PubMed · 14869748
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S C TRUELOVE, H M WHYTE. 1951-10-13. Acrosclerosis.. https://doi.org/10.1136/bmj.2.4736.873
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High-quality genomic DNA extraction from basidiomycete fungi remains challenging due to polysaccharide co-purification and nuclease-mediated DNA degradation. We systematically compared 22 experimental conditions combining five DNA extraction methods (one Dellaporta-based method, three CTAB-based variants, and one commercial kit) with five mycelium preparation techniques (scalpel scraping, fungal suspension, silica bead homogenization, liquid fermentation, and liquid nitrogen treatment) for Phanerodontia australis (BRM62389). DNA quality was assessed by 1% agarose gel electrophoresis, NanoDrop spectrophotometry, and Qubit fluorimetry. The optimal protocol combined liquid-fermentation mycelium with an adapted CTAB method, yielding Qubit-quantified concentrations of 34.2 and 62.6 ng/µL (samples S21 and S22), 260/280 ratios of 2.06 and 2.05, 260/230 ratios of 1.88 and 1.85, and no detectable DNA degradation by agarose gel electrophoresis. This protocol enabled whole-genome sequencing, yielding a 37.62 Mb assembly with 98.5% completeness as assessed by BUSCO v5 (basidiomycota_odb10 lineage dataset, n = 1,764). Liquid fermentation is associated with the production of younger, actively growing hyphae with reduced cell wall thickness, and the adapted CTAB method effectively removed residual polysaccharides. This protocol provides a reliable, cost-effective approach for obtaining sequencing-grade DNA from P. australis.
There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent.
The molecular properties and stability of a laccase from the white-rot fungus Trametes hirsuta (ThL) were studied to exploit the unique capability of electrospray ionization mass spectrometry (ESI-MS) to monitor conformational and molecular-based heterogeneities and metal ion binding simultaneously. Acid and organic solvents were applied as denaturing agents. In aqueous acidic solution, ThL existed in two major forms, distinguished by their mass difference; in addition to these, two other forms were detected. This molecular heterogeneity was due to the variable glycan content of ThL. Additionally, copper-depleted forms of laccase were observed in mass spectra measured from aqueous acidic solution. A small amount of organic solvent (acetonitrile, CH(3)CN) increased the loss of one Cu atom from folded states and led to unfolding. In the unfolded state, ThL was depleted of all four copper atoms, and the charge state distribution was shifted to lower mass-to-charge region. Thus, denaturation took place in two stages: first, the loss of one Cu resulting in an inactive form; second, complete denaturation with the loss of the three remaining Cu atoms. After all coppers were lost, ThL was unfolded, as was clearly seen in the increased number of charge states in the mass spectra. Different stabilities of the glycoforms were observed in the denaturation triggered in acid and organic solvents.