Pectin from citrus fruit

PME activity was assayed as described (Klavons and Bennett, 1986 ) with minor modifications. Fifty milligrams of tomato leaf tissue ground in liquid N₂ was incubated for 1h at 4 °C in extraction buffer (20mM Na₂HPO₄, 20mM citric acid, 1M NaCl, 0.1% (v/v) Tween 20, 0.2% (w/v) PVPP, adjusted to pH 7.0) under shaking. After centrifugation (16 000×g, 30min, 4 °C) the cleared supernatant was desalted by ultracentrifugation (10kDa MWCO, Vivaspin concentrators, Sartorius; Göttingen, Germany) using extraction buffer without PVPP and NaCl, and the protein concentration was determined. PME activity was assayed in a total volume of 300 µl reaction buffer containing 1 µg protein, 100 µg pectin from citrus fruit (≥85% esterified, Sigma-Aldrich; Taufkirchen, Germany), 0.025U alcohol oxidase (from Pichia pastoris, Sigma-Aldrich) in 50mM sodium phosphate buffer pH 7.5. After 30min at 28 °C, the reaction was stopped by the addition of the same volume of 2M ammonium acetate, 19.5mM acetylacetone and 49mM acetic acid and incubated at 68 °C for 15min. The absorbance was read at 420nm against a buffer-only blank to quantify PME activity as nmol methanol µg⁻¹ protein min⁻¹ using a reference curve of 0–175 nmol methanol.