PubMed · 5753775
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H Leuner. 1968. [Toxic ecstasy].. https://pubmed.ncbi.nlm.nih.gov/5753775/
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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.
Isocitrate lyase (EC 4.1.3.1), a key enzyme in the glyoxylate cycle, was purified 76-fold with 23% yield as an electrophoretically homogeneous protein from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose. The native enzyme has a molecular mass of 186 kDa, consisting of three identical subunits of 60 kDa. The K(m) for DL-isocitrate was found to be 1.6 mM at the optimum pH (7.0). The enzyme required Mg(2+) (K(m) 92 microM) and sulfhydryl compounds for optimal activity. The enzyme activity was strongly inhibited by oxalate and itaconate with a K(i) of 37 and 68 microM, respectively. The inhibition by the glycolysis and tricarboxylic acid cycle intermediates and related compounds suggested that the isocitrate lyase was a regulatory enzyme playing a crucial role in the fungal growth.
Laccase isozymes from the white-rot basidiomycete fungi Trametes versicolor and Pycnoporus cinnabarinus were purified to apparent iso-electric homogeneity and crystallised. T. versicolor laccase crystallises in two crystal forms, both with the orthorhombic space group P2(1)2(1)2(1), which diffract to 1.9 and 2.95 A resolution, respectively. The crystals of P. cinnabarinus laccase belong to the monoclinic space group C2 and diffract to at least 2.2 A resolution. All the laccase crystals are suitable for X-ray structure determination and contain a full complement of copper ions.