Xylanase

Leaves of fourteen days old TN-1 plants were infiltrated with one of the following: xylanase (2 U/ml, Sigma), pectinase (2 U/ml, Sigma), Flg22 (100 μM, Genescript) and water. For assaying response to wounding, leaves of fourteen days old TN-1 rice seedlings were pin pricked 8–10 times and sprayed with water [7 (link)]. Leaves were harvested 12 h post-treatment and processed for qRT-PCR as mentioned previously. For abiotic stress treatment, one-week-old TN-1 rice seedlings were dipped in each of the following: 150 mM NaCl solution (salinity stress), a 20% (w/v) PEG-6000 (drought stress) and water (mock). The treated seedlings were incubated in a greenhouse. Leaves were harvested after 12 h of treatment and processed for qRT-PCR as mentioned previously. In order to study the effect of OsWRKY42 on expression of JA response genes, transient overexpression of OsWRKY42 was carried out. For this, leaves of fourteen days old TN-1 rice seedlings were infiltrated either with LBA4404 or LBA4404/pH7FWG2::OsWRKY42. Leaves were harvested 12 h post treatment and processed for qRT-PCR analysis.

Enzymatic hydrolysis conversion efficiency was evaluated for each of the four feedstocks to determine biomass recalcitrance using purified and lyophilized enzymes. Purified cellulase from Trichoderma reesei ATCC 26921 (C8546; Sigma-Aldrich), xylanase from Trichoderma viride (X3876; Sigma-Aldrich), and beta-glucosidase from almonds (49290; Sigma-Aldrich) were used for hydrolysis. Prior to comparing enzymatic digestibility of a biomass substrate, protein content of the enzyme solution was determined using a Pierce bicinchoninic acid (BCA) protein quantification kit (BCA1; Sigma-Aldrich Co., LLC.). The BCA quantification assay was recommended for fungus-derived enzymes because cellulase produced by the species T. reesei contains roughly 50% cellobiohydrolase I (CBH I; Cel7A), and this cellulolytic enzyme and others within the same glycoside hydrolase family (GHF) 7 may react poorly to other calorimetric assays (Resch et al., 2015 ). Protein content of the enzyme cocktail determined from the Pierce BCA protein assay was 14.47 mg mL⁻¹ ± 1.17 mg mL⁻¹ for the mixed enzymes. Three samples from each of the four plant species studied here were subjected to enzymatic hydrolysis treatments using the mixed enzymes.

Cellulase from Trichoderma reesei ATCC 26921 (C8546, Sigma-Aldrich Corporation, St. Louis, MO, USA), β-glucosidase from almonds (49290, Sigma-Aldrich Corporation, St. Louis, MO, USA), and xylanase from Trichoderma longibrachiatum (X2629, Sigma-Aldrich) were used to hydrolyze raw and pretreated PH. Cellulase activity was represented with Filter paper unit (FPU/mL) and was measured using dinitrosalicylic acid reagent [65 (link)]. The activities of Cellulase and β-glucosidase used in this study were 0.538 FPU/mg and 6 U/mg, respectively. xylanase activity was defined as the amount of enzyme that produced 1 μmol of reducing sugar per 30 min, and Choi et al. have detected the activity of xylanase at 2.65 international units IU/mg [66 (link), 67 (link)].
The PHs as 1% (w/v) substrate were treated in 50 mM sodium citrate buffer (pH 4.8) supplemented with 0.01% (w/v) sodium azide. The enzymes, Cellulase, β-glucosidase, and xylanase, were loaded with 8 FPU/g, 12 U/g, and 300 IU/g of PH, respectively. All samples were completely suspended in rotary shaker at 200 rpm, 37 °C for 48 h. All enzymatic hydrolysis experiments were performed in triplicate. The concentration of reducing sugar was detected using dinitrosalicylic acid reagent [68 (link)]. The glucose concentration was determined using HPLC.

The pretreated residue was enzymatically hydrolyzed in a conical flask with phosphate buffer (pH 4.8), and the substrate was added at 2% (w/v). Sodium azide (10 mM) was also added to the solution to prevent microbial contamination. The enzymatic hydrolysis was conducted at 50 °C for 72 h in the presence of Cellic^® CTec2 cellulase (15 FPU/g substrate), β-glucosidase (30 CBU/g substrate) with/without xylanase (Sigma X2629-100 g) in a shaking incubator at 150 rpm.
The glucose enzymatic hydrolysis yield, xylose enzymatic hydrolysis yield, total glucose yield, and total xylose yield were calculated according to the equations as follows: $$\text{Glucose}\text{enzymatic}\text{hydrolysis}\text{yield}\left(\%\right)=100\times \left(0.9\times \text{Amount}\text{of}\text{released}\text{glucose}\text{after}\text{enzymatic}\text{hydrolysis}\right)/\text{Amount}\text{of}\text{glucan}\text{in}\text{residue}$$ $$\text{Xylose}\text{enzymatic}\text{hydrolysis}\text{yield}\left(\%\right)=100\times \left(0.88\times \text{Amount}\text{of}\text{released}\text{xylose}\text{after}\text{enzymatic}\text{hydrolysis}\right)/\text{Amount}\text{of}\text{xylan}\text{in}\text{residue}$$ $$\text{Total}\text{glucose}\text{yield}\left({\text{Total}}_{\text{Glu}.},\%\right)=100\times \left(0.9\times \text{Amount}\text{of}\text{glucose}\text{in}\text{residue}\times \text{glucose hydrolysis yield}\right)/\text{Amount}\text{of}\text{glucan}\text{in}\text{raw}\text{material}$$ $$\text{Total}\text{xylose}\text{yield}\left({\text{Total}}_{\text{Xyl}.},\%\right)=100\times \left(0.88\times \text{Amount}\text{of}\text{xylose}\text{in}\text{residue}\times \text{xylan}\text{hydrolysis}\text{yield}\right)/\text{Amount}\text{of}\text{xylan}\text{in}\text{raw}\text{material}$$

The hulless barley seeds were obtained from Qinghai Xinning Biological Technology Company (Xining, China). Seeds were thoroughly soaked in water and placed onto a seedling tray filled with a thin layer (0.5 cm) of nutrition-rich soil. After a few days of cultivation, the seeds germinated, and the young green leaves were harvested on the fifteenth day. The harvested leaves included the portion that was exposed above the ground. Leaf samples were washed thoroughly with deionized (DI) water to remove any soil and contaminants. After cleaning, leaves were freeze-dried for 24 h and then ground to green powders using a grinder and passed through a sieve (40 mesh). The protein, lipid, soluble and insoluble dietary fiber, and dietary fiber content of the young leaf powders (dry basis) were 28.5%, 12.9%, 1.6%, and 47.4%, respectively [17 (link)]. The α-amylase (EC 232-588-1, 30 U/mg), papain (EC 232-627-2, 10 U/mg), xylanase (EC 253-439-7, 2500 U/g), and cellulase (EC 232-734-4, 5000 U/g) were purchased from Sigma-Aldrich Co. Ltd. (Saint Louis, MO, USA). All other chemicals used in this study were of chemical grade and from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

The PHs as 1% (w/v) substrate were treated in 50 mM sodium citrate buffer (pH 4.8) supplemented with 0.01% (w/v) sodium azide. Each of the enzymes, celluclast (Novozymes, Denmark) and xylanase (endo-1,4-β-xylanase from Trichoderma longibrachiatum, Sigma-Aldrich, USA), were loaded with 30 FPU per gram of glucan and 300 international units (IU)/mL, respectively. All samples were completely suspended in rotary shaker at 200 rpm at 37°C for 48 h. All enzymatic hydrolysis experiments were performed in triplicate.

Enzymatic hydrolysis of WIS from pine wood, oak wood, and rice straw was carried out 2 % substrate consistency (2 g biomass per each 100 mL solution, 10 mM of sodium acetate buffer, pH 5.0) and 37 °C. The commercial enzymes used in this study were cellulase from Trichoderma longibrachiatum (C9748, Sigma-Aldrich) and xylanase from Trichoderma longibrachiatum (X2629, Sigma-Aldrich). Filter paper unit activity of cellulase was measured in terms of FPU/mL [43 ]. One filter paper unit (FPU) was defined as the amount of enzyme required to release 1 μmol of glucose from filter paper per min. xylanase activity was measured on the basis of xylose released from birch wood xylan as a substrate and was expressed in terms of international units (IU)/mL. One IU was defined as the amount of enzyme required to release 1 μmol of xylose from birch wood xylan per min [44 (link)]. The activities of cellulase and xylanase were 79 kFP U/mL and 592 IU/mL, respectively. Hydrolysate samples were collected at different time intervals during 24 h. Reducing sugar were measured using a 3,5-dinitrosalicylic acid reagent and a standard glucose curve [45 (link)].