Lysing matrix c tube

To determine the activity of the D-galacturonic acid metabolism, the first step in the enzymatic conversion, D-galacturonic acid to L-galactonic acid, was measured. D-galacturonic acid reductase assay based on conversion of NADPH to NADP⁺, previously described by Kuorelahti et al., was applied (Kuorelahti et al. [2005 (link)]). 20 mg of frozen mycelium was added to 400 μl of extraction buffer (100 mM phosphate buffer pH 7.0, 0.1 mM EDTA, 1 mM DTT and fungal protease inhibitor cocktail) in lysing matrix C tubes (MP biomedicals) and disrupted at level 6 for 30 seconds in a bead beater (MP fastprep-24). Cell debris was removed by centrifugation and supernatant was used as cell extract. 25 μl of cell extract was added to 200 μl of assay buffer (100 mM sodium phosphate buffer pH 7.0, 0.25 mM NADPH) and reaction was started by the addition of 25 μl of substrate (1 M D-galacturonic acid pH 6). Decrease of absorbance at 340 nm was measured on a platereader (Biotek Synergy) in parallel with sample controls, for which 25 μl of buffer was added. Background activity of sample control without D-galacturonic acid was substracted from sample measurements with D-galacturonic acid. Protein content was determined by photometric assay (Peterson [1977 (link)]).

Cells from CPCC 95 E. gracilis were removed from − 80 °C and resuspended in 2 mL of TRIzol™ Reagent (Invitrogen™). Samples were transferred to MP Biomedical Lysing Matrix C tubes and lysed using a MP Biomedical Fast Prep-24. The RNA was precipitated using 250  $\mu$ L of 0.8 M disodium citrate/1.2 M NaCl and 250 µL isopropanol. DNA was removed from the samples using a Dnase1 treatment before the RNA was precipitated again using the same RNA precipitation solution that was mentioned above and isopropanol. The quality of each sample was assessed by visualizing RNA following electrophoretic separation on a 1.5% BPTE agarose gel following glyoxal denaturation. Dnase-treated RNA and a ssRNA ladder were loaded on the gels (New England BioLabs, Whitby, Canada).

To extract the aflatoxins produced, 0.5 mL culture supernatant was mixed with an equal amount of chloroform, and the chloroform solution was collected and evaporated by air drying. The remaining residue was dissolved in 1 mL 90% aqueous acetonitrile and subjected to LC/MS analysis. For acetyl-CoA extraction, the harvested fungal mycelia were washed and lyophilized. The dried mycelia were transferred to a Lysing matrix C tube (MP Biomedicals, Irvine, CA, USA) and ground (FastPrep-24; MP Biomedicals). Trifluoroacetic acid (5%; 200 μL) was added to the cell debris, with vigorous mixing at 4°C. After centrifugation at 10,000 × g, 20 μL 25% ammonia aqueous solution was added and mixed to neutralize, and then centrifuged at 15,000 × g. The supernatant was filtered and subjected to LC/MS analysis.