Paddy is an important crop in Malaysia. There are various pathogens able to infect paddy causing a loss in yield's production. In this study, dual culture method, volatile organic compound (VOC) analysis, and non-volatile compound analysis were used to assess the ability of mushroom to control fungal rice pathogens including Curvularia lunata, Bipolaris panici-miliacei, and Nigrospora sp. Four mushroom isolates were further analysed for their antagonistic activity against rice pathogen. The highest percentage inhibition of radial growth (PIRG) was recorded between 45.55 and 73.68% observed in isolate 42b. The 4 isolates with the highest PIRG based on the dual culture analysis were then tested for their production of VOCs and non-volatile compound. Internal transcribed spacer (ITS) region analysis of the 4 mushroom isolates revealed their identity as Coprinellus disseminates (isolate 12b), Marasmiellus palmivorus (isolate 42b), Trametes maxima (isolate 56e), and Lentinus sajor-caju (isolate 60a). This study showed that mushroom isolates have the potential of antagonistic effect on various fungal rice pathogens tested by the production of secondary metabolites and mycoparasitic interaction.
Laccases hold great potential for biotechnological applications, particularly in environmental pollutant remediation. Laccase activity is governed by the solvent environment, and ionic liquids (ILs) emerge as a versatile solvent for activation or stabilization of enzymes. Herein, effects of cholinium-based ILs formulated with carboxylic acids, inorganic acid, and amino acids as anionic species, on the catalytic activity of laccase from Trametes versicolor were investigated by experimental and computational approaches. Experimental results showed that laccase activity was enhanced by 21.39 % in 0.5 M cholinium dihydrogen citrate ([Cho][DHC]), in relation to the laccase activity in phosphate buffer medium. However, cholinium aminoate ILs negatively affected laccase activity, as evidenced by the partial deactivation of laccase in both cholinium glycinate and cholinium phenylalaninate, at concentrations of 0.1 M and 0.5 M, respectively. Molecular dynamics studies revealed that the enhancement of laccase activity in [Cho][DHC] might be attributed to the highly stabilized and compact structure of laccase, facilitating a better internal electron transfer during the laccase-substrate interactions. Enhanced catalytic performance of laccase in [Cho][DHC] was postulated to be driven by the high accumulation level of dihydrogen citrate anions around laccase's surface. [Cho][DHC] holds great promise as a cosolvent in laccase-catalyzed biochemical reactions.
Basal stem rot due to a fungal pathogen, Ganoderma boninense, is one of the most devastating diseases in oil palm throughout the major palm oil producer countries. This study investigated the potential of polypore fungi as biological control agents against pathogenic G. boninense in oil palm. In vitro antagonistic screening of selected non-pathogenic polypore fungi was performed. Based on in planta fungi inoculation on oil palm seedlings, eight of the 21 fungi isolates tested (GL01, GL01, RDC06, RDC24, SRP11, SRP12, SRP17, and SRP18) were non-pathogenic. In vitro antagonistic assays against G. boninense revealed that the percentage inhibition of radial growth (PIRG) in dual culture assay for SRP11 (69.7%), SRP17 (67.3%), and SRP18 (72.7%) was relatively high. Percentage inhibition of diameter growth (PIDG) in volatile organic compounds (VOCs) in dual plate assay of SRP11, SRP17, and SRP18 isolates were 43.2%, 51.6%, and 52.1%, respectively. Molecular identification using the internal transcribed spacer gene sequences of SRP11, SRP17, and SRP18 isolates revealed that they were Fomes sp., Trametes elegans, and Trametes lactinea, respectively.
The study monitored the characteristics of the leachate collected from ten different landfills and presented the experimental work for the treatment of leachate by immobilized Trametes menziesii. Variation in biological oxygen demand (BOD), chemical oxygen demand (COD) and ammoniacal nitrogen (NH3-N) showed that the age of the leachate has a significant effect on its characteristics and composition. The BOD5/COD ratio tends to decrease as the age of leachate increases, varying from 0.71 for a relatively 'fresh' leachate to 0.62 for an older (more stabilized) one. Variations in the characteristics of the leachate suggested that these leachates are difficult to treat. The principal pollutants in the leachate samples were organic and ammonia loads. Treatment of leachate using immobilized Trametes menziesii achieved 89.14 and 2.11% removals for leachate BOD5 and COD, respectively. These findings suggested that using immobilized Trametes menziesii can remove promising percentage of BOD and COD leachate.
White rot fungi are good lignin degraders and have the potential to be used in industry. In the present work, Phellinus sp., Daedalea sp., Trametes versicolor and Pycnoporus coccineus were selected due to their relatively high ligninolytic enzyme activity, and grown on Acacia mangium wood chips under solid state fermentation. Results obtained showed that manganese peroxidase produced is far more compared to lignin peroxidase, suggesting that MnP might be the predominating enzymes causing lignin degradation in Acacia mangium wood chips. Cellulase enzyme assays showed that no significant cellulase activity was detected in the enzyme preparation of T. versicolor and Phellinus sp. This low cellulolytic activity further suggests that these two white rot strains are of more interest in lignin degradation. The results on lignin losses showed 20-30% of lignin breakdown at 60 days of biodegradation. The highest lignin loss was found in Acacia mangium biotreated with T. versicolor after 60 days and recorded 26.9%, corresponding to the percentage of their wood weight loss recorded followed by P. coccineus. In general, lignin degradation was only significant from 20 days onwards. The overall percentage of lignin weight loss was within the range of 1.02-26.90% over the biodegradation periods. Microscopic observations conducted using scanning electron microscope showed that T. versicolor, P. coccineus, Daedalea sp. and Phellinus sp. had caused lignin degradation in Acacia mangium wood chips.
Macrofungi of the order Polyporales are among the most important wood decomposers and caused economic losses by decaying the wood in standing trees, logs and in sawn timber. Diversity and distribution of Polyporales in Peninsular Malaysia was investigated by collecting basidiocarps from trunks, branches, exposed roots and soil from six states (Johor, Kedah, Kelantan, Negeri Sembilan, Pahang and Selangor) in Peninsular Malaysia and Federal Territory Kuala Lumpur. This study showed that the diversity of Polyporales were less diverse than previously reported. The study identified 60 species from five families; Fomitopsidaceae, Ganodermataceae, Meruliaceae, Meripilaceae, and Polyporaceae. The common species of Polyporales collected were Fomitopsis feei, Amauroderma subrugosum, Ganoderma australe, Earliella scabrosa, Lentinus squarrosulus, Microporus xanthopus, Pycnoporus sanguineus and Trametes menziesii.
e effects of biological pretreatment on the rubberwood (Hevea brasiliensis), was evaluated after cultivation of white rot fungi Ceriporiopsis subvermispora, Trametes versicolor, and a mixed culture of C. subvermispora and T. versicolor. The analysis of chemical compositions indicated that C. subvermispora had greater selectivity for lignin degradation with the highest lignin and hemicellulose loss at 45.06% and 42.08%, respectively, and lowest cellulose loss (9.50%) after 90 days among the tested samples. X-ray analysis showed that pretreated samples had a higher crystallinity than untreated samples. The sample pretreated by C. subvermispora presented the highest crystallinity of all the samples which might be caused by the selective degradation of amorphous components. Fourier transform infrared (FT-IR) spectroscopy demonstrated that the content of lignin and hemicellulose decreased during the biological pretreatment process. A study on hydrolysis of rubberwood treated with C. subvermispora, T. versicolor, and mixed culture for 90 days resulted in an increased sugar yield of about 27.67%, 16.23%, and 14.20%, respectively, as compared with untreated rubberwood (2.88%). The results obtained demonstrate that rubberwood is a potential raw material for industrial applications and white rot fungus C. subevermispora provides an effective method for improving the enzymatic hydrolysis of rubberwood.
The present work investigated the potential of fungal species from grain maize farms in Malaysia as antagonists against the indigenous mycotoxigenic fungal species and their subsequent mycotoxin production. Dual-culture assay was conducted on grain maize agar (GMA) with 12 strains of potential fungal antagonists namely Bjerkandra adusta, Penicillium janthinellum, Schizophyllum commune, Trametes cubensis, Trichoderma asperelloides, Trichoderma asperellum, Trichoderma harzianum, and Trichoderma yunnanense against seven mycotoxigenic strains namely Aspergillus flavus, Aspergillus niger, Fusarium verticillioides, and Fusarium proliferatum producing aflatoxins, ochratoxin A, and fumonisins, respectively. Based on fungal growth inhibition, Trichoderma spp. showed the highest inhibitory activity (73-100% PIRG, Percentage Inhibition of Radial Growth; 28/0 ID, Index of Dominance) against the tested mycotoxigenic strains. Besides, B. adusta and Tra. cubensis showed inhibitory activity against some of the tested mycotoxigenic strains. All fungal antagonists showed varying degrees of mycotoxin reduction. Aflatoxin B1 produced by A. flavus was mainly reduced by P. janthinellum, Tra. cubensis, and B. adusta to 0 ng/g. Ochratoxin A produced by A. niger was mainly reduced by Tri. harzianum and Tri. asperellum to 0 ng/g. Fumonisin B1 and FB2 produced by F. verticillioides was mainly reduced by Tri. harzianum, Tri. asperelloides, and Tri. asperellum to 59.4 and 0 µg/g, respectively. Fumonisin B1 and FB2 produced by F. proliferatum were mainly reduced by Tri. asperelloides and Tri. harzianum to 244.2 and 0 µg/g, respectively. This is the first study that reports on the efficacy of Tri. asperelloides against FB1, FB2, and OTA, P. janthinellum against AFB1, and Tra. cubensis against AFB1.
Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k1', value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ∼ 22 kJ mol(-1), while energy for laccase inactivation was 18 kJ mol(-1). Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (ΔT = 40 K) as opposed to fungal biomass (ΔT = 15 K). Comparison of entropy change (ΔS) values indicated metabolism of the dye by the biomass.
Laccases, oxidative copper-enzymes found in fungi and bacteria were used as the basis in the design of nona- and tetrapeptides. Laccases are known to be excellent catalysts for the degradation of phenolic xenobiotic waste. However, since solvent extraction of laccases is environmentally-unfriendly and yields obtained are low, they are less preferred compared to synthetic catalysts. The histidine rich peptides were designed based on the active site of laccase extracted from Trametes versicolor through RCSB Protein Data Bank, LOMETS and PyMol software. The peptides were synthesized using Fmoc-solid phase peptide synthesis (SPPS) with 30-40% yield. These peptides were purified and characterized using LC-MS (purities >75%), FTIR and NMR spectroscopy. Synthesized copper(II)-peptides were crystallized and then analyzed spectroscopically. Their structures were elucidated using 1D and 2D NMR. Standards (o,m,p-cresol, 2,4-dichlorophenol) catalysed using laccase from Trametes versicolor (0.66 U/mg) were screened under different temperatures and stirring rate conditions. After optimizing the degradation of the standards with the best reaction conditions reported herein, medications with phenolic and aromatic structures such as ibuprofen, paracetamol (acetaminophen), salbutamol, erythromycin and insulin were screened using laccase (positive control), apo-peptides and copper-peptides. Their activities evaluated using GC-MS, were compared with those of peptide and copper-peptide catalysts. The tetrapeptide was found to have the higher degradation activity towards salbutamol (96.8%) compared with laccase at 42.8%. Ibuprofen (35.1%), salbutamol (52.9%) and erythromycin (49.7%) were reported to have the highest degradation activities using Cu-tetrapeptide as catalyst when compared with the other medications. Consequently, o-cresol (84%) was oxidized by Tp-Cu while the apo-peptides failed to oxidize the cresols. Copper(II)-peptides were observed to have higher catalytic activity compared to their parent peptides and the enzyme laccase for xenobiotic degradation.
Carboxylic acid reductases (CARs) are attracting burgeoning attention as biocatalysts for organic synthesis of aldehydes and their follow-up products from economic carboxylic acid precursors. The CAR enzyme class as a whole, however, is still poorly understood. To date, relatively few CAR sequences have been reported, especially from fungal sources. Here, we sought to increase the diversity of the CAR enzyme class. Six new CAR sequences from the white-rot fungus Pycnoporus cinnabarinus were identified from genome-wide mining. Genome and gene clustering analysis suggests that these PcCAR enzymes play different natural roles in Basidiomycete systems, compared to their type II Ascomycete counterparts. The cDNA sequences of all six Pccar genes were deduced and analysis of their corresponding amino acid sequence showed that they encode for proteins of similar properties that possess a conserved modular functional tri-domain arrangement. Phylogenetic analyses showed that all PcCAR enzymes cluster together with the other type IV CARs. One candidate, PcCAR4, was cloned and over-expressed recombinantly in Escherichia coli. Subsequent biotransformation-based screening with a panel of structurally-diverse carboxylic acid substrates suggest that PcCAR4 possessed a more pronounced substrate specificity compared to previously reported CARs, preferring to reduce sterically-rigid carboxylic acids such as benzoic acid. These findings thus present a new functionally-distinct member of the CAR enzyme class.
In this study, laccase was immobilized on nylon 6,6/Fe(3+) composite (NFC) nanofibrous membrane and used for the detoxification of 3,3'-dimethoxybenzidine (DMOB). The average size and tensile strength of the NFC membrane were found to be 60-80 nm (diameter) and 2.70 MPa, respectively. The FTIR results confirm that the amine (N-H) group of laccase was attached with Fe(3+) particles and the carbonyl (C=O) group of NFC membrane via hydrogen bonding. The half-life of the laccase-NFC membrane storage stability was increased from 6 to 11 weeks and the reusability was significantly extended up to 43 cycles against ABTS oxidation. Enhanced electro-oxidation of DMOB by laccase was observed at 0.33 V and the catalytic current was found to be 30 µA. The DMOB-treated mouse fibroblast 3T3-L1 preadipocytes showed maximum (97 %) cell inhibition at 75 µM L(-1) within 24 h. The cytotoxicity of DMOB was significantly decreased to 78 % after laccase treatment. This study suggests that laccase-NFC membrane might be a good candidate for emerging pollutant detoxification.
The disposal of oil palm biomass is a huge challenge in Malaysian oil palm plantations. The aim of this study was to develop efficient solid-state cultivated (SSC) ligno-hemicellulolytic bio-degrader formulations of indigenous white-rot hymenomycetes (Trametes lactinea FBW and Pycnoporus sanguineus FBR) utilizing oil palm empty fruit bunches (EFB), rubber wood sawdust (SD) and vermiculite (V) either alone or in combination as substrates. Based on significant laccase (849.40 U mg-1 protein), xylanase (42.26 U g-1 protein) and amylase (157.49 U g-1 protein) production, SD+V (T5) and V (T3) were the optimum substrates for SSC of T. lactinea FBW. Whereas, utilizing EFB (T1) substrate for SSC of P. sanguineus FBR enhanced the production of MnP (42.51 U mg-1 protein), LiP (103.20 U mg-1 protein) and CMCase (34.39 U g-1 protein), enzymes. Apparently, this is the first study reporting on the protein profiles by T. lactinea FBW, producing two isoforms of un-purified laccase (~55 and 70 kDa) and MnP (~40 and 60 kDa) and a CMCase band (~60 kDa) during SSC on SD+V (T5) substrate. Interestingly, this is also the first report to document a single isoform of un-purified laccase (~50 kDa), MnP (~45 kDa), CMCase (~60 kDa) and xylanase (~55 kDa) by P. sanguineus FBR during SSC on empty fruit bunches substrate. The computed Principal Component Analysis (PCA) Biplot analysis elucidated the relationship between the solid substrate compositions, the hymenomycete strain, ligno-hemicellulolytic enzyme profiles, and cultivation time. Therefore, it is suggested to use PCA as a tool for multivariate analysis method for comprehensive selection and optimization of ligno-hemicellulolytic enzyme cocktails by the indigenous white rot hymenomycetes. These non-toxic (acute oral toxicity) formulations are safe to be used in field applications to efficiently degrade oil palm trunks and root mass that had been felled, chipped or pulverized under zero burning waste management program. This study could also serve as an alternative method for efficient utilization of agro-industrial waste as substrates for the development of cost-effective bio-degraders formulations for agro-waste management.
Laccases are industrially attractive enzymes and their applications have expanded to the field of bioremediation. The challenge of today's biotechnology in enzymatic studies is to design enzymes that not only have a higher activity but are also more stable and could fit well with the condition requirements. Laccases are known to oxidize non-natural substrates like polycyclic aromatic hydrocarbons (PAHs). We suppose by increasing the hydrophobicity of laccase, it would increase the chance of the enzyme to meet the hydrophobic substrates in a contamination site, therefore increasing the bioremediation efficacy of PAHs from environment. In this attempt, the applications of evolutionary trace (ET), molecular surface accessibility and hydrophobicity analysis on laccase sequences and laccase's crystal structure (1KYA) are described for optimal design of an enzyme with higher hydrophobicity. Our analysis revealed that Q23A, Q45I, N141A, Q237V, N262L, N301V, N331A, Q360L and Q482A could be promising exchanges to be tested in mutagenesis experiments.