Strains and growth conditions
Aspergillus fumigatus Z5 (CGMCC Accession No. 3309, China General Microbiology Culture Collection Center, Genome GenBank accession AZZA01000000) is stored in our lab and used for the study of lignocellulose degradation. The cultivation was performed according to Miao et al. (2015a). Briefly, 1 × 107 fresh conidia was added into 200 ml of Mandels’ salt solution supplemented with 2% (w/v) oat spelts xylan (Sigma, USA), and then incubated for 20 h at 50 °C and 150 rpm. The mycelia were then harvested and washed thoroughly with sterile water to be stored at − 80 °C for RNA extraction.
Pichia pastoris X33 (Invitrogen, USA) was used as the gene expression host. Plasmid construction and storage was based on the Escherichia coli Top10 (stored in our lab) cultivated in LLB (Low-salt Luria–Bertani) medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, pH7.0). YPDS medium (2% peptone, 1% yeast extract, 2% glucose, 1 M sorbitol, pH 6.0) was used for transformants screening. BMGY/BMMY (2% peptone, 1% yeast extract, 1.34% YNB, 4 × 10−5% biotin, 1% glycerol or 0.5% methanol, pH 6.0) was used as the growth/induction medium for enzyme production.
cDNA synthesis, plasmid construction and enzyme preparation
Xylan-induced mycelia of strain Z5 was grinded in liquid nitrogen for total RNA extraction using RNeasy Plant Mini Kit (Qiagen, Germany) combined with the RNase-Free DNase set (Qiagen, Germany) according to the manufacturer’s instructions. A 1 μg mass of total RNA with good quality was used as the template for cDNA synthesis using PrimeScript™ RT-PCR Kit (TAKARA, China).
The open reading frames (ORFs) of these xylanases encoding genes, excluding their native signal sequences, were amplified by PCR using the synthesized xylan-induced cDNA as the template. All primers are listed in Additional file 1: Table S1. PrimeSTAR HS DNA polymerase (TAKARA, China) was used for high-fidelity PCR amplifications. Gene fragments were inserted into pPICZαA/B using T4 DNA Ligase (TAKARA, China) and verified by DNA sequencing, the correct plasmids (pPICZαB-Xyn10A, pPICZαB-Xyn10AdC, pPICZαB-Xyn10AdLC, pPICZαA-Xyn10B and pPICZαB-Xyn10BaLC) were linearized with PmeI (New England Biolabs, China) and then transformed into P. pastoris X33 by electroporation (Gene Pulser Xcell™ Electroporation System #165-2660, Bio-Rad, USA) according to the manufacturer’s protocol. Transformants were screened in the YPDS plates containing 100 μg ml−1 of zeocin. The correct transformants (P. pastoris X33-Xyn10A, P. pastoris X33-Xyn10AdC, P. pastoris X33-Xyn10AdLC, P. pastoris X33-Xyn10B, P. pastoris X33-Xyn10BaLC) were verified by PCR, and then cultured in 100 ml BMGY medium in a 500 ml flask for 20 h at 30 °C and 200 rpm. The cultures were centrifuged for 5 min at 3000 rpm, and the supernatants were discarded, then P. pastoris cells were transferred into the fresh BMMY medium for enzyme inductions. Every 24 h, 100% methanol was added to a final concentration of 1% in the medium. After 96 h, the supernatants were collected by centrifugation and proteins were extracted by the 80% of ammonium sulfate. The transformants used for further study were finally confirmed by the extracellular enzyme activities and SDS-PAGE analysis. Enzyme purifications were carried out according to the procedure described previously (Miao et al. 2015b).
Enzyme activity assay
Xylanase activity was measured by DNS method (Linton and Greenaway 2004) with some modifications. 5 μg of each purified xylanase was incubated with 1 ml of substrate solution, consisting of 1% (w/v) oat spelts xylan in sodium acetate buffer (50 mM, pH 6.0), at each optimal reaction temperature for 10 min, then reaction was terminated by adding 1 ml of 3,5-dinitrosalicylic acid (DNS) and boiled for 10 min. The concentration of produced reducing sugars was determined by measuring the absorbance at 520 nm. d-Xylose (Sigma, China) was used as the control. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugars from the substrate in 1 min.
Characterization of the expressed xylanases
To determine the optimal reaction temperature of these expressed xylanases to the substrate of oat spelts xylan, the purified xylanase activities were detected as described above but at different temperatures ranging from 20 to 100 °C. Their thermostabilities were determined by incubating these enzymes at temperatures ranging from 20 to 100 °C for 6 h, then the residual enzyme activities were measured in each sampling time (10, 30 min, 1, 2, 4, 6 h).
The optimal pH for each enzyme was determined by incubating each xylanase with the substrate dissolved in an appropriate buffer at different pH values: 50 mM citrate buffer (pH 2.0–6.0), 50 mM PBS buffer (phosphate-buffered saline, pH 6.0–8.0), and 50 mM glycine–NaOH buffer (pH 8.0–11.0), at the optimal temperature for 10 min. Enzyme activity was determined as described above. For pH stability, the purified xylanases were pre-incubated in the different pH buffers for 1 h at 4 °C followed by activity determination at the optimal conditions.
Determination of the kinetic parameters for the purified xylanases was carried out under optimal conditions for 10 min using oat spelts xylan/xylan from beechwood at concentrations ranging from 2.5 to 20 mg ml−1. The reaction rate versus the substrate concentration was plotted, and the data were fitted to the Michaelis–Menten equation.
Protein assay, SDS-PAGE and zymogram analysis
Protein extraction was carried out by ammonium sulfate precipitation. The clear supernatant was slowly added to 80% (w/v) ammonium sulfate on ice while stirred at low speed on a magnetic stirrer simultaneously. After incubating at 4 °C for 16 h, the sample was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was decanted off. The protein pellet was resuspended in the sterile distilled water. Protein concentration was determined using a Micro BCA protein assay kit (Beyotime, China) according to the manufacturer’s instructions.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the description in Laemmli’s research (Laemmli 1970) on a 10% (w/v) polyacrylamide gel with a protein marker of PageRuler Prestained Protein Ladder (Fermentas, China) using a Mini-PROTEAN Tetra Cell Systems (Bio-Rad, USA). For zymogram analysis, protein samples were buffered by the loading buffer (16% (v/v) glycerol, 10% SDS, 250 mM Tris–HCl (pH 6.8) and 0.05 mg ml−1 Bromophenol Blue) without pre-heating, and then separated by the SDS-PAGE. After that, the gel was washed twice (30 min each) using phosphate-citrate buffer (pH 6.0) containing 25% isopropanol, followed by twice washing (30 min each) using the same buffer but without isopropanol. Then, the gel was covered on a xylan-containing agar plate (1% oat spelts xylan, 1.5% agar), and incubated at 50 °C for 30 min. the resulted agar plate was then stained by 0.5% congo red and destained by 1 M NaCl.
Thin-layer chromatographic analysis
To investigate the final products from hydrolyzing oligosaccharides by Xyn10A, thin-layer chromatography (TLC) was used for sugar separation as described in our previous article (Miao et al. 2015b). Briefly, the hydrolyzed products were separated by a solvent system consisting of n-butanol–acetic acid–water (3:2:2, v/v), and subsequently visualized by spraying with a mixture of methanol and sulfuric acid (9:1, v/v) and heated at 60 °C for 20 min. Xylobiose, xylotriose and xylopentaose were used as the oligosaccharides for test.