Strains and growth conditions
A. niger An76 (DDBJ accession no. BCMY00000000, DNA Data Bank of Japan) was used for enzyme gene amplifications. Cultivation of A. niger An76 was performed according to methods described previously [21, 36]. A 1% (w/v) solution of different carbon sources was added to the culture medium to induce the production of enzymes, including glycerol, xylose and XOS. Fresh conidia (1ⅹ106 /mL) were used to inoculate in 250 mL liquid medium in triplicate at 30℃ and 200 rpm. For qRT-PCR experiments, washed mycelia, which were cultivated in the presence of 1% (w/v) glycerol as carbon source for 24 h as a reference sample (0 h), were transformed into the fresh media containing 1% (w/v) xylose or XOS as the sole carbon source for induction. Mycelia samples were collected at different sampling times (0, 6, 12, 24 and 48 h) and stored at -80℃. Escherichia coli strains BL21 (DE3) and DH5α (DingGuo, Beijing, China) were used for protein expression and gene cloning, respectively.
RNA isolation and cDNA synthesis
Mycelia sampled at each time point were used to extract total RNA and synthesize cDNA. Total RNA was extracted using TRIzol reagent (Sangon, Shanghai, China) and other regents including chloroform, isopropyl alcohol and ethanol (Dingguo) using the TRIzol method [37]. The cDNA was synthesized using 1 μg RNA as template and purified using the HiScript III RT SuperMix + gDNA wiper (Vazyme, Nanjing, China) according to the manufacturer’s instructions.
qRT-PCR analysis of gene expression
qRT-PCR was performed using SYBR qPCR Master Mix (Vazyme) on LightCycler 480 instrument (Roche, Basel, Switzerland). The primers used to detect the expression levels of encoding genes are listed in Additional file 1: Table S1. Target genes included five xylanase genes (xynA, g9709.t1; xynB, g10033.t1; xynC, g1233.t1; xynD, g1345.t1; xynE, g3744.t1). The glyceraldehyde-3-phosphate dehydrogenase gene (gapdh, g7576.t1) was used as an internal reference gene. Error bars indicated the standard deviation. The concentration of cDNA template was 20 ng/μL. Relative transcription levels of target genes were calculated by the relative quantitation (2-ΔΔCT) method [38]. Three biological replicates and technical replicates were performed.
Gene cloning, enzyme expression and purification
Xylanase genes were cloned using PCR with synthesized cDNA as the template and primers listed in additional file 1: Table S1. PCR products were purified and cloned into a pLYJ-163 vector to construct recombinant plasmids, which were transformed into E. coli DH5α cells and confirmed by DNA sequencing (TSINGKE, Qingdao, China). The correct recombinant plasmids were transformed into E. coli BL21 (DE3) for protein expression. When the absorbance at 600nm (OD600) reached 0.6-0.8, strains were induced using 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG, Solarbio, Beijing, China) for 20 h at 16℃. Cells were centrifuged and resuspended in buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). After ultrasonic fragmentation, xylanases were purified using HisCap Co 6FF resin (Smart-life Sciences, Changzhou, China). The eluent was replaced with optimal buffer (50 mM Na2HPO4, 20 mM citrate, pH 5.0) by ultrafiltration (3 kDa membrane, Millipore, Billerica, MA) at 4 °C. Pure enzymes were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were determined using the Bradford method [39].
Enzymatic activity assay
Xylanase activity was assayed using the 3,5-dinitrosalicylic acid (DNSA) method [40]. A 40 μL volume of appropriately diluted XynA, XynB and XynD (5 μg/mL, 1 μg/mL and 5 μg/mL, respectively) were incubated with 60 μL 1% (w/v) xylan substrates. After incubation at 50 °C for 10 min, samples were mixed with 80 μL of DNSA reagent, boiled immediately for 10 min, and subsequently cooled on ice. After adding 820 μL water, the absorbance were measured at 540 nm. One unit of xylanase activity (IU) was defined as the amount of enzyme (mg) that released 1 μmol of xylose per minute under the optimal assay conditions.
Effects of pH and temperature on xylanases
The optimal pH of xylanases was determined in the range of pH 3.0-10.0 (50 mM sodium citrate buffer, pH 3.0-8.0; 50Mm Glycine-NaOH buffer, pH 9.0-10.0) by adding beechwood xylan substrate and incubating for 10 min at 50℃ using the above activity assay method. The activity at the optimal pH was defined as 100%. The optimal temperature of xylanases was determined at temperatures ranging from 30 to 70℃ in sodium citrate buffer (pH 5.0). The activity at the optimal temperature was defined as 100%.
The temperature stability of xylanases was determined by measuring the remaining activity after incubating the enzymes at various times (0, 20, 40, 60, 120, 180 and 240 min) at 50°C in the absence of beechwood xylan in 50 mM sodium citrate buffer (pH 5.0). The activity without pre-incubation was defined as 100%. To assess pH stability, xylanases were incubated in pH 6.0 buffers at different times (0, 30, 60, 150, 240 min) before the addition of xylan to initiate the action. The remaining enzyme activities were determined under optimal conditions. These experiments were repeated three times.
Substrate specificity and determination of kinetic parameters
Substrate specificity of xylanases was investigated using different xylan substrates at 1% concentration, including beechwood xylan, wheat arabinoxylan (Megzyme, Wicklow, Ireland), xylan from wheat bran and corn cob extracted by alkaline pretreatment [41]. Solid material was washed, 0.7% NaOH was added at a ratio of 1:7, and samples were incubated at 60℃ for 2 h. The resulting mixtures were filtered and the filtrate was neutralized with HCl. Xylan was precipitated with absolute ethanol, dried, ground into a powder, and stored at room temperature for subsequent experiments. Reactions were performed in 50 mM sodium citrate buffer (pH 5.0) at 50℃ for 10 min.
The kinetic parameters (Km and Vmax) of the enzymes were determined by incubating with different concentrations of beechwood xylan and wheat arabinoxylan (0.2-2.4%) at 50℃ for 5 min. Parameters were calculated using nonlinear regression of the Michaelis-Menten equation with GraphPad Prism 8.0 [42]. All experiments were performed in triplicate.
Analysis of hydrolysis products.
Hydrolysis products of different xylan substrates were determined by fluorescence-assisted carbohydrate electrophoresis (FACE) experiments [43]. Hydrolysis products were obtained following the reaction of enzymes (20 IU/mL) with 1% substrates at a ratio of 1:1 (v/v) at 50℃. Samples were removed at different times (0, 5, 15, 30 and 60 min) and inactivated at 105℃ for 10 min to terminate the reaction. The supernatant were collected by centrifugation at 12,000 rpm for subsequent electrophoresis analysis. Xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), xylohexaose (X6) were used as standards.
Synergistic hydrolysis experiments
To investigate the synergistic effect among xylanases, for each xylanase, the amount of the enzyme with equal xylanase activity (0.3 IU/ml) towards beechwood xylan was used to experience. XynA, XynB and XynD were added singly or in pairs to 1% beechwood xylan. The release of reducing sugars were determined by the DNS method after incubation at 50℃ for 24 h. Samples were removed at intervals, and samples without enzyme served as controls.
Another method was used to determine synergy between XynA and XynB [22]. XynA was added into the substrate and incubated at 50℃ for 2 h, then XynA and XynB with equal xylanase activity were added to separate solutions to give XynA+ XynB and XynA+ XynA. The reducing sugars released under different conditions were detected upon incubation for 24 h. Samples without enzyme under the same conditions served as controls. All the above hydrolysis assays were performed in triplicate.
Bioinformatics analysis
Signal peptides were predicted using SignalP website (http://www.cbs.dtu.dk/services/SignalP/). Multiple sequence alignments were performed using CLUSTAL (http://www.clustal.org/). Phylogenetic analysis was performed using MEGA 5.0 with the Neighbor-Joining method and 1000 bootstrap replicates [44]. Homology modeling was carried out using SWISS-MODEL (https://swissmodel.expasy.org/). Suitable templates were identified, Aspergillus niger CBS 513.88 (PDB ID: 2QZ2), Talaromyces funiculosus IMI-134756 (PDB ID: 1TE1), Talaromyces cellulolyticus CF-2612 (PDB ID: 3WP3) were selected for XynA, XynB and XynD, respectively. Models were then generated based on each of the individual templates. The ligand in the enzyme-ligand complex of GH11 and GH10 was obtained from TrXyn11A (PDB: 4HK8) and Geobacillus stearothermophilus (PDB: 4PRW), respectively. Amino acid residues surrounding the substrate within 5 Å were selected to analyse the interactions between enzymes and substrates.