbial protein synthesis during in vitro ruminal incubation. This paper demonstrated that rLeXyn11A could strengthen the cellulase hydrolysis and in vitro ruminal fermentation of soybean straw by degrading xylan and changing fiber structure, showing its potential for improving the utilization of soybean straw in ruminants.

Characteristics of a recombinant Lentinula edodes endoglucanase and its potential for application in silage of rape straw.

An experiment was conducted to determine the characteristics of recombinant endoglucanase and its effects on rape straw silage. The endoglucanase from Lentinula edodes (LeCel12A) was produced in Pichia pastoris and shown maximum activity at 40 °C and pH 3.0. The LeCel12A exhibited preferential hydrolysis of carboxymethylcellulose. The activity of LeCel12A could be enhanced by MnCl2 in dose-dependent manners. Trp22 was a key amino acid affecting LeCel12A activity. The LeCel12A enhanced the hydrolysis of rape straw, rice straw, wheat straw, and corn straw. Supplemental LeCel12A increased lactic acid concentration and reduced lignocellulosic content of the rape straw silage. Though an increase in the saccharification efficiency of LeCel12A-treated rape straw silage was observed when the fibrolytic enzyme loading of hydrolysis system was enough, supplemental LeCel12A did not dramatically enhance the saccharification of rape straw silage in the current study. This study demonstrates that LeCel12A may be useful for improving the utilization of rape straw silage as an additive, but its supplemental dose, cost benefit, and consequent application possibility in biofuel production require careful consideration and further investigation.

Isolation and identification of the umami peptides from shiitake mushroom by consecutive chromatography and LC-Q-TOF-MS.

Umami is critical to the taste of shiitake mushroom. To isolate and identify umami peptides, fractions from hydrolyzed dried shiitake mushroom were separated by ultrafiltration, gel filtration chromatography (GFC), and reversed-phase high-performance liquid chromatography (RP-HPLC). Separations were combined with sensory evaluations (grading and taste dilution analysis) and analysis of electronic tongue, which were used to identify the most umami component in shiitake mushroom. Low-molecular-weight fractions (MW < 3 kDa) have the strongest flavor in the shiitake mushroom hydrolysate. In the 3 subfractions separated from low-molecular-weight fractions (MW < 3 kDa) by GFC, the second subfraction (F2) was selected for RP-HPLC analysis. The first peak (G1) in RP-HPLC was identified by LC-Q-TOF-MS, and 2 tripeptides and 3 dipeptides were identified. The amino acid sequence of these peptides were Gly-Cys-Gly, Glu-Pro-Glu, Cys-Met, Val-Phe, and Gly-Glu.

Expression of a recombinant Lentinula edodes cellobiohydrolase by Pichia pastoris and its effects on in vitro ruminal fermentation of agricultural straws.

An experiment was conducted to determine the effects of recombinant cellobiohydrolase on the hydrolysis and in vitro rumen microbial fermentation of agricultural straws including rice straw, wheat straw, and corn straw. The cellobiohydrolase from Lentinula edodes (LeCel7A) was produced in Pichia pastoris. The optimal temperature and pH for LeCel7A were 60 °C and 5.0, respectively. The recombinant protein enhanced the hydrolysis of three straws. During in vitro rumen fermentation of three straws, the fiber digestibility, concentration of acetate and total volatile fatty acids, and fermentation liquid microbial protein were increased by LeCel7A. High throughput sequencing and real-time PCR data showed that the effects of LeCel7A on ruminal microbial community depended on the fermentation substrates. The relative abundances of Prevotellaceae_UCG_003 and Saccharofermentans were increased by LeCel7A regardless of agricultural straws. With rice straw, LeCel7A increased the relative abundances of Desulfovibrio, Ruminococcaceae and its some genus. With wheat straw, LeCel7A increased the relative abundances of Succiniclasticum, Ruminococcus flavefaciens, and Ruminococcus albus. With corn straw, Succiniclasticum, Christensenellaceae_R_7_group and Desulfovibrio were increased by LeCel7A. This study demonstrates that LeCel7A could enhance the hydrolysis and in vitro ruminal fermentation of agricultural straws, showing the potential of LeCel7A for improving the utilization of agricultural straws in ruminants.

Overexpression and repression of the tyrosinase gene in Lentinula edodes using the pChG vector.

Tyrosinase is an industrially useful enzyme, however, it causes gill browning of Lentinula edodes fruiting bodies during preservation. In this study, we constructed two vectors, pChG-gTs and pChG-gTa, expressing sense and antisense tyrosinase gene of L. edodes, respectively, using promoters derived from the glyceraldehyde-3-phosphate dehydrogenase gene. The host strain SR-1 of L. edodes was selected because of its fast growth, high protoplast yield, and high regeneration rate. Upon transformation of the host strain SR-1 with the pChG-gTs vector, a clone with 3.6-fold and 14.5-fold higher tyrosinase activity in vegetative mycelia and in fresh gills, respectively, than that of the host strain was obtained from nine transformants. Similarly, two clones containing the pChG-gTa vector with effectively repressed tyrosinase gene expression in vegetative mycelia and gills during the late stage of post-harvest preservation of fruiting bodies were obtained from 10 transformants. However, it remained unclear whether repression of the tyrosinase gene prevented gill browning, as the host strain also showed less browning than a commercial strain. Thus, this study highlights the usefulness of the pChG vector in expressing homologous enzyme coding genes in the vegetative mycelia and fruiting bodies of L. edodes.

A protease-resistant α-galactosidase characterized by relatively acid pH tolerance from the Shitake Mushroom Lentinula edodes.

A monomeric α-galactosidase with a molecular weight of 64 kDa was purified from fresh fruiting bodies of Lentinula edodes. The purification protocol involved ion-exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a final gel-filtration on Superdex 75. The purified α-galactosidase (LEGI) was identified by LC-MS/MS. It demonstrated the optimum pH of 5.0 and temperature optimum of 60 °C towards pNPGal. It was inhibited by Cd2+, Fe3+, Pb2+, Zn2+, Al3+, Hg2+, Cr2+, Ba2+. The LEGI activity was strongly abolished by the chemical modification N-bromosuccinimide (NBS) at 1 mM, while significantly enhanced by the thiol-reducing agents dithiothreitol (DTT). Moreover, LEGI showed strong resistance to protease pepsin, papain, acid protease and neutral protease. LEGI demonstrated hydrolysis towards melibiose (13.27%), raffinose (4.75%), stachyose (2.58%), locust bean gum (0.82%) and guar gum (1.29%). The Km values of LEGI for pNPGal, stachyose, raffinose, and melibiose were found to be 1.08, 17.24, 13.80 and 8.05 mM, respectively. Results suggest that LEGI demonstrates potential for elimination of indigestible oligosaccharides.

Site-Directed Mutagenesis of a ß-Glycoside Hydrolase from Lentinula Edodes.

The ß-glycoside hydrolases (LXYL-P1-1 and LXYL-P1-2) from Lentinula edodes (strain M95.33) can specifically hydrolyze 7-ß-xylosyl-10-deacetyltaxol (XDT) to form 10-deacetyltaxol for the semi-synthesis of Taxol. Our previous study showed that both the I368T mutation in LXYL-P1-1 and the T368E mutation in LXYL-P1-2 could increase the enzyme activity, which prompted us to investigate the effect of the I368E mutation on LXYL-P1-1 activity. In this study, the ß-xylosidase and ß-glucosidase activities of LXYL-P1-1I368E were 1.5 and 2.2 times higher than those of LXYL-P1-1. Most importantly, combination of I368E and V91S exerted the cumulative effects on the improvement of the enzyme activities and catalytic efficiency. The ß-xylosidase and ß-glucosidase activities of the double mutant LXYL-P1-1V91S/I368E were 3.2 and 1.7-fold higher than those of LXYL-P1-1I368E. Similarly, the catalytic efficiency of LXYL-P1-1V91S/I368E on 7-ß-xylosyl-10-deacetyltaxol was 1.8-fold higher than that of LXYL-P1-1I368E due to the dramatic increase in the substrate affinity. Molecular docking results suggest that the V91S and I368E mutation might positively promote the interaction between enzyme and substrate through altering the loop conformation near XDT and increasing the hydrogen bonds among Ser91, Trp301, and XDT. This study lays the foundation for exploring the relationship between the structure and function of the ß-glycoside hydrolases.

Bioavailability of Compounds Susceptible to Enzymatic Oxidation Enhances Growth of Shiitake Medicinal Mushroom (Lentinus edodes) in Solid-State Fermentation with Vineyard Prunings.

Grapes are widely produced in northwestern Mexico, generating many wood trimmings (vineyard prunings) that have no further local use. This makes vineyard prunings a very attractive alternative for the cultivation of white-rot medicinal mushrooms such as Lentinus edodes. This type of wood can also offer a model for the evaluation of oxidative enzyme production during the fermentation process. We tested the effect of wood from vineyard prunings on the vegetative growth of and production of ligninolytic enzymes in L. edodes in solid-state fermentation and with wheat straw as the control substrate. The specific growth rate of the fungus was 2-fold higher on vineyard pruning culture (µM = 0.95 day-1) than on wheat straw culture (µM = 0.47 day-1). Laccase-specific production was 4 times higher in the vineyard prunings culture than on wheat straw (0.34 and 0.08 mU · mg protein-1 · ppm CO2-1, respectively), and manganese peroxidase production was 3.7 times higher on wheat straw culture than on vineyard prunings (2.21 and 0.60 mU · mg protein-1 · ppm CO2-1, respectively). To explain accurately these differences in growth and ligninolytic enzyme activity, methanol extracts were obtained from each substrate and characterized. Resveratrol and catechins were the main compounds identified in vineyard prunings, whereas epigallocatechin was the only one detected in wheat straw. Compounds susceptible to enzymatic oxidation are more bioavailable in vineyard prunings than in wheat straw, and thus the highest L. edodes growth rate is associated with the presence of these compounds.

Purification and Antithrombotic Potential of a Fibrinolytic Enzyme from Shiitake Culinary- Medicinal Mushroom, Lentinus edodes GNA01 (Agaricomycetes).

We purified Lentinus edodes GNA01 fibrinolytic enzyme (LEFE) and identified it as a novel metalloprotease. LEFE was purified to homogeneity through a 2-step procedure, with an 8.28-fold increase in specific activity and 5.3% recovery. The molecular mass of LEFE was approximately 38 kDa, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its optimal pH, optimal temperature, pH stability, and thermal stability were 5, 30°C, 6-7, and 40°C, respectively. LEFE was inhibited by zinc and magnesium ions, and by EDTA and EGTA, indicating that LEFE is a metalloprotease. The protease exhibited fibrinolytic activity and a degradative effect on clot formation and blood clots. The protease prolonged activated partial thromboplastin time, prothrombin time, and coagulation time as induced by platelet aggregators (collagen and epinephrine). Taken together, our results indicate that L. edodes GNA01 produces a metalloprotease/fibrinolytic enzyme and that this enzyme might be applied as a new thrombolytic and antithrombotic agent for thrombosis-related cardiovascular disorders.

Brown Mycelial Mat as an Essential Morphological Structure of the Shiitake Medicinal Mushroom Lentinus edodes (Agaricomycetes).

We show here, to our knowledge for the first time, that the brown mycelial mat of the xylotrophic shiitake medicinal mushroom, Lentinus edodes, not only performs a protective function owing to significant changes in the ultrastructure (thickening of the cell wall, increased density, and pigmentation of the fungal hyphae) but also is a metabolically active stage in the development of the mushroom. The cells of this morphological structure exhibit repeated activation of expression of the genes lcc4, tir, exp1, chi, and exg1, coding for laccase, tyrosinase, a specific transcription factor, chitinase, and glucanase, which are required for fungal growth and morphogenesis. This study revealed the maximum activity of functionally important proteins with phenol oxidase and lectin activities, and the emergence of additional laccases, tyrosinases, and lectins, which are typical of only this stage of morphogenesis and have a regulatory function in the development and formation of fruiting bodies.

Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes.

Lentinula edodes (shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain.

Comparative secretomic analysis of lignocellulose degradation by Lentinula edodes grown on microcrystalline cellulose, lignosulfonate and glucose.

Lentinula edodes has the potential to degrade woody and nonwoody lignocellulosic biomass. However, the mechanism of lignocellulose degradation by L. edodes is unclear. The aim of this work is to explore the profiling of soluble secreted proteins involved in lignocellulose degradation in L. edodes. For that, we compared the secretomes of L. edodes grown on microcrystalline cellulose, cellulose with lignosulfonate and glucose. Based on nanoliquid chromatography coupled with tandem mass spectrometry of whole-protein hydrolysate, 230 proteins were identified. Label-free proteomic analysis showed that the most abundant carbohydrate-active enzymes involved in polysaccharide hydrolysis were endo-ß-1,4-glucanase, α-galactosidase, polygalacturonase and glucoamylase in both cellulosic secretomes. In contrast, enzymes involved in lignin degradation were most abundant in glucose culture, with laccase 1 being the predominant protein (13.13%). When the cellulose and cellulose with lignosulfonate secretomes were compared, the abundance of cellulases and hemicellulases was higher in cellulose with lignosulfonate cultures, which was confirmed by enzyme activity assays. In addition, qRT-PCR analysis demonstrated that the expression levels of genes encoding cellulases and hemicellulases were significantly increased (by 32.2- to 1166.7-fold) when L. edodes was grown in cellulose with lignosulfonate medium. BIOLOGICAL SIGNIFICANCE: In this article, the secretomes of L. edodes grown on three different carbon sources were compared. The presented results revealed the profiling of extracellular enzymes involved in lignocellulose degradation, which is helpful to further explore the mechanism of biomass bioconversion by L. edodes.

Alteration in the ultrastructural morphology of mycelial hyphae and the dynamics of transcriptional activity of lytic enzyme genes during basidiomycete morphogenesis.

The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological-biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was activated. These genes encode enzymes such as tyrosinase, chitinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.

Hydrolytic enzyme activities in shiitake mushroom (Lentinula edodes) strains cultivated on coffee pulp.

Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate.

Actividad de las enzimas hidrolíticas en cepas del hongo shiitake (Lentinula edodes) cultivadas en pulpa de café / Hydrolytic enzyme activities in shiitake mushroom (Lentinula edodes) strains cultivated on coffee pulp

Se estudió la producción de enzimas hidrolíticas (celulasas, laminarinasas y xilanasas) en cultivos de Lentinula edodes en pulpa de café estéril. Se tomaron muestras de sustrato colonizado por el micelio después de 7, 14, 21, 28 y 35 días de incubación a 25°C (W1 a W5) y durante el período de fructificación en diferentes etapas: formación de primordios (PF), primera cosecha (H) y una semana después de la primera cosecha (PH). La actividad enzimática fue menor al inicio del crecimiento micelial y mostró mayores niveles en la formación y el desarrollo de basidiomas. Durante la etapa reproductiva del hongo, las muestras se sometieron a un tratamiento de remojo. Sin embargo, no fue posible relacionar este tratamiento con el aumento de la producción de enzimas. Los niveles de actividad enzimática sugieren que la secreción de las enzimas estudiadas no influye en la capacidad de adaptación de las cepas al sustrato

Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate

[Role of the NO Synthase System in Response to Abiotic Stress Factors for Basidiomycetes Lentinula edodes and Grifola frondosa].

Effect of stressors (unfavorable pH and temperature or carbon and nitrogen limitation) on the synthesis of the components of the NO synthase signaling system was studied in submerged cultures of xylotrophic basidiomycetes Lentinula edodes and Grifola frondosa. Marker compounds of the NO synthase signaling system were found in both cultures. A simultaneous increase of the concentrations of NO and citrulline in the culture liquid of the basidiomycetes grown at superoptimal pH and in nitrogen-limited medium indicates the activation of the NO synthase signaling system under such stress conditions.

Application of residual polysaccharide-degrading enzymes in dried shiitake mushrooms as an enzyme preparation in food processing.

OBJECTIVE: To examine the activities of residual enzymes in dried shiitake mushrooms, which are a traditional foodstuff in Japanese cuisine, for possible applications in food processing. RESULTS: Polysaccharide-degrading enzymes remained intact in dried shiitake mushrooms and the activities of amylase, ß-glucosidase and pectinase were high. A potato digestion was tested using dried shiitake powder. The enzymes reacted with potato tuber specimens to solubilize sugars even under a heterogeneous solid-state condition and that their reaction modes were different at 38 and 50 °C. CONCLUSION: Dried shiitake mushrooms have a potential use in food processing as an enzyme preparation.

Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii.

Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, ß-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.