Apple pectin

Polygalacturonic acid sodium salt, D-galacturonic acid monohydrate, digalacturonic acid, trigalacturonic acid, citrus pectin (esterification degree of 34% and 90%), apple pectin (esterification degree of 75%), and chromatographic resins were all purchased from Sigma. citrus pectin (esterification of 72%) was kindly donated by CP Kelco (Limeira, SP, Brazil).

To detect and quantify enzymatic activities on plant-derived carbohydrates in the culture filtrates of carbon-starved cultures, filtrates of duplicate cultures were collected 2, 4, 6 and 9 h after transfer to medium without carbon source. Culture filtrates were concentrated 20-fold in the presence of protease inhibitors as described in Section 2.5. Culture filtrate (400 μl) was incubated for 6 h at 37 °C in a total volume of 1 ml with 0.05% sodium azide, 50 mM sodium acetate–citrate buffer pH 4.8 and with 0.3% (w/v) of the following substrates: sugar beet arabinan, wheat flour medium viscosity arabinoxylan, guar medium viscosity galactomannan, tamarind xyloglucan (all from Megazyme), beechwood xylan, 2-hydroxyethyl cellulose and apple pectin (Sigma Aldrich). Enzymes were inactivated through heating (10 min, 100 °C) and incubations were stored at −20 °C. Reducing sugars in the incubations were quantified using Nelson Somogyi assay method (Nelson, 1944 , Somogyi, 1952 (link)) using glucose as a standard.

Paenibacillus amylolyticus was grown aerobically in tryptic soy broth without dextrose (TSB; 17 g pancreatic digest of casein, 3 g enzymatic digest of soybean meal, 5 g NaCl, 2.5 g dipotassium phosphate per liter) at 37 °C with shaking unless stated otherwise. Tryptic soy agar (15 g pancreatic digest of casein, 5 g papaic digest of soybean meal, 5 g NaCl, 15 g agar per liter) was used for solid media. All media were purchased from Becton–Dickinson (Franklin Lakes, NJ). Polygalacturonic acid (PGA), citrus pectin, apple pectin, galacturonic acid (GalA), glucose, crystalline cellulose (Sigmacell), oat spelt xylan, and CaCl₂ were purchased from Sigma-Aldrich (St. Louis, MO). RG-I from soy was purchased from Megazyme (Bray, County Wicklow, Ireland).

Bacterial cellulose was produced by culturing Komagataeibacter sucrofermentans (ATCC^® 700178™, Promochem, London, UK) in 100 mL YGC medium (50 g L⁻¹ glucose, 5.0 g L⁻¹ yeast extract, 12.5 g L⁻¹, CaCO₃) in 250 mL Erlenmeyer flasks in static conditions for at least 120 h at 26 °C. A long incubation was necessary to ensure that the pellicles were dense enough to exclude PEG of Mw 6000 (PEG 6000). Composites of bacterial cellulose with pectin were produced by the addition of 0.5% (w/v) pectin to the YGC medium, and composites of bacterial cellulose with pectin and xyloglucan by addition of 0.25% (w/v) pectin and 0.25% (w/v) xyloglucan to the YGC medium (apple pectin, Sigma-Aldrich, Dorset, UK, cat. No. 76282, 70–75% esterified; tamarind seed xyloglucan, Megazyme International, Ireland, Cat. No. P-XYGLN).

C. phytofermentans was cultured in a modified form of a previously described anaerobic medium [48 (link)] containing the following (g/l): yeast extract, 6.0; urea, 2.1; KH₂PO4, 4.0; Na₂HPO₄, 6.5; trisodium citrate dihydrate, 3.0; L-cysteine hydrochloride monohydrate, 2.0; resazurin, 1; with pH adjusted to 7.0 using KOH. This medium was supplemented with 0.3% (wt/vol) of the following substrates: glucose, cellobiose, xylose, L-arabinose, birchwood xylan, and apple pectin (Sigma-Aldrich) as well as cellulose and “plant biomass” (Brachypodium distachyon). Substrates were added as a filter-sterilized solution to the sterile medium if soluble or autoclaved with the medium if insoluble. Duplicate liquid cultures were incubated at 30°C under anaerobic conditions (in an atmosphere of N₂) as described by Hungate [49 ]. Growth on soluble substrates was determined spectrophotometrically by monitoring changes in optical density at 660 nm. Growth on solid substrates was estimated by visually monitoring and marking the reduction in biomass levels in the test tubes.

The relative viscosity of nostoglycan was determined using an Ubbelohde capillary viscometer (ShenLi Glass Labware, Shanghai, China) as previously described [60 (link)]. Briefly, 20 mg of nostoglycan was dissolved in 20 mL of water and passed through a 0.45 μm Millipore membrane filter. Then 10 mL of the nostoglycan solution or water was transferred into the viscometer, and the efflux time of nostoglycan solution and water was measured respectively. The relative viscosity was calculated as the ratio of the efflux time of the polysaccharide solution to that of water. Apple pectin and agar (Sigma) were used for comparison.

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)).