ABSTRACT
Dye-decolorizing peroxidases (DyPs) belong to a novel superfamily of heme peroxidases that can oxidize recalcitrant compounds. In the current study, the GlDyP2 gene from Ganoderma lucidum was heterologously expressed in Escherichia coli, and the enzymatic properties of the recombinant GlDyP2 protein were investigated. The GlDyP2 protein could oxidize not only the typical peroxidase substrate ABTS but also two lignin substrates, namely guaiacol and 2,6-dimethoxy phenol (DMP). For the ABTS substrate, the optimum pH and temperature of GlDyP2 were 4.0 and 35 °C, respectively. The pH stability and thermal stability of GlDyP2 were also measured; the results showed that GlDyP2 could function normally in the acidic environment, with a T50 value of 51 °C. Moreover, compared to untreated controls, the activity of GlDyP2 was inhibited by 1.60 mM of Mg2+, Ni2+, Mn2+, and ethanol; 0.16 mM of Cu2+, Zn2+, methanol, isopropyl alcohol, and Na2EDTA·2H2O; and 0.016 mM of Fe2+ and SDS. The kinetic constants of recombinant GlDyP2 for oxidizing ABTS, Reactive Blue 19, guaiacol, and DMP were determined; the results showed that the recombination GlDyP2 exhibited the strongest affinity and the most remarkable catalytic efficiency towards guaiacol in the selected substrates. GlDyP2 also exhibited decolorization and detoxification capabilities towards several dyes, including Reactive Blue 19, Reactive Brilliant Blue X-BR, Reactive Black 5, Methyl Orange, Trypan Blue, and Malachite Green. In conclusion, GlDyP2 has good application potential for treating dye wastewater.
Subject(s)
Coloring Agents , Enzyme Stability , Escherichia coli , Guaiacol , Recombinant Proteins , Reishi , Temperature , Coloring Agents/metabolism , Coloring Agents/chemistry , Reishi/genetics , Reishi/enzymology , Reishi/metabolism , Hydrogen-Ion Concentration , Escherichia coli/genetics , Escherichia coli/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/chemistry , Guaiacol/metabolism , Guaiacol/analogs & derivatives , Biodegradation, Environmental , Kinetics , Benzothiazoles/metabolism , Substrate Specificity , Lignin/metabolism , Oxidation-Reduction , Peroxidase/genetics , Peroxidase/metabolism , Peroxidase/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungal Proteins/chemistry , Peroxidases/genetics , Peroxidases/metabolism , Peroxidases/chemistry , Water Pollutants, Chemical/metabolism , Azo Compounds/metabolism , Wastewater/microbiology , Wastewater/chemistry , Sulfonic Acids/metabolism , Anthraquinones , Rosaniline DyesABSTRACT
Ganoderma lingzhi is a traditional Chinese medicine that has been used to improve health and longevity for thousands of years. It is usually cultivated on hardwood log- or sawdust-based formulations. Conversely, in this study, we used Miscanthus sacchariflorus (MSF), M. floridulus, and M. sinensis (MSS), fast-growing perennial grasses widely distributed in China, for G. lingzhi cultivation. Mycelial growth rate, activities of lignin-degrading enzymes on colonized mushroom substrates, and expression levels of CAZymes and laccase genes based on different substrates were analyzed. Total triterpenoids, sterols, and polysaccharides content of fruiting bodies obtained from different substrates were investigated. The activities of laccase and manganese peroxidase in mycelia increased in the MSF- and MSS-based formulations compared with that in the sawdust-based formulation. The results of mycelial growth- and cultivation-related experiments showed that the Miscanthus substrates could be used as the substrates for cultivating G. lingzhi. The content of active ingredients, namely triterpenoids, sterols, and polysaccharides, in fruiting bodies cultivated on the Miscanthus substrates did not decrease compared with those in substrate obtained from the sawdust-based formulation. Therefore, the present study provides alternative substrates for the cultivation of G. lingzhi, and a reference for better utilization of inexpensive substrate in future.
Subject(s)
Reishi , Triterpenes , Laccase/genetics , Laccase/metabolism , Reishi/metabolism , Poaceae , Polysaccharides/metabolism , Sterols/metabolismABSTRACT
Ganoderma lucidum is an edible mushroom highly regarded in the traditional Chinese medicine. To better understand the molecular mechanisms underlying fruiting body development in G. lucidum, transcriptome analysis based on RNA sequencing was carried out on different developmental stages: mycelium (G1); primordium (G2); young fruiting body (G3); mature fruiting body (G4); fruiting body in post-sporulation stage (G5). In total, 26,137 unigenes with an average length of 1078 bp were de novo assembled. Functional annotation of transcriptomes matched 72.49% of the unigenes to known proteins available in at least one database. Differentially expressed genes (DEGs) were identified between the evaluated stages: 3135 DEGs in G1 versus G2; 120 in G2 versus G3; 3919 in G3 versus G4; and 1012 in G4 versus G5. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs identified in G1 versus G2 revealed that, in addition to global and overview maps, enriched pathways were related to amino acid metabolism and carbohydrate metabolism. In contrast, DEGs identified in G2 versus G3 were mainly assigned to the category of metabolism of amino acids and their derivatives, comprising mostly upregulated unigenes. In addition, highly expressed unigenes associated with the transition between different developmental stages were identified, including those encoding hydrophobins, cytochrome P450s, extracellular proteases, and several transcription factors. Meanwhile, highly expressed unigenes related to meiosis such as DMC1, MSH4, HOP1, and Mek1 were also analyzed. Our study provides important insights into the molecular mechanisms underlying fruiting body development and sporulation in G. lucidum.