Search

Low fertilizer quality and remediation ability are considered the major factors hampering the land application of anaerobic digestate. Therefore, the role of Myrothecium verrucaria treatment in enhancing the fertilizer quality and remediation ability of digestate for land application was explored. Higher content of humic acid (7.5 g/L) with a higher degree of humification index and oxygen-containing functional groups was observed in the digestate receiving Myrothecium verrucaria treatment. Likewise, humic acid formed from Myrothecium verrucaria treatment had a higher capacity of heavy metal binding. Moreover, the viable and culturable count of S. faecalis, S. typhi, C. perfringens, and E. coli pathogens in the digestate decreased to approximately 12.50%, 41.70%, 18.87%, and 50.00% and 25.97%, 64.44%, 37.51%, and 75.27%, respectively, after treatment with Myrothecium verrucaria. This study provided a novel strategy to enhance the fertilizer quality, remediation ability, and biological safety of anaerobic digestate for land application.

Nematicidal activity of heat-sterilizing filtrate culture of Myrothecium verrucaria was evaluated against Meloidogyne incognita in Vitro and the metabolites formulated as granules of M. verrucaria were prepared and tested on sugar beet plants infested with M. incognita comparison with DiTera™ and Rugby 10%G, under greenhouse conditions. Laboratory results showed that sterilized culture filtrate of the fungus M. verrucaria either by syringe or by autoclaving significantly reduced the percentages of egg hatching and increased the percentage of 2nd stage juveniles (J2) mortality of M. incognita. The most effective reductions of hatching were obtained at 50% concentration with the sterilized filtrate by autoclaving (94.23%), while the most effective reached 96.70% increasing in the percentage of mortality of J2 after 3 days from treating by 50% concentration in case the sterilized filtrate by syringe. Results of greenhouse experiment showed that, all treatments by the prepared fungal formulae of M. verrucaria or DiTerawith different rats significantly reduced the number of galls on sugar beet roots and final nematode population (J2) in soil compared with control (nematode only). The higher rate (0.2 g /pot) was achieved the highest reduction in root-galling (86%) with DiTera treatment and the highest reduction in (J2) with fungal formulae treatment (92.2%). Improvement in plant growth parameters were noticed by treatments. Analysis by GC-MS of the fungus metabolites was identified compounds to have antimicrobial and nematicidal activities. The prepared formulae from the Egyptian isolate of M. verrucaria can be used as safety bio-nematicide against M. incognita on sugar beet.

The oxidation activity of multicopper-oxidases overlaps with different substrates of laccases and bilirubin oxidases, thus in the present study an integrated approach of bioinformatics using homolo...

This study shows for the first time the effect of the fermentation filtrate of Myrothecium verrucaria, strain ZW-2, on four different species of plant-parasitic nematodes. Our results suggested that the fermentation filtrate of M. verrucaria inhibited hatching of Meloidogyne incognita and Heterodera glycines eggs. The lowest egg hatching rates of H. glycines and M. incognita were 6.3% and 2.0%, respectively, after 15 days of incubation with a fermentation filtrate of M. verrucaria. A 2-week fermentation filtrate had lethal effects on of M. incognita (second stage juveniles), H. glycines and Bursaphelenchus xylophilus. After 72 h of incubation, the following mortality rates were observed: M. incognita J2s, 100%; Hirschmanniella spp., 8.4%; H. glycines J2s, 82.4%; and B. xylophilus, 96.1%. A pot test showed that the mean suppressive rates of H. glycines after one- and two-week fermentation treatments were 78.8% and 91.2%, respectively. There were 0.8 nematodes per tomato root system at seven days after inoculation (DAI) and 4.4 galls and 933.3 eggs per root system at 30 DAI, which were significantly lower than those of the tap water controls (8.0 nematodes, 31.4 galls and 14,622.5 eggs per root system). Our studies confirmed that the fermentation filtrate of strain ZW-2 has nematicidal effects against three plant-parasitic nematodes.

A mycelial formulation of the bioherbicidal fungus Myrothecium verrucaria (Alb. & Schwein.) Ditmar: Fr. (MV) was tested alone and in combination with a commercially available glyphosate [N-(phosphonomethyl)glycine] (GLY) product for controlling the invasive vines, redvine [Brunnichia ovata (Walt.) Shinners], and trumpet creeper [Campsis radicans (L.) Seem. ex Bureau] in field experiments conducted near Stoneville, MS. Several application timing regimens were evaluated (Fall, Spring, Fall + Spring, and Spring + Fall). We found that a Fall + Spring application of MV + GLY controlled redvine and trumpet creeper by 95%, 12 days after the second treatment, through a synergistic interaction of the fungus and glyphosate. Disease symptomatology was characterized by rapid necrosis of leaf and stem tissues, with mortality occurring within 72 h. Neither glyphosate alone, nor MV alone, effectively controlled either weed species under any application timing regimen. No visual disease or herbicide damage occurred on glyphosate-resistant soybean plants in the treated test plots. These results suggest that some formulations of glyphosate, mixed with the bioherbicide MV, can effectively control redvine and trumpet creeper, two of the most troublesome weeds in the row crops of the Mississippi Delta region in the mid-southern U.S.

Bilirubin oxidases (BODs), which catalyze the oxidation of bilirubin to biliverdin, are multi-copper oxidases that can be used in clinical diagnoses. Given their different reported enzymatic characterizations from laccases, BODs have gained considerable attention for their application in healthcare and environment management. In this study, a BOD gene from Myrothecium verrucaria was chemically synthesized and optimized to heterogeneous expression in Arabidopsis thaliana. The optimized DNA sequence showed 80.1% similarity to the original sequences. The gene was successfully transferred into A. thaliana, and the crude extracts of transgenic plants showed higher BOD activity compared with that of untransformed plants. BOD expressed in transgenic A. thaliana also exhibited an activity on the typical substrate (ABTS) of laccase and was able to decolorize the textile dyes crystal violet and malachite green. The results indicated that the M. verrucaria BOD gene could be transgenically exploited in plants for dye decolorization.

Previously we found two biotypes of Amaranthus palmeri (Palmer amaranth) in a population of this economically important weed that were resistant to glyphosate but differed with respect to pigmentation. One biotype was typically red-pigmented (betacyanin) while the other was green, with no visual appearance of red hue on any plant part at any growth stage. We have also reported that a strain of Myrothecium verrucaria (MV) exhibited bioherbicidal activity against several important weeds including glyphosate-resistant Palmer amaranth. In greenhouse tests, MV was applied to these two biotypes (red and green) at two ages (3-week- and 6-week-old) and effects of this fungus monitored over a 5-day time course. Initial symptoms of MV (16 to 24 h after inoculation) were: epinastic curvature, wilting and development of lesions on leaves and stems. Generally, the younger plants tended to be more sensitive to MV than older plants. Bioherbicidal damage increased with time leading to necrosis and plant mortality and increasing disease progress. Severe loss of fresh weight occurred in both biotypes as compared to untreated plants. Results indicated that MV was effective on both biotypes, but effects on growth reduction and disease progression were more rapid and generally greater in the green biotype, suggesting that compounds responsible for red pigmentation may be more potent as defense against pathogen attack.

The fungal genus Myrothecium was once polyphyletic but a recent reconsideration of the family Stachybotryaceae spilt it into several genera. The ex-neotype specimen of the species Myrothecium verrucaria is now recognized as Albifimbria verrucaria. The well-studied plant pathogen and candidate bioherbicide CABI-IMI 368023, previously identified as M. verrucaria, was analyzed morphologically and genetically and found to be most consistently aligned with the other representatives of A. verrucaria.

In China, the production of crop straw has been estimated to be approximately 800 Million tons yearly of which about 40% was burned. Corn stover is one of the main agricultural wastes in China. It has shown that lignin in corn stover could be effectively removed byMyrothecium verrucaria. The effects of the pretreatment of corn strover by Myrothecium verrucaria on compost were studied. The results showed that corn stover pretreatment by Myrothecium verrucaria, the Cellulose, Hemicellulose, and lignin were degraded and the results were 33.43%, 11.53% and 18.70% respectively. Scanning electron microscope (SEM) analysis showed that the surface structure of corn stover was changed. Fourier transform infrared spectroscopy (FTIR) analysis showed that the degradation products of lignin were increased. The exposed area of cellulose and hemicellulose was increased. Compared with the control group, the pH value was stable and the temperature was higher. The content of nitrogen in the material decreased, while the contents of total phosphorus and total potassium increased.The C/N ratio of materials decreased after composting.The results showed that the pretreatment of Myrothecium verrucaria improve the degradation of lignocelluloses, a great contribution was made to reduce the causes loss of plant nutrient and to fight against environmental pollution.

To report a case of keratomycosis caused by a very rare pathogen, Myrothecium verrucaria. This is a case report. A 53-year-old man complaint of left eye redness, irritation, intermittent pain after ashes entered his left eye. The patient was examined by slit lamp, anterior segment OCT and in vivo confocal microscopy. The HRT III-RCM image showed massive interlocking white thin lines in the cornea stroma. Corneal scrapings were collected for pathogen culture and PCR test. M. verrucaria was isolated and identified. Hourly topical natamycin (5%) and voriconazole (10 mg/ml) was given as well as intravenous fluconazole (200 mg per day). Treatment was continued with oral itraconazole, 200 mg/day, topical natamycin (5%), 4 times/day, and pranoprofen, 4 times/day. The therapy was tapered off over one and half a month. The cornea lesion healed with scar formation two months later. This is the first case report of M. verrucaria keratomycosis in China. We are the first to show the characteristic of M. verrucaria on cornea with In vivo confocal microscopy. A combination treatment of tropical natamycin, voriconazole and systemic fluconazole was effective in the treatment of M. verrucaria.

High-performance autotolerant bioelectrodes should be ideally suited to design implantable bioelectronic devices. Because of its high redox potential and ability to reduce oxygen directly to water, human ceruloplasmin, HCp, the only blue multicopper oxidase present in human plasma, appears to be the ultimate biocatalyst for oxygen biosensors and also biocathodes in biological power sources. In comparison to fungal and plant blue multicopper oxidases, e.g. Myrothecium verrucaria bilirubin oxidase and Rhus vernicifera laccase, respectively, the inflammatory response to HCp in human blood is significantly reduced. Partial purification of HCp allowed to preserve the native conformation of the enzyme and its biocatalytic activity. Therefore, electrochemical studies were carried out with the partially purified enzyme immobilised on nanostructured graphite electrodes at physiological pH and temperature. Amperometric investigations revealed low reductive current densities, i.e. about 1.65 µA cm−2 in oxygenated electrolyte and in the absence of any mediator, demonstrating nevertheless direct electron transfer based O2 bioelectroreduction by HCp for the first time. The reductive current density obtained in the mediated system was about 12 µA cm−2. Even though the inflammatory response of HCp is diminished in human blood, inadequate bioelectrocatalytic performance hinders its use as a cathodic bioelement in a biofuel cell. Swedish Knowledge Foundation 20170168 Ministerio de Economía y Competitividad BES-2015-071247

Fumigant use in perennial crops can be reduced through prolonging the life of existing orchards. The longer an orchard remains healthy and productive, the less often it will be terminated, fumigated, and replanted. Two trials were conducted to determine the effectiveness of DiTera, a toxin produced by the fungus (Myrothecium verrucaria) and Nema-Q, an extract of the soap bark tree (Quillaja saponaria) for management of root-lesion (Pratylenchus vulnus) and ring (Mesocriconema xenopla) nematodes on walnuts. In the first trial, spring and fall treatments of DiTera were applied each year for four years to variety ‘Chandler’ scion on seedling ‘Paradox’ rootstock, and to own-rooted ‘Chandler’ trees. On ‘Paradox’ rootstock, both DiTera and Nema-Q increased walnut yields (P ≤ 0.05) and produced more vigorous trees (P ≤ 0.05) without significant reductions in nematode populations (P ≤ 0.05). A second trial was conducted with three rates of DiTera and four rates of Nema-Q, combinations of the two products, and an untreated control on ‘Chandler’ scion on ‘Paradox’ rootstock. The highest rate of Nema-Q (P ≤ 0.05), and a combination treatment of DiTera plus Nema-Q (P ≤ 0.05) increased trunk circumference. Several treatments showed reductions in root-lesion and ring nematodes (P ≤ 0.05). Bionematicides can improve yield, growth, and vigor in walnut orchards infested with plant-parasitic nematodes.

Beneficial microbes used for the control of crop pests have one or more modes of action against their target arthropods, nematodes, pathogens, or weeds. Metabolites, enzymes, volatile compounds, and other bioactive materials help them to antagonize and suppress target pests. The pesticidal activity of several microbial metabolites has been extensively studied, but only a few have commercial potential. While microbes themselves, rather than their metabolites, are primarily used for plant disease control, metabolites such as anisomycin, avermectins, bialophos, and spinosad are some examples of successful commercialization for insect, mite, nematode, and weed control. Biopesticides based on fermentation solids and solubles, without live microbes, are also available in the market. Pesticides based on Burkholderia rinojensis and Chromobacterium subtsugae for insect and mite control and Myrothecium verrucaria for nematode control are some of such examples that do not have viable microbes. Microbial biopesticides play a critical role in integrated pest management and maintaining crop productivity. This chapter focuses on microbial metabolites and metabolite-producing microbes that are commercialized and briefly discusses the potential of others.

Laccase has the ability to act on a broad range of substrates, as a result of which multiple compounds have been used for laccase assay and numerous applications have been discovered over time. Thus, considering the above points, in the present study, white and blue laccase from Myrothecium verrucaria ITCC-8447 and Pleurotus ostreatus were used for laccase assay using various substrates which includes 2,2′ azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,6-dimethoxyphenol (2, 6 DMP), guaiacol (GCL), and syringaldazine (SYZ). The most effective substrate of the four substrates was ABTS for white and blue laccase. The white and blue laccase were further used for the synthesis of bio-material via fungal-assisted treatment of wheat bran (WB) and sugarcane bagasse (SB) followed by micro-wave pre-treatment and the addition of starch and glycerol to the treated agro-residue in the ratio of 5:1:1. The thickness swelling and water absorption percentage was minimum for wheat bran synthesized bio-material using white laccase in comparison to the other synthesized bio-material.

The construction of bias- and donor-free photobioelectrochemical cells for the generation of light-triggered electrical power is presented. The developed oxygen reduction biocathodes are based on bilirubin oxidase (BOD) that originates from Myrothecium verrucaria (MvBOD) and a thermophilic Bacillus pumilus (BpBOD). Methods to entrap the BOD with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) redox molecules in a polydopamine layer are presented. A pH-independent, positively charged pyrenebetaine linker was synthesized, utilized, and led to a threefold improvement to the bioelectrocatalytic current. Both the developed polydopamine/ABTS/MvBOD and the pyrenebetaine/BpBOD biocathodes were further coupled with BiVO4 /cobalt phosphate water-oxidation photoanodes to construct biotic/abiotic photobioelectrochemical cells, which generated power outputs of 0.74 and 0.85 mW cm-2 , respectively. The presented methods are versatile, show the strength of biotic/abiotic hybrids, and can be further used to couple different redox enzymes with electrodes.

Bilirubin oxidase (BOD) belongs to the family of blue multicopper oxidases, and catalyzes the concomitant oxidation of bilirubin to biliverdin and the reduction of molecular oxygen to water via a four-electron reduction system. The active sites of BOD comprise four copper atoms; type I copper (T1Cu) forms a mononuclear site, and a cluster of three copper atoms forms a trinuclear center. In the present study, we determined the high-resolution crystal structures of BOD from the fungus Myrothecium verrucaria. We investigated wild-type (WT) BOD and a BOD mutant called Met467Gln, which is inactive against bilirubin. The structures revealed that a novel post-translational crosslink between Trp396 and His398 is formed in the vicinity of the T1Cu site in WT BOD, whereas it is absent in the Met467Gln mutant. Our structural and computational studies suggest that the His-Trp crosslink adjusts the redox potential of T1Cu to that of bilirubin to efficiently abstract electrons from the substrate.

In this work, corn stover was studied as a lignocellulosic material with the potential of serving as a feedstock in the field of bioenergetics. To improve the enzymatic digestibility of carbohydrates in the lignocellulosic biomass, corn stover was pretreated using Myrothecium verrucaria to deplete the lignin, combined with a solid acid pretreatment method to deplete the hemicellulose. The effects of lignin degradation conditions and solid acid concentration on cellulose conversion were investigated. The two-stage pretreatment with M. verrucaria and solid acid under optimal conditions gave 81.54% cellulose conversion yield, 214.22% higher than that of untreated corn stover. Significant lignin and hemicellulose removal rates were observed with the two-stage pretreatment. Scanning electron microscope (SEM), X-ray diffractometer (XRD) and Fourier transform infrared spectroscopy (FTIR) analyses showed major structural changes in the pretreated corn stover. This study provided an efficient strategy to enhance the enzymatic saccharification of cellulose in corn stover.

Multicopper oxidases such as bilirubin oxidase (BOD) from Myrothecium verrucaria and laccase (LC) from the basidial fungus Trametes hirsuta have been used as catalysts in dihydroquercetin (DHQ) oxidative polymerization. The conditions selected enabled good yields of DHQ oligomers, which were then analyzed using UV-vis, FTIR, 1Н and 13С NMR spectroscopy. DHQ oligomers synthesized using both enzymes showed higher thermostability as compared with the monomer. Depending on the oxidase, the products of DHQ polymerization differed in physicochemical properties, and as shown by NMR studies, had different structures.

The widespread use of synthetic nematicides has caused significant problems to the environment as well as human health. To address this issue, eco-friendly control measures, such as microbial nematicides, are being developed. During the screening of Myrothecium strains with nematicidal activity against the root-knot nematode (RKN) Meloidogyne incognita, we found that the acetone extract of Myrothecium sp. KACC 40321 was highly effective against hatched juveniles of M. incognita at 7 days after exposure. The fungus was identified as Meloidogyne verrucaria. Two macrocyclic trichothecenes verrucarin A and roridin A were isolated and identified as major active metabolites by bioassay-guided fractionation and instrumental analysis. When the second-stage juveniles were treated with the chemicals, no juvenile mortality was observed. However, they effectively killed juveniles from treated eggs. The hatched juvenile mortality was used to evaluate the in vitro nematicidal activity of the compounds against M. incognita. The median effective concentrations were 1.88 μg/mL for verrucarin A and 1.50 μg/mL for roridin A. Among various liquid media, commercial malt extract broth (cMEB) was found to be the best for the production of verrucarin A and roridin A, followed by potato dextrose broth. The cMEB culture filtrate effectively reduced the formation of galls and egg masses on tomato roots in a pot experiment. In addition, the culture filtrate reduced the formation of galls on the roots of melon plants and the number of RKNs in the soils under field conditions. These results suggest that M. verrucaria KACC 40321 can be used as a biocontrol agent against RKNs in various crops. To the best of our knowledge, this is the first study to report the effectiveness of verrucarin A and rorridin A against hatched juveniles of M. incognita.

This paper presents an optimally bound bilirubin oxidase (BOD) (Myrothecium verrucaria) on palladium nanoparticles (Pd NPs) for enhanced oxygen reduction reaction (ORR). Theoretical modeling of BOD on Pd demonstrated that Pd has strong preferential binding to BOD via T1 copper (Cu) site because of its high adsorption energy. This preferential binding was accompanied by a reduction in distance between the Cu active sites and Pd which would result in an increase in electron transfer rate (kcat) and an enhancement in catalytic activity of BOD. Inspired by the computational results, a biocathode comprising carbon nanotube (CNT), Pd NPs, and BOD (CNT-Pd-BOD) was facilely fabricated using an electroless deposition method. The CNT-Pd-BOD biocathode exhibited higher catalytic activity (1.52 times) and kcat (1.71 times) when compared with CNT-BOD only biocathode. These results demonstrate Pd NPs as a suitable substrate for preferential binding with BOD to increase catalytic activity.

A potential pulse-assisted approach was used to immobilize Myrothecium verrucaria bilirubin oxidase at planar and nanoporous gold electrodes (NPG) containing pores of ca. 20 nm and ca. 40 nm in diameter. An increase in the current due to the bioelectrocatalytic reduction of oxygen by MvBOD-modified gold electrodes obtained from a 20 μL drop by the proposed pulse-assisted approach was observed when compared to the response obtained with electrodes modified by drop-casting. This increase likely arises from a preferential orientation of MvBOD molecules at the planar gold surface obtained by fast switching of the potential pulses between opposite charges. The concomitant ion stirring effect induces the attraction of the enzymes to the charged gold surface and forces access to the internal pore volume of the NPG. Immobilization of MvBOD using the potential pulse-assisted approach significantly increases current densities by facilitating the electron transfer between the enzyme and the electrode surface.

Previously we found that a strain of Myrothecium verrucaria (MV) exhibited bioherbicidal activity against several important weeds, and that some commercial formulations of glyphosate applied with MV resulted in synergistic interactions that improved weed control efficacy. We also found that MV had bioherbicidal activity against glyphosate-resistant Palmer amaranth. We have also reported that some commercial formulations are inhibitory to MV. Our objectives were to test the effect of unformulated glyphosate (high purity, technical-grade glyphosate) alone and in combination with MV for bioherbicidal activity on glyphosate-susceptible and -resistant Palmer amaranth biotypes under greenhouse conditions and to examine technical-grade glyphosate on the growth of this bioherbicide. High purity glyphosate (without adjuvants/surfactants) was not toxic to MV growth and sporulation at concentrations up to 2.0 mM when grown on agar supplemented with the herbicide. Both biotypes were injured by MV and MV plus glyphosate treatments as early as 19 h after application (3 h after a dew period of 16 h). These injury effects increased and were more evident through the 6-day time course, when after 120 h the MV plus glyphosate treatment had killed all glyphosate-susceptible and -resistant plants. The interaction of glyphosate plus MV was synergistic toward the control of Palmer amaranth. Data strongly suggest that the active ingredient is responsible for the synergy previously found when this bioherbicide was combined with some commercial formulations of glyphosate. Results demonstrated that MV can control both glyphosate-resistant and -susceptible Palmer amaranth seedlings and act synergistically with high-purity glyphosate to provide improved weed control.

We isolated three laccase-producing fungus strains from Taxus rhizosphere. Myrotheium verrucaria strain DJTU-sh7 had the highest laccase activity of 216.2 U/ml, which was increased to above 300 U/ml after optimization. DJTU-sh7 had the best decolorizing effect for three classes of reactive dyes. The DJTU-sh7-containing fungal consortium displayed the robust decolorizing ability. Both color removal efficiency and chemical oxygen demand were increased in the consortium mediated biotransformation. Transcriptome changes of M. verrucaria elicited by azo dye and phenolic were quantified by the high throughput transcriptome sequencing, and the activities of the selected oxidases and reductases were determined. The possible involvement of oxidases and reductases, especially laccase, aryl alcohol oxidase, and ferric reductase in the biotransformation of dye and phenolic compounds was revealed at both transcriptomic and phenotypic levels. Revealing the transcriptomic mechanisms of fungi in dealing with organic pollutants facilitates the fine-tuned manipulation of strains in developing novel bioremediation and biodegradation strategies. We isolated three laccase-producing fungus strains from Taxus rhizosphere. Myrotheium verrucaria strain DJTU-sh7 had the highest laccase activity of 216.2 U/ml, which was increased to above 300 U/ml after optimization. DJTU-sh7 had the best decolorizing effect for three classes of reactive dyes. The DJTU-sh7-containing fungal consortium displayed the robust decolorizing ability. Both color removal efficiency and chemical oxygen demand were increased in the consortium mediated biotransformation. Transcriptome changes of M. verrucaria elicited by azo dye and phenolic were quantified by the high throughput transcriptome sequencing, and the activities of the selected oxidases and reductases were determined. The possible involvement of oxidases and reductases, especially laccase, aryl alcohol oxidase, and ferric reductase in the biotransformation of dye and phenolic compounds was revealed at both transcriptomic and phenotypic levels. Revealing the transcriptomic mechanisms of fungi in dealing with organic pollutants facilitates the fine-tuned manipulation of strains in developing novel bioremediation and biodegradation strategies.

Laboratory and nursery experiments were conducted to identify the causal agent of a needle blight of Pinus wallichiana, a species native to the Western Himalayas. The pathogen was identified as Myrothecium verrucaria, on the basis of morphological, cultural and molecular characterization. BLAST analysis of ITS sequences of the pathogen revealed maximum sequence identity of 99% with M. verrucaria. The sequence is the first of this fungus from P. wallichiana. Phylogenetic analysis grouped all M. verrucaria isolates in a single clade; M. roridum and M. inundatum clustered in separate clades. The pathogen grew optimally at 25 ± 1 °C on oat meal agar, pH 5.5. Inoculation experiments with M. verrucaria demonstrated pathogenicity on Pinus halepensis, Cedrus deodara and Cryptomeria japonica, in addition to Pinus wallichiana.

The shelf-life of a bioherbicide product is an important factor with regard to its commercial potential. The bioherbicidal efficacy of freshly fermented Myrothecium verrucaria (strain IMI 368023) (MV) mycelia formulations and MV mycelia preparations that had been freeze-dried and then stored at -20°C for 8 years was compared. Two concentrations of each formulation (1.0x and 0.5x) were tested, utilizing bioassays on seedlings of the weed, hemp sesbania (Sesbania exaltata) under greenhouse conditions or in darkness utilizing hydroponically grown seedlings. Freeze drying of freshly prepared MV mycelium produced a light, brownish-colored powder. Efficacy tests of this reconstituted 8-year-old dried material showed that some bioherbicidal activity was lost during long-term storage, i.e., ~20% and ~60% seedling dry weight reduction at the 1.0x and 0.5x rate, respectively. Although plant mortality was greater in the fresh mycelial preparations treatments versus the freeze-dried and stored samples at all time points in the time-course, the stored material still caused >80% mortality, 15 days after treatment. Comparative disease progression ratings also showed a similar trend. Overall results show that freeze-drying MV is a useful method to reduce the bulk and cumbersomeness of storing heavy liquid fermentation product, while retaining bioherbicidal activity. These findings increase the utility of this bioherbicide and offer the potential to use the dried material in soil treatments or in a more concentrated form than attainable via the fermented product.

Chitin, a crucial structural and functional component of insects and fungi, serves as a target for pest management by utilizing novel chitinases. Here, we report the biocontrol potential of recombinant Myrothecium verrucaria endochitinase (rMvEChi) against insect pest and fungal pathogens. A complete ORF of MvEChi (1185 bp) was cloned and heterologously expressed in Escherichia coli. Structure based sequence alignment of MvEChi revealed the presence of conserved domains SXGG and DXXDXDXE specific for GH-18 family, involved in substrate binding and catalysis, respectively. rMvEChi (46.6 kDa) showed optimum pH and temperature as 7.0 and 30 °C, respectively. Furthermore, rMvEChi remained stable within the pH range of 6.0 to 8.0 and up to 40 °C. rMvEChi exhibited kcat/Km values of 129.83 × 103 [(g/L)−1 s−1] towards 4MU chitotrioside. Hydrolysis of chitooligosaccharides with various degrees of polymerization (DP) using rMvEChi indicated the release of DP2 as main end product with order of reaction as DP6 > DP5 > DP4 > DP3. Bioassay of rMvEChi against Helicoverpa armigera displayed potent anti-feedant activity and induced mortality. In vitro antifungal activity against plant pathogenic fungi (Ustilago maydis and Bipolaris sorokiniana) exhibited significant inhibition of mycelium growth. These results suggest that MvEChi has significant potential in enzyme-based pest and pathogen management.

Trichoderma reesei NRRL 11236, Trichoderma viride ATCC 32630, Trichoderma viride ATCC 32098 and Myrothecium verrucaria ATCC 9095 were evaluated for the production of toxic substances with the fertile egg tests. Strains NRRL 11236, ATCC 32630, and ATCC 9095 produced no detectable toxic substance. However, when an oil extract was made of fermented corn cobs, strain ATCC 32098 produced a significant kill of 23.1% of the embryos. Cellulases (C1,Cx) and beta glucosidase production were produced using different ammonium salts as additional nitrogen sources to what the corn cobs contained. Temperatures (23° and 30°C) had no effect on beta glucosidase except for T. viride ATCC 32630 which produced significantly smaller quantities at 30°C than at 23°C.

El nematodo agallador (Meloidogyne sp.) tiene importancia económica potencial en diversos cultivos, en su combate casi siempre se han utilizado diferentes productos de agroquímicos, por lo que se necesita evaluar nuevos productos que sean una mejor alternativa en su manejo y en el cuidado al medio ambiente. En el presente trabajo se evaluó la eficacia biológica de diferentes extractos del quitosano un biopolímero natural con efecto nematicida y regulador de poblaciones de estos fitonematodos y Fluensulfone un nematicida de amplio espectro, el estudio se realizó In vitro, se colectaron muestras de suelo y se extrajeron los nematodos mediante el método de embudo de Baermann, se colocaron en cajas petri en diluciones con 30-35 nematodos por unidad experimental, en donde para su control se utilizó quitosano extraído de langostino de rio y quitosano comercial a diferentes concentraciones (0. 25, 0.50, 0.75), así mismo un nematicida comercial con extracto de quitosano Nematrol Plus , esto comparado con otros nematicidas comerciales Ditera (Myrothecium verrucaria ), Nemover (Extracto de pino) y Nimitz (Fluensulfone) .Se encontró que el efecto nematicida del quitosano en dosis media (0.50) fue mejor posterior a las 24 horas después de la aplicación con una mortalidad de 95.83 a 100 % de nematodos, y una eficacia del 100 %, esto comparado con los nematicidas comerciales.

We report the controlled orientation of bilirubin oxidases (BOD) from Myrothecium verrucaria on multiwalled carbon nanotubes (MWCNTs) functionalised by electrografting of 6-carboxynaphthalenediazonium and 4-(2-aminoethyl)benzenediazonium salts. On negatively charged naphthoate-modified MWCNTs, a high-potential (0.44 V vs. SCE) oxygen reduction electrocatalysis is observed, occurring via the T1 copper centre. On positively charged ammonium-modified MWCNTs, a low-potential (0.15 V) oxygen reduction electrocatalysis is observed, occurring through a partially oxidised state of the T2/T3 trinuclear copper cluster. Finally, chemically modified naphthoate MWCNTs exhibit high bioelectrocatalytic current densities of 3.9 mA cm(-2) under air at gas-diffusion electrode.

Among the most important and visible weeds in the Southeastern USA is the exotic invasive vine, kudzu (Pueraria montana var. lobata). Efforts to eradicate it typically involve many years of application of restricted-use pesticides. Recent availability of effective, non-restricted-use pesticides and developments with the application of the bioherbicide Myrothecium verrucaria has made possible new control programmes for kudzu management. Field trials at three sites over two years with aminocyclopyrachlor, aminopyralid, fluroxypyr, metsulfuron methyl and combinations of these herbicides achieved 99–100% reduction in aboveground kudzu biomass. Additionally, programmes were developed that eradicated kudzu while simultaneously establishing native vegetation. One of these successful programmes integrated bioherbicide application, mechanical removal of kudzu biomass and planting switchgrass (Panicum virgatum) in an entirely chemical herbicide-free system. These field tests demonstrate a variety of methods...

Comparative studies were conducted on mycelial preparations of the bioherbicide, Myrothecium verrucaria (MV) strain IMI 361690 and a recently discovered sector (MV-Sector BSH) of this fungus. The whitish sector was discovered, isolated, grown in pure culture on PDA and found to be a stable, non-spore producing mutant when cultured over several months under conditions that cause circadian sporulation during growth of its MV parent. Application of MV and MV-Sector BSH mycelial preparations to intact plants (hemp sesbania and sicklepod) and leaf discs (kudzu and glyphosate-resistant Palmer amaranth) showed that the sector efficacy was generally equal to, or slightly lower than MV. Bioassays of MV and this sector on seed germination and early growth of sicklepod and hemp sesbania seeds demonstrated that hemp sesbania seeds were slightly more sensitive to the fungus than sicklepod seeds and that the sector bioherbicidal activity was slightly less than that of MV. SDS-PAGE protein profiles of cellular extracts of MV and the sector and their respective culture supernatants showed several differences with respect to quantity and number of certain protein bands. Overall results showed that the isolate was a non-spore producing mutant with phytotoxicity to several weeds (including weeds tolerant or resistant to glyphosate), and that the phytotoxic effects were generally equivalent to those caused by MV treatment. Results of this first report of a non-sporulating MV mutant that suggest additional studies on protein analysis, and an extended weed host range under greenhouse and field conditions are needed in order to further evaluate its possible bioherbicidal potential.

Myco-valorization (Myco-Valtn) concept was explored using Myrothecium verrucaria ITCC-8447 via column bioreactor to remove two anthraquinone (ARq) dyes. Among the three myco-valtn approaches free cell dependent enabled more than 80% removal of Anthraquinone violet R (AVR) and Alizarin cyanine green (ACG) after 48 h at 30 ± 2 °C. Enzymatic treatment removed more than 50% AVR and ACG after 10 min. However, synthetic and natural support entrapment on Scotch-Brite® (SB) and Luffa cylindrica (LC) enabled more than 80% removal in SB-ACG and LC-ACG and more than 60% in SB-AVR and LC-AVR in 6th cycle. The regenerated column enhanced the removal of dye and allowed the column to run for multiple cycles. Further after FTIR XRD and LCMS Q-TOF, analysis it was observed that ACG and AVR were completely degraded and laccase played the key roles in the degradation of ACG and AVR and the degraded metabolites of anthraquinone dyes (ACG and AVR) exhibited much less toxicity than the parent dye using phytotoxicity assays.

In the present work attempts have been made to mimic the natural system of biomass delignification using laccase from Myrothecium verrucaria ITCC-8447 and synergistic effect of cellulase and xylanase from Schizophyllum commune NAIMCC-F-03379 and Aspergillus oryzae ITCC-8571 respectively for enhanced saccharification and subsequent bioethanol production. The laccase mediated pretreatment resulted in 6.7% delignification of rice straw (RS). The structural analysis of enzymatic pretreated RS using XRD and FTIR suggested high crystallinity index (CrI) and improved accessibility of cellulose and hemicelluloses as compared to untreated RS. The single step simultaneous delignification, saccharification and fermentation (SDSF) of RS using partially purified in-house enzyme cocktail with Saccharomyces cerevisiae MTCC-173 resulted in sugar yield of 26.7 ± 0.2 g/L with an experimental ethanol yield of 6.47 ± 0.16 g/L which is comparable to maximum sugar and ethanol yield of 31.2 ± 0.7 g/L and 7.34 ± 0.39 g/L respectively using commercial enzyme cocktail with S. cerevisiae MTCC-173. The SDSF was obtained as a suitable single step process for the ethanol production from RS.

Multicopper oxidases (MCOs) has a unique feature of having the presence of four Cu atoms arranged into three (Type I, II and III) spectral classification. MCOs laccase due to its broad range of substrate specificity has numerous biotechnological applications. The two types of laccases include the typical blue and the atypical white, yellow laccases which have been isolated from diverse geographical locations globally. In the present study laccases were identified using Liquid Chromatograph Mass Spectrometer Studies (LCMS) study where blue laccase exhibited homology with Trametes villosa Q99044 and Q99046 and white, yellow laccase exhibited homology with Myrothecium verrucaria OX = 1859699; Q12737 and Trametes versicolor Q12717 respectively. The spectral comparison between laccases were determined via spectroscopic analysis where UV-spectra of blue laccase from Trametes versicolor had a peak at 605 nm (Type I Cu atom) whereas in case of white and yellow laccases the peak was absent and in addition had an absorption peak at 400nm. It was followed by X-Ray Diffraction (XRD) analysis of proteins where α-helix (10°) and β-sheet (22°) structure were observed in case of all the three laccases. However, the intensity of α-helix in white and yellow laccase was stronger as compared to the blue laccase whereas the intensity of β-sheet was stronger in case of blue laccase as compared to other two laccases. Further, Fourier-transform infrared spectroscopy (FTIR) analysis was performed which enabled the analysis of proteins where α-helix (1650–1658 cm−1), β-sheets (1620–1640 cm−1), amide I (1700−1600 cm−1) amide II (bands at under 1400 cm−1) and amide A, B (bands above 3000 cm−1)., Bioinformatics; Microbiology; Structural biology; Mycology; Microorganism; Enzymology; Proteomics; Spectroscopy; Laccase; LCMS; UV-Spectra; XRD; FTIR

Made available in DSpace on 2019-10-06T15:33:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-05-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Regio and stereoselective activation of sp 3 C–H bonds remain one of the major advantages of biocatalysis over traditional chemocatalytic methods. Herein, we describe the oxy-functionalization of halimane diterpenoid 1 by whole cells of three filamentous fungi, aiming to obtain derivatives with desirable biological properties. After incubating 1 with Fusarium oxysporum, Myrothecium verrucaria, and Rhinocladiella similis at different concentrations and incubation times, four known (3, 5, 6, and 7) and three new (2, 4, and 8) halimane derivatives were obtained and characterized. F. oxysporum catalyzed the hydroxylation of positions C-2 (2) and C-7 (4), while R. similis simultaneously mediated the 2-oxo-functionalization and the hydration of 13,14-(C–C)double bond belonging to an α,β-unsaturated carbonyl system (8). Compounds 1–7 were non-cytotoxic against HCT-116 and MCF-7 cancer cell lines at tested concentrations. However, substrate 1 displayed moderate reduction ability against biofilm produced by Staphylococcus epidermidis ATCC35984 (84% at 1.6 mM), and this effect was retained to some extent by derivatives 4 and 7. These results emphasize the prominent potential of filamentous fungi associated with the microbiota of medicinal plants as versatile catalysts for singularly useful reactions through their complex enzymatic machinery, as well as the high susceptibility of halimane-diterpenoid substrates. Núcleo de Bioensaios Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE) Universidade Estadual Paulista (UNESP) Instituto de Química Departamento de Química Orgância, Francisco Degni, 55 São Carlos Institute of Physics University of São Paulo, PO Box 369 Universidade de São Paulo (USP) Instituto de Ciências Biomédicas, Av. Lineu Prestes, 1524 Núcleo de Bioensaios Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE) Universidade Estadual Paulista (UNESP) Instituto de Química Departamento de Química Orgância, Francisco Degni, 55 CAPES: Finance code 001 CAPES: PROEX – 1427484

The white laccase was produced from Myrothecium verrucaria ITCC-8447 under submerged fermentation. The media components were optimized by response surface methodology (CCD-RSM). The nutritional components (glucose and peptone) and physical parameters (pH and temperature) were optimized by response surface methodology for enhanced laccase production by Myrothecium verrucaria ITCC-8447. The enzyme activity under optimum condition exhibited 1.45 fold increases in laccase activity. The white laccase was subjected to ion exchange chromatography with 6 fold purification. The molecular weight of white laccase was ~63-75kDa as estimated by SDS-PAGE followed by the activity staining with ABTS where green bands confirmed the presence of laccase. The enzyme was stable over an alkaline pH range of 7-9 and the temperature range of 30-40°C. The characterization of white laccase was done by CD spectra, UV-visible absorption, FTIR and XRD. The Km and Vmax values of the purified laccase were 2.5mM and1818.2μmol/min/L. The delignification capability of the white laccase was determined by reduction in Kappa number (58.8%) and Klason lignin (64.7%) of wheat straw after 12h of incubation. Further the delignification was confirmed FTIR and XRD.

Herein, the direct electrochemistry of bilirubin oxidase from Magnaporthe orizae (MoBOD) was studied on CNTs functionalized by electrografting several types of diazonium salts. The functionalization induces favorable or unfavorable orientation of MoBOD, the latter being compared to the well-known BOD from Myrothecium verrucaria (MvBOD). On the same nanostructured electrodes, MoBOD can surpass MvBOD in terms of both current densities and minimal overpotentials. Added to the fact that MoBOD is also highly active at the gas-diffusion electrode (GDE), these findings make MoBOD one of the MCOs with the highest catalytic activity towards the oxygen reduction reaction (ORR).

Three new tetralol analogs, myrochromanols A-C (1–3), together with 11 known trichothecenes (4–14), were isolated from a soil fungus Myrothecium verrucaria HL-P-1. The structures of the three new compounds were elucidated by extensive spectroscopic analysis including HRESIMS, NMR, and ECD calculation. All of the new compounds were tested for their anti-inflammatory activity and cytotoxicity. Compounds 1 and 3 inhibited lipopolysaccharide (LPS)-induced NO production in BV2 cells with IC50 values of 26.04 and 25.80 μM, respectively. Three new tetralol analogs were isolated from a soil fungus Myrothecium verrucaria HL-P-1. All of them were tested for their anti-inflammatory and cytotoxicity activity.

Italy is one of the leading countries for the production and consumption of ready‐to‐eat salads. This has led to a steady increase in the area used over the last decade for the growth of leafy vegetables under intensive systems. The recent new diseases that affect lettuce, wild and cultivated rocket, lamb's lettuce, spinach and basil are reported here: Plectosphaerella cucumerina on wild rocket, endive and lamb's lettuce, Fusarium equiseti on wild and cultivated rocket and lettuce, Myrothecium verrucaria on spinach and wild rocket, Myrothecium roridum on lamb's lettuce, Allophoma tropica on lettuce and Alternaria sp. on basil and rocket are among the new foliar pathogens in Italy. Among the soil‐borne pathogens, Pythium aphanidermatum, Pythium irregulare and Pythium Cluster B2a have recently been isolated in Italy on spinach and Swiss chard, lamb's lettuce and lettuce, while Fusarium oxysporum f. sp. lactucae, which causes lettuce wilt, is gradually spreading to new countries. Some of the new pathogens have been found to be transmitted by seed and typical of tropical climate, and are thus favoured by the increases in temperature linked to climate change. The globalization of markets, climate change and intensive cultivation are among the factors responsible for the proliferation and spread of some of these new pathogens that are ‘alien’ to Italian production systems.

Replicated field plots were established and monitored for two years to evaluate management practices for kudzu. The bioherbicidal plant pathogen, Myrothecium verrucaria, several herbicides and a variety of integrated control programmes achieved a high level of kudzu suppression, although no system tested reliably achieved eradication in this time frame.

Options for control of northern root-knot nematode (NRKN, Meloidogyne hapla ) on vegetables are very limited currently. In this study, we characterized the parasitism of Myrothecium verrucaria strain X-16, a new nematophagous fungus, on NRKN at the stages of eggs, J2, and adult females and evaluated its biocontrol efficacy in the greenhouse. Strain X-16 produced conidia that geminated and invaded in 80 h after in contact with eggs, causing the shrinkage and depression of egg shell and blastocolysis of the embryo. The strain also attacked 2nd-stage juveniles by producing developing surface networks of hyphae on the nematode body wall. Strain X-16 attacked adult females by producing dense networks of hyphae on the nematode body wall in 120 h. Strain X-16 had lethal effects (22–71% mortality) against NRKN J2 at the concentration as low as 3.1 × 10 7 conidia/ml and with the incubation treatment time as short as 24 h. The lethal effects linearly increased with the increase of conidial concentration, with the estimated LC 50 values as low as 1.0 × 10 8 conidia/ml. Soil treatments with strain X-16 at 1%, 2% or 4% (wt/wt) induced significant reductions in J2 nematode counts in 100 g of dry soil, Pf/Pi ratios and root-know index in cucumber in the greenhouse evaluations. These studies are the first to demonstrate that M . verrucaria is able to parasitize NRKN and strain X-16 can be a potential biocontrol agent for management of NRKN.

A green efficient strategy for the combination of biodegradation and adsorption methods is urgently desired nowadays to degrade dyes in an economical and effective way. In this study, a kind of novel, environmentally friendly and efficient hybrid for the fungus and chitosan–Fe membrane has been fabricated by the alginate approach. Myrothecium verrucaria I-5 and C-1, deuteromycete fungi capable of producing laccase to degrade dyes, have been used. It is found that the chitosan–Fe membrane can excellently immobilize the fungi mycelia and enzyme, and after the immobilization, the fungi and enzyme still maintain a high activity after the dye decolorization. Also, the immobilized fungi membranes have been proved to have a high efficiency for dye decolorization. Simultaneously, the decolorization ability of the crude enzymes, which are extracted from I-5 and C-1 medium, and the decolorization ability of chitosan–Fe membrane are also investigated, respectively. The results demonstrate that the dyes can be removed by the fungi and the membrane synergistically. In addition, different kinds of dyes are successfully removed with chitosan–Fe immobilized fungi membranes. These results demonstrate that dyes are first adsorbed on the chitosan–Fe membrane, decreasing the toxicity to the fungus, and then bio-degraded by the laccase produced from the fungi on the membrane.

Direct electron transfer (DET) between cellobiose dehydrogenase from Humicola insolens ascomycete (HiCDH) and gold nanoparticles (AuNPs) was achieved by modifying AuNPs with a novel, positively charged thiol N-(6-mercapto)hexylpyridinium (MHP). The DET enabled the use of the HiCDH enzyme as an anodic biocatalyst in the design of a mediatorless carbohydrate/oxygen enzymatic fuel cell (EFC). A biocathode of the EFC was based on bilirubin oxidase from Myrothecium verrucaria (MvBOx) directly immobilised on the surface of AuNPs. The following parameters of the EFC based on Au/AuNP/MHP/HiCDH bioanode and Au/AuNP/MvBOx biocathode were obtained in quiescent air saturated PBS, pH 7.4, containing: (i) 5 mM glucose-open-circuit voltage (OCV) of 0.65 ± 0.011 V and the maximal power density of 4.77 ± 1.34 μW cm−2 at operating voltage of 0.50 V; or (ii) 10 mM lactose-OCV of 0.67 ± 0.006 V and the maximal power density of 8.64 ± 1.91 μW cm−2 at operating voltage of 0.50 V. The half-life operation times of the EFC were estimated to be at least 13 and 44 h in air saturated PBS containing 5 mM glucose and 10 mM lactose, respectively. Among advantages of HiCDH/MvBOx FCs are (i) simplified construction, (ii) relatively high power output with glucose as biofuel, and (iii) the absence of the inhibition of the HiCDH based bioanode by lactose, when compared with the best previously reported CDH based bioanode.

In a laboratory experiment, insects larvae were infected with spores of a strain of M. verrucaria to establish the fungal sensitivity of the larvae. The fungus was reisolated from 30% of dead Scotia sp., 6,6% O. brumata, 10% P. ribesii, 20% Anobidae sp., 10% A. pomorum. Investigated strain of M. verrucaria seems to be potential insect pathogens.

Different modification procedures to stabilize and control the orientation of Myrothecium verrucaria bilirubin oxidase (MvBOD) on 3D carbon nanotube/carbon microfiber-modified graphite electrode surfaces were evaluated for the development of biofuel-cell cathodes. The surface properties of different linkers for covalent binding of BOD were investigated by using atomic force microscopy-based techniques. For all immobilization strategies, the maximal current response was obtained at a pH value of 6.5 with temperatures between 20 and 35 °C. The biocathode based on MvBOD immobilized through an imino bond to the electrode showed the highest current density (1600 μA cm−2) and was resistant to the presence of chloride ions. A biofuel cell was constructed, and it exhibited a maximal power of 54 μW cm−2 at 350 mV with an open-circuit voltage of about 600 mV by using a cellobiose dehydrogenase based bioanode and glucose as the fuel.

Myrothecium verrucaria NF-05 is a deuteromycete fungus capable of producing a white laccase. The optimal concentration of Cu2+ for laccase production by this strain is 0.2 mM (43.23 ± 1.16 U mL− 1). A comprehensive investigation of the induction demonstrated that NF-05 laccase production could be synergistically enhanced by various inducers, including aromatic phenols, amines and recalcitrant dyes, in the presence of 0.2 mM Cu2+. Sixteen phenols, fourteen amines and four dyes exhibited significant inductive effects on laccase production. The best inducer was 3, 3’-dimethylbenzidine, which increased laccase production to 258.1 ± 11.1 U mL− 1. These results suggest that M. verrucaria NF-05 is a promising industrial laccase producer. Based on the increased production, purified NF-05 laccase was used to decolorize dyes of various structural types in the presence of six redox mediators. Among the 26 tested dyes, the decolorization rate of six azo dyes, chromotrope 2R, orange G6, Congo red, Ponceau S, amaranth ...

Here we present the very first study of an enzymatic fuel cell (EFC) in a cell culture. An EFC with Corynascus thermophilus cellobiose dehydrogenase (CDH) based bioanode and Myrothecium verrucaria bilirubin oxidase (BOx) based biocathode was constructed at the bottom of a medusa cell culture plate. The constructed EFC had a power density of up to 25 μW cm(-2) at 0.5 V potential in simple buffer solution and in cell culturing medium. L929 murine fibroblast cells were seeded on top of the EFC and possible effects of the EFC on the cells and vice versa were studied. It was shown that on average the power of the EFC drops by about 70% under a nearly confluent layer of cells. The EFC appeared to have a toxic effect on the L929 cell line. It was concluded that the bioanode, consisting of CDH, produced hydrogen peroxide at toxic concentrations. However, the toxic effect was circumvented by co-immobilizing catalase on the bioanode.

Two blue multicopper oxidases (MCOs) (viz. Trametes hirsuta laccase (ThLc) and Myrothecium verrucaria bilirubin oxidase (MvBOx)) were immobilized on bare polycrystalline gold (Au) surfaces by direct adsorption from both dilute and concentrated enzyme solutions. The adsorption was studied in situ by means of null ellipsometry. Moreover, both enzyme-modified and bare Au electrodes were investigated in detail by atomic force microscopy (AFM) as well as electrochemically. When adsorbed from dilute solutions (0.125 and 0.25 mg mL⁻¹ in the cases of ThLc and MvBOx, respectively), the amounts of enzyme per unit area were determined to be ca. 1.7 and 4.8 pmol cm⁻², whereas the protein film thicknesses were determined to be 29 and 30 Å for ThLc and MvBOx, respectively. A well-pronounced bioelectrocatalytic reduction of molecular oxygen (O₂) was observed on MvBOx/Au biocathodes, whereas this was not the case for ThLc-modified Au electrodes (i.e., adsorbed ThLc was catalytically inactive). The initially observed apparent k(cat)(app) values for adsorbed MvBOx and the enzyme in solution were found to be very close to each other (viz. 54 and 58 s⁻¹, respectively (pH 7.4, 25 °C)). However, after 3 h of operation of MvBOx/Au biocathodes, kcatapp dropped to 23 s⁻¹. On the basis of the experimental results, conformational changes of the enzymes (in all likelihood, their flattening on the Au surface) were suggested to explain the deactivation of MCOs on the bare Au electrodes.