Pectin Methylesterase Activity Assay

PME activity was assayed according to the method of Solecka (Solecka et al., 2008 (link)). The reaction mixture contained 0.5% (w/v) citrus pectin (Sigma, Germany), 0.2 M NaCl and 0.15% (w/v) Methyl Red (Sigma, Germany), pH 6.8. Protein sample (50 μL) was added to 950 μL of reaction mixture in a micro cuvette. Protein sample was determined by the Bradford protein assay method (Bradford, 1976 (link)). The change in color from yellow to red after incubating at 25°C for 20 min was measured spectrophotometrically at 525 nm. A calibration curve was obtained by adding 1–6 x 10^-3 mM HCl to the reaction mixture. Recombinant AtPMEI1 was expressed in P. pastoris (Raiola et al., 2004 (link)). B. cinerea PME activity was induced in the B. cinerea culture by adding 0.5% apple pectin (wt / vol; 76,282; Sigma-Aldrich) and after 12 hr the culture filtrate was collected (Lionetti, 2015 (link)).