PubMed · 5750416
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H Mlodecki, W Lasota, Z Wlodarczyk. 1968. [Tryptophan content in some edible mushrooms].. https://pubmed.ncbi.nlm.nih.gov/5750416/
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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.
It has recently been discovered that lignin peroxidase isozyme H2 (LiPH2) has the ability to oxidize Mn2+ (Khindaria et al., 1995). Furthermore, at pH 4.5, the physiological pH of Phanerochaete chrysosporium, LiPH2 oxidizes Mn2+ at a much faster rate (25 times) than veratryl alcohol (VA). The ability of Mn2+ to act as a redox mediator for indirect oxidations catalyzed by LiPH2 was therefore investigated. In the presence of physiologically relevant levels of oxalate and Mn2+, the rate of LiPH2-catalyzed oxidation of all substrates studied was dramatically increased. Up to 10-fold stimulations were observed compared to the rates of oxidation of substrate in either the presence or absence of VA. We propose that the stimulation is due to the ability of LiPH2 to oxidize Mn2+, producing the strong oxidant Mn3+, at a high rate. The rates of oxidation of the substrates showed a hyperbolic dependence on Mn2+ in the presence of oxalate, a chelator which was required for maximal activity. The oxalate dependence of the oxidation rates correlated well with the concentration of the 1:1 complex of Mn(2+)-oxalate. The relative concentrations of the substrates and H2O2 and the rate constants for their reactions with Mn3+ determined which chemical was oxidized by the enzymatically produced Mn3+. The importance of the ability of Mn(2+)-oxalate to stimulate the oxidation of chemicals by LiPH2 is discussed.
We have investigated the lignin peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. Pre-steady-state kinetic analyses demonstrated that guaiacol is a good substrate for both compounds I and II, the two- and one-electron oxidized enzyme intermediates, respectively, of lignin peroxidase. The rate constant for the reaction with compound I is 1.2 x 10(6) M-1s-1. The reaction of guaiacol with compound II exhibits a Kd of 64 microM and a first-order rate constant of 17 s-1. Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with a Km of 160 microM and a kcat of 7.7 s-1. Veratryl alcohol, a secondary metabolite of ligninolytic fungi, is capable of mediating the oxidation of guaiacol. This was shown by steady-state inhibition studies. Guaiacol completely inhibited the oxidation of veratryl alcohol, whereas veratryl alcohol had no corresponding inhibitory effect on guaiacol oxidation. In fact, at low guaiacol concentrations, veratryl alcohol stimulated the rate of guaiacol oxidation. These results collectively demonstrate that veratryl alcohol can serve as a mediator for phenolic substrates in the lignin peroxidase reaction.