2.9.1. Protein extraction for proteomics
Proteins were extracted from solid agar plates using a methodology modified from Tan, and successfully utilised by Casey (Casey et al., 2010; Tan, 2007).
MM agar plates were inoculated with 5 µL of 1x106 spores, or in the case of the non-sporulating mutants, with mycelial material in suspension. Fungal strains were grown in the dark for 9 days post inoculation (pre-sporulation time point) or 13 days post inoculation (post-sporulation time point). At each of the time points, mycelia from the fungi were harvested by scraping of the plates with number 24 scalpel blades and collection in 15 mL Falcon tubes. The tubes were snap frozen in liquid nitrogen and then lyophilised in a LABCONCO Freezone 2.5 Plus (Labconoco Corp., USA) depressurised with a JLT-10 high vacuum pump (JAVAC Pty. Ltd., Australia).
Following lyophilisation the dried mycelia was homogenised by mortar and pestle with an equal volume of glass beads (Ø106 µm) and 4 mL of 10 mM Tris-HCl (pH 7.6) containing 1 mM PMSF. The crude homogenate was transferred into 2 x 2mL micro- centrifuge tubes and placed on ice.
The crude homogenate was then transferred to an orbital mixer for 10 minutes at 4°C. Following mixing, tubes were centrifuged at 16000 x g for 15 minutes at 4°C. The supernatant was collected into a 15 mL Falcon tube and the tubes containing the remaining crude homogenate were once more centrifuged at 16000 x g for 15 minutes. Following centrifugation the supernatant was transferred to the 15 mL falcon tube and incubated with DNase (10 U) and RNase (10 U) for 1 hour at 25°C. Following DNase/RNase treatment the sample was transferred to 2 mL micro-centrifuge tubes and frozen at -80°C.
64 2.9.2. Protein extraction for enzyme activity determination
Spores were collected from the S. nodorum strains of interest following the procedure (Chapter 2, section 2.4.3). Replicate 250 mL flasks each containing 100 mL of sterile MM with either sucrose of mannitol as the sole carbon source, at a concentration of 30 g/L, were inoculated with the spores from the strain of interest at a concentration of 1 x107 spores/mL. The flasks were then incubated in the dark at 20°C with shaking at 140 rpm for three days.
Following growth of the fungi the contents of each flask were divided into two 50 mL centrifuge tubes and centrifuged at 3000 x g for 10 minutes, the supernatant was discarded and the mycelia washed with 50 mL of Tris buffer (50 mM Tris-HCl, pH 7.5). The mycelia were re-centrifuged at 3500 x g for a further 10 minutes and the supernatants were discarded. The mycelia pellets were then frozen in liquid nitrogen and then freeze dried overnight.
Following lyophilisation the dried mycelia were homogenised in an ice cold mortar and pestle and re-suspended in 2 mL of Tris buffer (50 mM Tris-HCl, pH 7.5). The homogenised mycelia was then transferred into 2 mL microcentrifuge tubes and centrifuged at 20000 x g for 15 minutes. The supernatant was collected into a clean microcentrifuge tube, re-labelled as crude protein extract, and stored on ice.
Protein concentration determination (Bradford assay), and enzyme activity assays were performed on the crude protein extracts soon as possible following protein extraction.
2.9.3. Protein concentration assay (Bradford assay)
Protein concentration determinations were carried out within 96 well clear bottom plates using the Bradford protein assay kit (Bio-Rad, Australia). Briefly, 300 µL protein standards were prepared at concentrations 0, 0.02, 0.04, 0.06, 0.08 and 0.1 µg/µL of bovine serum albumin in Milli-Q water. Protein samples, 300 µL each, were prepared as diluted samples at ratios of 1:5, 1:20, and 1:50 in Milli-Q water. Samples and standards were dispensed in triplicate, 80 µL per well into a 96 well clear flat bottom
65 plate. The samples were acidified by the addition of 10 µL of 1 M hydrochloric acid to each sample and mixed briefly. 20 µL of undiluted Bradford reagent was added to each well, the plate mixed thoroughly on an orbital shaker and then incubated at room temperature in the dark for between 15-30 minutes. The absorbance of each well was then read at 595nm on a Fluorostar Ultima (BMG, Germany). Protein concentration were recorded in µg/µL and determined by calculating against the linear section of the protein concentration curve.
2.9.4. Enzyme assays for mannitol metabolism
Paired quartz cuvettes were used for all spectrophotometric assays. And all readings were performed in triplicate using a Cary300 UV-Vis spectrophotometer (Agilent, Australia). The oxidation of NADH/NADPH was measured at 340 nm to determine activity in terms of U/mg of protein. One unit of activity was defined as the amount of enzyme required to oxidise 1 µmole of NADH/NADPH in 1 minute at 30°C.
2.9.4.1. Mannitol-1-phosphate dehydrogenase activity
Mannitol-1-phosphate dehydrogenase enzymatic activity was performed by monitoring the oxidation of NADH at 340 nm. The reaction contained 0.25 mM NADH, 2 µM fructose-6-phosphate and 50 µL of crude enzyme extract in a volume made up to 1 mL with 50 mM Tris-HCl (pH 7.5).
The reaction components were allowed to equilibrate before the substrate, fructose-6-phosphate, was added to initiate the reaction.
2.9.4.2. Mannitol dehydrogenase activity
Mannitol dehydrogenase enzymatic activity was performed by monitoring the oxidation of NADPH at 340 nm. The reaction contained 0.25 mM NADPH, 0.8 M fructose and 50 µL of crude enzyme extract in a volume made up to 1 mL with 50 mM Tris-HCl (pH 7.5).
The reaction components were allowed to equilibrate before the substrate, fructose, was added to initiate the reaction.
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