Pullulanase

Proteins (HvPho1, HvPho1ΔL78 and HvPho1Asp383Ala) were produced in E. coli and purified to high homogeneity as described above. To ensure that no dextrin impurities are present in the reaction mix, the G1P stock (500 mM) was incubated overnight with α-amylase (Sigma A3403) and pullulanase (SigmaP2986). This mixture was then boiled at 100°C for 5 min and centrifuged at 22000 g to separate the proteins from the G1P solution. HvPho1 and HvPho1ΔL78 (5 ml of 50 μg∙ml^-1) were dialyzed with 20 changes of dialysis buffer in 250 ml dialysis buffer (50 mM sodium citrate pH 6.5, 25 mM NaCl). The dialysis buffer was prepared according to the following procedure: 1 l of 250 mM sodium citrate, 125 mM NaCl pH 5.0 was incubated with 5 ml pullulanase (Sigma P2986) at 22°C for 1 h. After incubation, the pH was increased to pH 6.9 and 5 ml of α-amylase (Sigma A3403) were added and the mixture was incubated for an additional hour at 22°C. The pH of the buffer was then adjusted to pH 6.5 before it was diluted to 5 L and stored at 4°C. This dextrin free buffer was then used as dextrin free dialysis buffer.

Cellulose was extracted from leaf tissue taken from 6–7 week-old plants. Ten samples were taken from each transgenic line and from wt tobacco plants. Alcohol-insoluble residues were prepared from frozen leaf tissue by grinding 50 mg of sample to a fine powder under liquid nitrogen, and isolating the plant cell walls by washing with different organic solvents [39 (link)]. Starch was removed by enzymatic digestion with α-amylase and pullulanase (Sigma-Aldrich, Seelze, Germany). The remaining AIR was extracted with acetone, dried and weighed. Matrix polysaccharide composition was determined with GC/MS measurement according to Foster et al. [38 (link)]. The crystalline cellulose content was determined [41 (link)] after hydrolyzing the non-crystalline cellulose with acetic and nitric acid. The remaining crystalline cellulose residues were hydrolyzed with 72% (w/v) sulfuric acid allowing the remaining glucose to be measured using the anthrone assay [64 (link)].
Significant differences from wt were determined using a Student t-test (p value ≤ 0.01).

The purified EPS or the standard pullulan (Sigma-Aldrich Corp., St. Louis, MO, USA) of 0.1 g was dissolved in 10 mL distilled water at 80 °C. EPS solution of 1 mL was mixed with 0.9 mL of 50 mM sodium acetate buffer (pH 4.5) and 0.1 mL pullulanase, which was purchased from Sigma. Then, the mixture was incubated at 60 °C for 15 min in a water bath, and the reducing sugar released was detected using the Nelson–Somogyi method [29 (link)].

Three colored (red:black:white = 1:1:1) whole quinoa flour (WQ) produced in the year of 2019 were purchased from Sanjiang Fertile Soil Co., Ltd. (Qinghai, China). Total starch assay, glucose oxidase/peroxidase (GOPOD), dietary fiber, and β-glucan assay kits were provided by Megazyme International Ireland Ltd. (Bray, Ireland). Pullulanase (EC 3.2.1.41), amyloglucosidase (EC 3.2.1.3), α-amylase (EC 3.2.1.1), pepsin (EC 3.4.23.1), and trypsin (EC 3.4.4.4) were provided by Sigma Chemical Co. (St. Louis, MO, USA). Other chemical reagents obtained the standards of the analysis grade.
Hydrochloric acid-potassium chloride with pH = 2.0 was used to dissolve pepsin (10.0 mg/mL), then the miscible liquid was stored at 4 °C. Suitable amounts of α-amylase and amyloglucosidase were dissolved in 0.2 M sodium acetate buffer (pH = 5.6, mixed with 40 mM calcium chloride), and the concentrations were 290 and 60 U/mL, respectively.

Senesced stem plus leaf sheath tissue from 12 biological replicates per inbred line was ball-milled, water and solvent extracted, and then destarched to generate alcohol-insoluble residue (AIR) cell wall material. Destarching was performed in a pH 5.0-buffered solution of 1.2 μg/mL amylase (Sigma–Aldrich) and 12.5 U/mL pullulanase (Sigma–Aldrich) at 37°C overnight, as previously described (Foster et al., 2010 (link)). Matrix polysaccharide composition and crystalline cellulose content was determined as described in Albersheim et al. (1967) (link) and Foster et al. (2010) (link). Briefly, polysaccharide composition was determined by GC-MS separation on the alditol acetates resulting from a 2 M trifluoroacetic acid (TFA) hydrolysis, reduction (NaBH₄) of the neutral monosaccharides present the hydrolysate supernatant, and subsequent acetylation. Crystalline cellulose content was determined by treating the cell wall material not hydrolyzed by TFA with Updegraff reagent, an acid mix that results in further stripping of hemicelluloses and amorphous glucan from the cell wall material (Updegraff, 1969 (link)). The residual crystalline cellulose was hydrolyzed with sulfuric acid (Selvendran and O’Neill, 1987 ) and the resulting monosaccharide (glucose) was quantified using a colorimetric anthrone assay.

Waxy potato starch (approximately 2% amylose and 98% amylopectin) was purchased from Holland AVEBE Company. Pullulanase (EC 3.2.1.41) (4461.6 NPUN/g) was supplied by Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Insulin was supplied by Guangzhou Les Biological Technology Co., Ltd. (Guangzhou, China). All the reagents used were of analytical grade.