Novel cobiomass degradation of NSAIDs by two wood rot fungi, Ganoderma applanatum and Laetiporus sulphureus: Ligninolytic enzymes induction, isotherm and kinetic studies.

A novel study on biodegradation of 30 mg L-1 of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) mixture (celecoxib, diclofenac and ibuprofen) by two wood-rot fungi; Ganoderma applanatum (GA) and Laetiporus sulphureus (LS) was investigated for 72 h. The removal efficiency of celecoxib, diclofenac and ibuprofen were 98, 96 and 95% by the fungal consortium (GA + LS). Although, both GA and LS exhibited low removal efficiency (61 and 73% respectively) on NSAIDs. However, 99.5% degradation of the drug mixture (NSAIDs) was achieved on the addition of the fungal consortium (GA + LS) to the experimental set-up. Overall, LS exhibited higher degradation efficiency; 92, 87, 79% on celecoxib, diclofenac and ibuprofen than GA with 89, 80 and 66% respectively. Enzyme analyses revealed significant induction of 201, 180 and 135% in laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP) by the fungal consortium during degradation of the NSAIDs respectively. The experimental data showed the best goodness of fit when subjected to Langmuir (R2 = 0.980) and Temkin (R2 = 0.979) isotherm models which suggests monolayer and heterogeneous nature exhibited by the mycelia during interactions with NSAIDs. The degradation mechanism followed pseudo-second-order kinetic model (R2 = 0.987) indicating the strong influence of fungal biomass in the degradation of NSAIDs. Furthermore, Gas Chromatography-Mass Spectrometry (GCMS) and High-Performance Liquid Chromatography (HPLC) analyses confirmed the degraded metabolic states of the NSAIDs after treatment with GA, LS and consortium (GA + LS). Hence, the complete removal of NSAIDs is best achieved in an economical and eco-friendly way with the use of fungi consortium.

Effect of fungal co-cultures on ligninolytic enzyme activities, H2O2 production, and orange G discoloration.

In this study, the effects of Aspergillus niger in coculture with the basidiomycetes, Trametes versicolor, T. maxima, and Ganoderma spp., were studied to assess H2O2 production and laccase (Lac), Lignin Peroxidase (LiP), and manganese peroxidase (MnP) activities. The results indicated that maximum discoloration was of 97%, in the T. maxima and A. niger coculture, where the concentration of H2O2 was 5 mg/L and 6.3 mg/L in cultures without and with dye, respectively. These concentrations of H2O2 were 1.6- and 1.8-fold higher than monocultures of T. maxima (3.37 mg/L) and A. niger (3.87 mg/L), respectively. In the same coculture, the LiP and MnP enzyme activities also increased 12-fold, (from 0.08 U/mg to 0.99 U/mg), and 67-fold, (from 0.11 U/mg to 7.4 U/mg), respectively. The Lac activity increased 1.7-fold (from 13.46 U/mg to 24 U/mg). Further, a Box-Behnken experimental design indicated a 1.8-fold increase of MnP activity (from 7.4 U/mg to 13.3 U/mg). In addition, dye discoloration regression model obtained from the Box-Behnken experimental design showed a positively correlation with H2O2, (R2 = 0.58) and a negatively correlation with Lac activity (R2 = -0.7).

Biodegradation of polycyclic aromatic hydrocarbons by native Ganoderma sp. strains: identification of metabolites and proposed degradation pathways.

Since polycyclic aromatic hydrocarbons (PAHs) are mutagenic, teratogenic, and carcinogenic, they are of considerable environmental concern. A biotechnological approach to remove such compounds from polluted ecosystems could be based on the use of white-rot fungi (WRF). The potential of well-adapted indigenous Ganoderma strains to degrade PAHs remains underexplored. Seven native Ganoderma sp. strains with capacity to produce high levels of laccase enzymes and to degrade synthetic dyes were investigated for their degradation potential of PAHs. The crude enzymatic extracts produced by Ganoderma strains differentially degraded the PAHs assayed (naphthalene 34-73%, phenanthrene 9-67%, fluorene 11-64%). Ganoderma sp. UH-M was the most promising strain for the degradation of PAHs without the addition of redox mediators. The PAH oxidation performed by the extracellular enzymes produced more polar and soluble metabolites such as benzoic acid, catechol, phthalic and protocatechuic acids, allowing us to propose degradation pathways of these PAHs. This is the first study in which breakdown intermediates and degradation pathways of PAHs by a native strain of Ganoderma genus were determined. The treatment of PAHs with the biomass of this fungal strain enhanced the degradation of the three PAHs. The laccase enzymes played an important role in the degradation of these compounds; however, the role of peroxidases cannot be excluded. Ganoderma sp. UH-M is a promising candidate for the bioremediation of ecosystems polluted with PAHs.

Inhibition and kinetic studies of lignin degrading enzymes of Ganoderma boninense by naturally occurring phenolic compounds.

AIM: Lignolytic (lignin degrading) enzyme, from oil palm pathogen Ganoderma boninense Pat. (Syn G. orbiforme (Ryvarden)), is involved in the detoxification and the degradation of lignin in the oil palm and is the rate-limiting step in the infection process of this fungus. Active inhibition of lignin-degrading enzymes secreted by G. boninense by various naturally occurring phenolic compounds and estimation of efficiency on pathogen suppression was aimed at. METHODS AND RESULTS: In our work, 10 naturally occurring phenolic compounds were evaluated for their inhibitory potential towards the lignolytic enzymes of G. boninense. Additionally, the lignin-degrading enzymes were characterized. Most of the peholic compounds exhibited an uncompetitive inhibition towards the lignin-degrading enzymes. Benzoic acid was the superior inhibitor to the production of lignin-degrading enzymes, when compared between the 10 phenolic compounds. The inhibitory potential of the phenolic compounds towards the lignin-degrading enzymes are higher than that of the conventional metal ion inhibitor. The lignin-degrading enzymes were stable in a wide range of pH but were sensitive to higher temperature. CONCLUSION: The study demonstrated the inhibitor potential of 10 naturally occurring phenolic compounds towards the lignin-degrading enzymes of G. boninense with different efficacies. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has shed a light towards a new management strategy to control basal stem rot disease in oil palm. It serves as a replacement for the existing chemical control.

Inhibition and kinetic studies of cellulose- and hemicellulose-degrading enzymes of Ganoderma boninense by naturally occurring phenolic compounds.

AIM: Ganoderma sp, the causal pathogen of the basal stem rot (BSR) disease of oil palm, secretes extracellular hydrolytic enzymes. These play an important role in the pathogenesis of BSR by nourishing the pathogen through the digestion of cellulose and hemicellulose of the host tissue. Active suppression of hydrolytic enzymes secreted by Ganoderma boninense by various naturally occurring phenolic compounds and estimation of their efficacy on pathogen suppression is focused in this study. METHODS AND RESULTS: Ten naturally occurring phenolic compounds were assessed for their inhibitory effect on the hydrolytic enzymes of G. boninense. The enzyme kinetics (Vmax and Km ) and the stability of the hydrolytic enzymes were also characterized. The selected compounds had shown inhibitory effect at various concentrations. Two types of inhibitions namely uncompetitive and noncompetitive were observed in the presence of phenolic compounds. Among all the phenolic compounds tested, benzoic acid was the most effective compound suppressive to the growth and production of hydrolytic enzymes secreted by G. boninense. The phenolic compounds as inhibitory agents can be a better replacement for the metal ions which are known as conventional inhibitors till date. The three hydrolytic enzymes were stable in a wide range of pH and temperature. CONCLUSION: These findings highlight the efficacy of the applications of phenolic compounds to control Ganoderma. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has proved a replacement for chemical controls of G. boninense with naturally occurring phenolic compounds.

Potential of selected fungal species to degrade wheat straw, the most abundant plant raw material in Europe.

BACKGROUND: Structural component of plant biomass, lignocellulose, is the most abundant renewable resource in nature. Lignin is the most recalcitrant natural aromatic polymer and its degradation presents great challenge. Nowadays, the special attention is given to biological delignification, the process where white-rot fungi take the crucial place owing to strong ligninolytic enzyme system. However, fungal species, even strains, differ in potential to produce high active ligninolytic enzymes and consequently to delignify plant biomass. Therefore, the goals of the study were characterization of Mn-oxidizing peroxidases and laccases of numerous mushrooms as well as determination of their potential to delignify wheat straw, the plant raw material that, according to annual yield, takes the first place in Europe and the second one in the world. RESULTS: During wheat straw fermentation, Lentinus edodes HAI 858 produced the most active Mn-dependent and Mn-independent peroxidases (1443.2 U L-1 and 1045.5 U L-1, respectively), while Pleurotus eryngii HAI 711 was the best laccase producer (7804.3 U L-1). Visualized bends on zymogram confirmed these activities and demonstrated that laccases were the dominant ligninolytic enzymes in the studied species. Ganoderma lucidum BEOFB 435 showed considerable ability to degrade lignin (58.5%) and especially hemicellulose (74.8%), while the cellulose remained almost intact (0.7%). Remarkable selectivity in lignocellulose degradation was also noted in Pleurotus pulmonarius HAI 573 where degraded amounts of lignin, hemicellulose and cellulose were in ratio of 50.4%:15.3%:3.8%. CONCLUSIONS: According to the presented results, it can be concluded that white-rot fungi, due to ligninolytic enzymes features and degradation potential, could be important participants in various biotechnological processes including biotransformation of lignocellulose residues/wastes in food, feed, paper and biofuels.

Ornithine Decarboxylase-Mediated Production of Putrescine Influences Ganoderic Acid Biosynthesis by Regulating Reactive Oxygen Species in Ganoderma lucidum.

Putrescine is an important polyamine that participates in a variety of stress responses. Ornithine decarboxylase (ODC) is a key enzyme that catalyzes the biosynthesis of putrescine. A homolog of the gene encoding ODC was cloned from Ganoderma lucidum In the ODC-silenced strains, the transcript levels of the ODC gene and the putrescine content were significantly decreased. The ODC-silenced strains were more sensitive to oxidative stress. The content of ganoderic acid was increased by approximately 43 to 46% in the ODC-silenced strains. The content of ganoderic acid could be recovered after the addition of exogenous putrescine. Additionally, the content of reactive oxygen species (ROS) was significantly increased by approximately 1.3-fold in the ODC-silenced strains. The ROS content was significantly reduced after the addition of exogenous putrescine. The gene transcript levels and the activities of four major antioxidant enzymes were measured to further explore the effect of putrescine on the intracellular ROS levels. Further studies showed that the effect of the ODC-mediated production of putrescine on ROS might be a factor influencing the biosynthesis of ganoderic acid. Our study reports the role of putrescine in large basidiomycetes, providing a basis for future studies of the physiological functions of putrescine in microbes.IMPORTANCE It is well known that ODC and the ODC-mediated production of putrescine play an important role in resisting various environmental stresses, but there are few reports regarding the mechanisms underlying the effect of putrescine on secondary metabolism in microorganisms, particularly in fungi. G. lucidum is gradually becoming a model organism for studying environmental regulation and metabolism. In this study, a homolog of the gene encoding ODC was cloned in Ganoderma lucidum We found that the transcript level of the ODC gene and the content of putrescine were significantly decreased in the ODC-silenced strains. The content of ganoderic acid was significantly increased in the ODC-silenced strains. Further studies showed that the effect of the ODC-mediated production of putrescine on ROS might be a factor influencing the biosynthesis of ganoderic acid. Our study reports the role of putrescine in large basidiomycetes, providing a basis for future studies of the physiological functions of putrescine in microbes.

Valorization of spent oyster mushroom substrate and laccase recovery through successive solid state cultivation of Pleurotus, Ganoderma, and Lentinula strains.

Spent mushroom substrate (SMS) of Pleurotus ostreatus was supplemented with wheat bran and soybean flour in various proportions to obtain C/N ratios of 10, 20, and 30, and their effect was evaluated in successive cultivation of Pleurotus ostreatus, Pleurotus pulmonarius, Ganoderma adspersum, Ganoderma resinaceum, and Lentinula edodes strains with respect to mycelium growth rate, biomass concentration, recovery of the enzyme laccase and crude exopolysaccharides, and also with additional fruiting body production. All fungi showed the highest growth rate on unamended SMS (C/N 30), with G. resinaceum being the fastest colonizer (Kr = 9.84 mm day-1), while biomass concentration maximized at C/N 10. Moreover, supplementation affected positively laccase activity, with P. pulmonarius furnishing the highest value (44,363.22 U g-1) at C/N 20. On the contrary, L. edodes growth, fruiting, and laccase secretion were not favored by SMS supplementation. Fruiting body formation was promoted at C/N 30 for Ganoderma and at C/N 20 for Pleurotus species. Exopolysaccharide production of further studied Pleurotus strains was favored at a C/N 20 ratio, at the initial stage of SMS colonization. The obtained results support the potential effective utilization of supplemented SMS for laccase production from Ganoderma spp. and for new fruiting body production of Pleurotus spp.

Edible fungus degrade bisphenol A with no harmful effect on its fatty acid composition.

Bisphenol A (BPA) is an endocrine-disrupting chemical that is ubiquitous in the environment because of its broad industrial use. The authors report that the most widely cultivated mushroom in the world (i.e., white-rot fungus, Pleurotus ostreatus) efficiently degraded 10mg/L of BPA in 7 days. Extracellular laccase was identified as the enzyme responsible for this activity. LC-MS analysis of the metabolites revealed the presence of both low- and high-molecular-weight products obtained via oxidative cleavage and coupling reactions, respectively. In particular, an analysis of the fatty acid composition and chemical structure of the fungal mycelium demonstrated that exposure to BPA resulted in no harmful effects on this edible fungus. The results provide a better understanding of the environmental fate of BPA and its potential impact on food crops.

Adding-value to Ganoderma lingzhi by producing enzymes and antioxidant compounds under submerged fermentation using different culture media.

Ganoderma lingzhi is widely reported for its medicinal properties, presenting several bioactive substances with potential pharmaceutical and industrial application. This study aimed to evaluate the production of mycelial biomass, extracellular enzymes and antioxidant compounds by G. lingzhi under submerged fermentation. G. lingzhi was cultured in Polysaccharide (POL) and Melin-Norkrans (MNM) media for 7 days. The cellulases, xylanases, pectinases, laccases, and proteases activities were quantified in the culture broth, while the antioxidant potential was evaluated for the mycelial biomass. G. lingzhi showed higher biomass production in MNM. However, it exhibited similar microstructural characteristics in both culture media. In the POL there was greater activity of CMCase (0.229 U/mL), xylanase (0.780 U/mL), pectinase (0.447 U/mL) and proteases (16.13 U/mL). FPase did not differ (0.01 U/mL), and laccase was detected only in MNM (0.122 U/mL). The biomass water extract from MNM showed high levels of phenolic compounds (951.97 mg AGE/100 g). DPPH• inhibition (90.55%) and reducing power (0.456) were higher in MNM medium, while ABTS•+ inhibition (99.95%) and chelating ability (54.86%) were higher in POL. Thus, the MNM medium was more favorable to the production of mycelial biomass and phenolic compounds, while the POL medium favored the synthesis and excretion of hydrolytic enzymes.

Ligninolytic enzymes from Ganoderma spp: current status and potential applications.

White-rot fungal species belonging to Ganoderma have long been used as medicinal mushrooms in many Asian countries. In recent years, however, attention is not just being paid to their pharmacological properties, but to their other potentially valuable features as well, including their secretion of enzymes which decompose lignin. The current literature regarding lignin-modifying enzymes from the genus Ganoderma, their potential uses, and the components, structures and processes of lignocellulose degradation are discussed. The ligninolytic enzymes from the genus Ganoderma, as well as the number of additional enzymes that participate in lignin degradation, are summarized; further, the potential applications of these enzymes are analyzed and probed in this article. This review will provide insight on the valuable applications of Ganoderma spp. and will serve as a useful reference on the use of lignocellulose degradation as a means of environmental protection.

Amperometric determination of total phenolic content in wine by laccase immobilized onto silver nanoparticles/zinc oxide nanoparticles modified gold electrode.

A method is described for construction of a highly sensitive amperometric biosensor for measurement of total phenolic compounds in wine by immobilizing laccase covalently onto nanocomposite of silver nanoparticles (AgNPs)/zinc oxide nanoparticles (ZnONPs) electrochemically deposited onto gold (Au) electrode. Scanning electron microscopy, X-ray diffraction, and electrochemical impedance spectroscopy were applied for characterization of the surface morphology of the modified electrode, and cyclic voltammetry was used to investigate the electrochemical properties of the proposed electrode toward the oxidation of guaiacol. The linearity between the oxidation current and the guaiacol concentration was obtained in a range of 0.1 to 500µM with a detection limit of 0.05µM (signal-to-noise ratio (S/N)=3) and sensitivity of 0.71µAµM(-1)cm(-2). The electrode showed increased oxidation and reduced reduction current with the deposition of AgNPs/ZnONPs on it. R(CT) values of ZnONPs/Au, AgNPs/ZnONPs/Au, and laccase/AgNPs/ZnONPs/Au electrode were 220, 175, and 380Ω, respectively. The biosensor showed an optimal response within 8s at pH 6.0 (0.1M acetate buffer) and 35°C when operated at 0.22V against Ag/AgCl. Analytical recovery of added guaiacol was 98%. The method showed a good correlation (r=0.99) with the standard spectrophotometric method, with the regression equation being y=1.0053x-3.5541. The biosensor lost 25% of its initial activity after 200 uses over 5months.

Optimization, purification and characterization of laccase from Ganoderma leucocontextum along with its phylogenetic relationship.

The aim of this work to study an efficient laccase producing fungus Ganoderma leucocontextum, which was identified by ITS regions of DNA and phylogenetic tree was constructed. This study showed the laccase first-time from G. leucocontextum by using medium containing guaiacol. The growth cultural (pH, temperature, incubation days, rpm) and nutritional (carbon and nitrogen sources) conditions were optimized, which enhanced the enzyme production up to 4.5-folds. Laccase production increased 855 U/L at 40 °C. The pH 5.0 was suitable for laccase secretion (2517 U/L) on the 7th day of incubation at 100 rpm (698.3 U/L). Glucose and sucrose were good carbon source to enhance the laccase synthesis. The 10 g/L beef (4671 U/L) and yeast extract (5776 U/L) were the best nitrogen source for laccase secretion from G. leucocontextum. The laccase was purified from the 80% ammonium sulphate precipitations of protein identified by nucleotides sequence. The molecular weight (65.0 kDa) of purified laccase was identified through SDS and native PAGE entitled as Glacc110. The Glacc110 was characterized under different parameters. It retained > 90% of its activity for 16 min incubation at 60 °C in acidic medium (pH 4.0). This enzyme exerted its optimal activity at pH 3.0 and temperature 70 °C with guaiacol substrate. The catalytic parameters Km and Vmax was 1.658 (mM) and 2.452 (mM/min), respectively. The thermo stability of the laccase produced by submerged fermentation of G. leucocontextum has potential for industrial and biotechnology applications. The results remarked the G. leucocontextum is a good source for laccase production.

Bioprospecting of the agaricomycete Ganoderma australe GPC191 as novel source for L-asparaginase production.

L-Asparaginase is a therapeutically and industrially-competent enzyme, acting predominantly as an anti-neoplastic and anti-cancerous agent. The existing formulations of prokaryotic L-asparaginase are often toxic and contain L-glutaminase and urease residues, thereby increasing the purification steps. Production of L-glutaminase and urease free L-asparaginase is thus desired. In this research, bioprospecting of isolates from the less explored class Agaricomycetes was undertaken for L-asparaginase production. Plate assay (using phenol red and bromothymol blue dyes) was performed followed by estimation of L-asparaginase, L-glutaminase and urease activities by Nesslerization reaction for all the isolates. The isolate displaying the desired enzyme production was subjected to morphological, molecular identification, and phylogenetic analysis with statistical validation using Jukes-Cantor by Neighbour-joining tree of Maximum Likelihood statistical method. Among the isolates, Ganoderma australe GPC191 with significantly high zone index value (5.581 ± 0.045 at 120 h) and enzyme activity (1.57 ± 0.006 U/mL), devoid of L-glutaminase and urease activity was selected. The present study for the first-time reported G. australe as the potential source of L-glutaminase and urease-free L-asparaginase and also is one of the few studies contributing to the literature of G. australe in India. Hence, it can be postulated that it may find its future application in pharmaceutical and food industries.

Kinetics Analysis of the Inhibitory Effects of Alpha-Glucosidase and Identification of Compounds from Ganoderma lipsiense Mycelium.

The studies on natural compounds to diabetes mellitus treatment have been increasing in recent years. Research suggests that natural components can inhibit alpha-glucosidase activities, an important strategy in the management of blood glucose levels. In this work, for the first time in the literature, the compounds produced by Ganoderma lipsiense extracts were identified and evaluated on the inhibitory effect of these on alpha-glucosidase activity. Four phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to crude extract from G. lipsiense grown in red rice medium (RCE) and synthetic medium (SCE), being syringic acid identified in both extracts. Gas chromatography-mass spectrometry (GC-MS) analysis showed fatty acids and their derivatives, terpene, steroid, niacin, and nitrogen compounds to SCE, while RCE was rich in fatty acids and their derivatives. Both extracts demonstrated alpha-glucosidase inhibition (RCE IC50 = 0.269 ± 8.25 mg mL-1; SCE IC50 = 0.218 ± 9.67 mg mL-1), and the purified hexane fraction of RCE (RHEX) demonstrated the highest inhibition of enzyme (81.1%). Studies on kinetic inhibition showed competitive inhibition mode to RCE, while SCE showed uncompetitive inhibition mode. Although the inhibitory effects of RCE and SCE were satisfactory, the present findings identified some unpublished compounds to G. lipsiense in the literature with important therapeutic properties.

Degradation of Aflatoxin B1 and Zearalenone by Bacterial and Fungal Laccases in Presence of Structurally Defined Chemicals and Complex Natural Mediators.

Aflatoxin B1 (AFB1) and zearalenone (ZEN) exert deleterious effects to human and animal health. In this study, the ability of a CotA laccase from Bacillus subtilis (BsCotA) to degrade these two mycotoxins was first investigated. Among the nine structurally defined chemical compounds, methyl syringate was the most efficient mediator assisting BsCotA to degrade AFB1 (98.0%) and ZEN (100.0%). BsCotA could also use plant extracts, including the Epimedium brevicornu, Cucumis sativus L., Lavandula angustifolia, and Schizonepeta tenuifolia extracts to degrade AFB1 and ZEN. Using hydra and BLYES as indicators, it was demonstrated that the degraded products of AFB1 and ZEN using the laccase/mediator systems were detoxified. Finally, a laccase of fungal origin was also able to degrade AFB1 and ZEN in the presence of the discovered mediators. The findings shed light on the possibility of using laccases and a mediator, particularly a natural plant-derived complex mediator, to simultaneously degrade AFB1 and ZEN contaminants in food and feed.

Multiple Reaction Monitoring for quantitative laccase kinetics by LC-MS.

Laccases (EC 1.10.3.2) are enzymes known for their ability to catalyse the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Lignin is a natural phenylpropanoids biopolymer whose degradation in nature is thought to be aided by enzymatic oxidation by laccases. Laccase activity is often measured spectrophotometrically on compounds such as syringaldazine and ABTS which poorly relate to lignin. We employed natural phenolic hydroxycinnamates having different degree of methoxylations, p-coumaric, ferulic and sinapic acid, and a lignin model OH-dilignol compound as substrates to assess enzyme kinetics by HPLC-MS on two fungal laccases Trametes versicolor laccase, Tv and Ganoderma lucidum laccase, Gl. The method allowed accurate kinetic measurements and detailed insight into the product profiles of both laccases. Both Tv and Gl laccase are active on the hydroxycinnammates and show a preference for substrate with methoxylations. Product profiles were dominated by the presence of dimeric and trimeric species already after 10 minutes of reaction and similar profiles were obtained with the two laccases. This new HPLC-MS method is highly suitable and accurate as a new method for assaying laccase activity on genuine phenolic substrates, as well as a tool for examining laccase oxidation product profiles.

Investigation of lignocellulolytic enzymes during different growth phases of Ganoderma lucidum strain G0119 using genomic, transcriptomic and secretomic analyses.

Ganoderma lucidum is a medicinal mushroom that is well known for its ability to enhance human health, and products made from this fungus have been highly profitable. The substrate-degrading ability of G. lucidum could be related to its growth. CAZy proteins were more abundant in its genome than in the other white rot fungi models. Among these CAZy proteins, changes in lignocellulolytic enzymes during growth have not been well studied. Using genomic, transcriptomic and secretomic analyses, this study focuses on the lignocellulolytic enzymes of G. lucidum strain G0119 to determine which of these degradative enzymes contribute to its growth. From the genome sequencing data, genes belonging to CAZy protein families, especially genes involved in lignocellulose degradation, were investigated. The gene expression, protein abundance and enzymatic activity of lignocellulolytic enzymes in mycelia over a growth cycle were analysed. The overall expression cellulase was higher than that of hemicellulase and lignin-modifying enzymes, particularly during the development of fruiting bodies. The cellulase and hemicellulase abundances and activities increased after the fruiting bodies matured, when basidiospores were produced in massive quantities till the end of the growth cycle. Additionally, the protein abundances of the lignin-modifying enzymes and the expression of their corresponding genes, including laccases and lignin-degrading heme peroxidases, were highest when the mycelia fully spread in the compost bag. Type I cellobiohydrolase was observed to be the most abundant extracellular lignocellulolytic enzyme produced by the G. lucidum strain G0119. The AA2 family haem peroxidases were the dominant lignin-modifying enzyme expressed during the mycelial growth phase, and several laccases might play roles during the formation of the primordium. This study provides insight into the changes in the lignocellulose degradation ability of G. lucidum during its growth and will facilitate the discovery of new approaches to accelerate the growth of G. lucidum in culture.

Redox potentials of the blue copper sites of bilirubin oxidases.

The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was >650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.

Solid-state fermentation for enhanced production of laccase using indigenously isolated Ganoderma sp.

Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using wheat bran as substrate with 70% initial moisture content at 25 degrees C and the seven agar plugs as the inoculum. Further enhancement in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using SSF. The process also promises economic utilization and value addition of agro-residues.