Xylans

Wild-type and mutant strains were grown in 10 ml of modified VM cellobiose medium or 50 ml of MTC medium. Carbon substrates in these defined media were 5.0 g/l D-glucose, 5.0 g/l D-cellobiose, 5.0 or 10 g/l Avicel PH-105 crystalline cellulose (FMC BioPolymer, Philadelphia, PA), 5.0 g/l D-xylose, 5.0 g/l xylan from birch wood or 10 g/l switchgrass (dry mass; pretreated with diluted sulfuric acid). For fermentation product analyses cultures were sampled after incubation for 5 to 14 days, when fermentation was complete. The samples were filtered through 0.2 μm filters, acidified and analyzed for primary fermentation products (lactate, acetate and ethanol) using HPLC [42 (link)]. These data from four strains were compared using the KaleidaGraph program (v. 4.1.2, Synergy Software, Reading, PA) to perform one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test (0.05 significance level).
The extent of substrate conversion to primary fermentation products was calculated from the molar carbon atom ratio of primary fermentation products to substrates, assuming a stoichiometric ratio of 2 lactate, 2 acetate + 2 CO₂ or 2 ethanol + 2 CO₂ per glucose equivalent. For switchgrass analysis, conversion efficiency and sugar composition was determined using quantitative saccharification [43 ].
More detailed metabolic profiles were obtained by GC-MS. Supernatant and cell pellet samples for metabolomic analysis were collected from duplicate stationary phase cultures grown in defined VM medium with 5.0 g/l cellobiose. Aliquots containing 250 μL of supernatant or cell lysate and 10 μL of sorbitol (0.1% w/v) were transferred by pipette to a vial and stored at -20°C overnight. The samples were thawed and concentrated to dryness under a stream of N₂. The internal sorbitol standard was added to correct for subsequent differences in derivatization efficiency and changes in sample volume during heating. Trimethylsilyl derivatives were prepared from each sample for analysis by GC-MS [42 (link)]. The GC-MS data indicated the presence of a number of 2-hydroxyacids. To confirm whether these were induced from 2-oxoacids with reactive carbonyl groups, a test sample was additionally prepared using a double derivatization protocol that preferentially protects carbonyl groups [44 (link)]. Briefly, 200 μL of methoxamine reagent was added to the test sample, which was heated at 30°C with stirring for 90 min. Then, 800 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide + 1% trichloromethylsilane was added and the sample was heated at 37°C for 30 minutes. The sample was analyzed by GC-MS after 2 h storage at room temperature and again after 1 day.

irx3-7 seeds were kindly donated by Simon Turner (University of Manchester). Seeds were surface sterilized and sown on 0.8% (w/v) agar, 0.5 9 Murashige and Skoog salts including vitamins (Sigma, http://www.sigma.com) and sucrose (1% w/v). Following stratification for 48 h at 4 °C in the dark, plates were transferred to a growth room (20 °C, 100 μmol m⁻² s⁻¹, 24 h light, 60% humidity). Optimaxx fibre rockwool (Cultilene, Netherlands) was cut into slabs and laid ∼8 cm deep in a growth tray. Hydroponics solution (2 mM MgSO₄, 2 mM CaNO₃, 50 μM FeEDTA, 5 mM KNO₃, 2.5 mM K₂HPO₄/KH₂PO₄ pH 5.5, 70 μM H₃BO₄, 14 μM MnCl₂, 0.5 μM CuSO₄, 1 μM ZnSO₄, 0.2 μM NaMoO₄, 10 μM NaCl and 0.1 μM CoCl₂) was poured into the tray until half-way up. The rockwool was then covered with foil. Holes were pierced into the foil and rockwool and seedling were placed in, ensuring that roots made direct contact with rockwool. A growth chamber was constructed following Chen et al.43 (link) illustrated in Supplementary Fig. 3. Compressed air was scrubbed of CO₂ using calcium oxide before ¹³CO₂ was mixed back in at a concentration of 500 p.p.m. before entering the growth chamber. Plants were grown in a 50% humidity and 24 °C environment for 6–8 weeks. ¹³C enrichment was measured by analysing xyloglucan oligosaccharides yielded from xyloglucan endoglucanase digestion of stems using matrix-assisted laser desorption/ionization–time of flight mass spectrometry. Typical enrichments were 90–95%. Roughly 35 mg of the bottom third parts of five to ten stems were chopped and packed into a 3.2 mm Magic-Angle Spinning NMR rotor. For wild-type, five biological replicates (of at least five plants) were grown, each of which was analysed at least once; for irx3, two biological replicates (of at least five plants) were grown, each of which was analysed at least once. On the basis of the relative content of cellulose, pectin, xyloglucan and xylan observed in the wild-type and irx3 samples by NMR, we estimate the stem material contained a substantial but minority proportion of primary cell walls.

The binding abilities of CBMs for soluble polysaccharides were investigated through affinity electrophoresis according to an established method [46 (link)]. In brief, five micrograms of the recombinant CBMs as well as bovine albumin (BSA) were respectively mixed with loading buffer and then used for non-denaturing polyacrylamide gel electrophoresis (PAGE) in a 10% gel. Soluble polysaccharides (arabinoxylan, glucuronoxylan, carboxymethyl xylan, arabinan or arabinogalactan) were added to a final concentration of 0.1% (w/v) when making separating gels. The electrophoresis was carried out in ice bath for 2 h with a voltage of 150 V, followed by Coomassie blue staining. The relative mobility (r) of CBM (distance migrated by CBM band divided by the distance migrated by dye) was calculated to show the CBM affinity for soluble polysaccharides. To determine the K_d of CrCBM13 and CrCBM2, the affinity electrophoresis was carried out using the gels containing arabinoxylan or glucuronoxylan with a gradient concentration (0/0.1/0.2/0.4/0.8 g/L for arabinoxylan and 0/0.75/1.25/2.5/5 g/L for glucuronoxylan). The plots of 1/r versus ligand concentration were then used to determine of K_d by regression analysis (Additional file 1), and the unit of K_d was finally converted from g/L to μM according to the average molecular weights of arabinoxylan and glucuronoxylan.
To investigate the affinity of CBMs for insoluble substrates, the microcrystalline cellulose, corncob xylan, delignified corncob or Carolina poplar were ground to the powders with particle sizes below 75# mesh. The CBMs and substrates were then dispersed in 1 mL of Na₂HPO₄-NaH₂PO₄ buffer (100 mM, pH 7.0) to a final concentration of 0.5 mg/mL and 10 mg/mL respectively in a 2 mL low-binding tube (Eppendorf, Germany). Subsequently, the tube was incubated in a shaker at 37 °C with a rotational speed of 200 rpm for 1 h. The supernatant was then collected through centrifugation, followed by the determination of protein concentration using Bradford assay. The concentration of the protein solution without any substrate is defined as 100%.

To investigate the specific activities of the enzymes, the recombinant xylanases and soluble polysaccharides were dissolved in the disodium hydrogen phosphate—citric acid buffer (pH 6.0, 200 mM). After that, 50 μL of recombinant xylanase solution (5 μM) was mixed with 100 μL of polysaccharide solution (0.5%) and incubated at 50 °C for 10 min. Afterwards, 200 μL of dinitrosalicylic acid (DNS) reagent was added, and the mixture was incubated in a boiling water bath for 5 min [47 (link)]. Subsequently, 1 mL of deionized water was added to dilute the solution. The supernatant was collected after centrifugation, and its absorbance at 520 nm was measured to calculate the reducing sugar concentration and the enzyme activity according to the calibration curve using corresponding monosaccharides as standard. The amount of enzyme required to produce 1 μmol of product per minute is defined as one enzyme activity unit. When insoluble corncob xylan was employed as substrates, the recombinant xylanases were dissolved to 10 μM in the disodium hydrogen phosphate—citric acid buffer (pH 6.0, 200 mM). Then, 20 mg of insoluble corncob xylan was added to 1 mL of the xylanase solution and incubated at 37 °C for 6 h in a shaker at 200 rpm. Afterwards, 150μL of the supernatant was taken for the determination of enzyme activity using DNS reagent as mentioned above.
To investigate the optimal temperature for catalysis, the recombinant xylanases and glucuronoxylan were dissolved in the disodium hydrogen phosphate—citric acid buffer (pH 6.0, 200 mM), respectively. After that, 50 μL of recombinant xylanase solution (2 μM) was mixed with 100 μL of glucuronoxylan solution (0.2%) and incubated at gradient temperature (20–90 °C) for 20 min. Enzyme activities were then measured using DNS reagent as previously described. To investigate the optimal pH value for catalysis, the recombinant xylanases and glucuronoxylan were dissolved in the disodium hydrogen phosphate—citric acid buffer (pH 4.0–8.0, 200 mM) and the glycine—sodium hydroxide buffer (pH 8.0–11.0, 100 mM). After that, 50 μL of recombinant xylanase solution (2 μM) was mixed with 100 μL of glucuronoxylan solution (0.2%) and incubated at 50 °C for 20 min. The enzyme activities were then measured using the DNS reagent as described above.
To investigate the thermostability of the enzymes, the recombinant xylanases and glucuronoxylan were dissolved in the disodium hydrogen phosphate—citric acid buffer (pH 6.0, 200 mM), respectively. After that, 50 μL of recombinant xylanase solution (2 μM) was incubated at 40 °C, 50 °C, 60 °C or 70 °C for 1 h and then mixed with 100 μL of glucuronoxylan solution (0.2%), followed by the incubation at 50 °C for 20 min. The enzyme activities were then measured with DNS reagent as mentioned above.
To investigate the kinetic parameters, the recombinant xylanases and soluble xylans were dissolved in the disodium hydrogen phosphate—citric acid buffer (pH 6.0, 200 mM), respectively. After that, 50 μL of recombinant xylanase solution (2 μM) were mixed with 100 μL of xylan solution with gradient concentration (from 0.2 to 4%), followed by the incubation at 50 °C for 10 min. The enzyme activities were then measured using the DNS reagent as described above. The K_m, V_max and k_cat values were calculated from the nonlinear regression curves using the OriginLab software package. All of these experiments were carried out in triplicate.