Strains and Plasmids
T. leycettanus JCM12802 as the donor strain was purchased from Japan Collection of Microorganisms RIKEN BioResource Center (Tsukuba, Japan). Escherichia coli Trans I-T1 (TransGen, Beijing, China) was used for routine gene cloning. T. reesei AST1116 and P. pastoris GS115 (Invitrogen, Carlsbad, CA, USA) were both used as the host for gene expression. The plasmids of pPIC9 (Invitrogen) and pTrEno were used for driving Tlswo gene expression in P. pastoris and in T. reesei. The plasmid construction of pTrEno was according to the description of Linger et al. .
The DNA and amino acid sequences of TlSWO were analyzed using the BLASTx and BLASTp programs (http://www.ncbi.nlm.nih.gov/BLAST), respectively . The introns and exons were predicted using the GENSCAN Web Server (http://genes.mit.edu/GENSCAN.html) . SignalP 3.0 was used to predict the signal peptide sequence (http://www.cbs.dtu.dk/services/SignalP) . The potential N-glycosylation sites were predicted online (http://www.cbs.dtu.dk/services/NetNGlyc). Sequence assembly and estimation of the molecular mass and pI of the mature peptide were performed using the Vector NTI Suite 10.0 software (Invitrogen). Protein molecular weight and molar extinction coefficients were estimated at the ExPASy tools page (http://www.expasy.org/tools). Multiple sequence alignments were performed with Clustal W program from MEGA software 4.0. PROSITE (http://br.expasy.org/prosite) analysis the protein domains and functional sites . DiANNA web server (http://clavius.bc.edu/~clotelab/DiANNA) is used to predicting the disulfide bond topology in protein .
Gene cloning and recombinant protein expression
T. leycettanus JCM12802 was cultured at 42 ℃ for 3-5 days in medium containing 5.0 g/L (NH4)2SO4, 1.0 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 0.2 g/L CaCl2, 10.0 mg/L FeSO4·7H2O, 30.0 g/L wheat bran, 30.0 g/L soybean meal, and 30.0 g/L corncob. Genomic DNA of T. leycettanus JCM12802 was extracted with the DNA isolation kit (Tiangen) following the manufacturer instructions and was used as a template for PCR amplification. Total RNA isolation and first strand cDNA synthesis were carried out as previously described . PCR amplification was carried out using Fastpfu DNA polymerase and which purchased from TransGen. The fragment of Tlswo corresponding to the 21–503 amino acid sequence was amplified with primers P1 (5’- GCTCGTGCTCAGAGCAGCTGTGCAGG-3’) and P2 (5’- CTAGATTACCTAGGTAAACTGCACC-3’). The amplified PCR product was cloned into pPIC9 and pTrEno vector using Gibson Assembly (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol for cloning and expression. Escherichia coli Trans I-T1 (TransGen, Beijing, China) which used for routine gene cloning was grown at 37 °C overnight in Luria-Bertani (LB) medium supplemented with 50 mg/mL of ampicillin (Sigma, St. Louis, MO).
When using P. pastoris GS115 as the expression host, the recombinant plasmids of pPIC9-Tlswo was linearized with BglII (New England Biolabs, UK) and transformed into the expression host via electroporation. The positive transformants were screened on minimal dextrose medium (MD) at 30 ℃ for 3 or 4 days until the single colonies appeared and placed into shaking tubes for enzyme production according to the protocol described in the Pichia Expression Kit (Invitrogen). The large-scale fermentation was performed as the previously described . The recombinant P. pastoris GS115 containing pPIC9-Tlswo was grown at 30 ℃ in 400 mL BMGY medium in a 1 L shaking flask for 48 hours. Cells were collected and re-suspended again with 200 mL buffered methanol-complex medium (BMMY) medium with 0.5% (v/v) methanol and cultured at 30 °C for 72 h with shaking (200 rpm). Methanol was added into the medium every 24 hours.
When using T. reesei AST1116 as the expression host, the recombinant plasmids of pTrEno-Tlswo were linearized with SbfI (New England Biolabs, UK) and then transformed into T.reesei AST1116 via electroporation. Potato dextrose (PD) plates were used for spore production and PDHX plates (PD plates with hygromycin and TritonX-100 at a final concentration of 100 μg/mL and 0.1% respectively) were used for the screening of potential T. reesei which allowed to grow for 2 to 3 days at 30 °C. Growth medium for TlSWO expression was Mandels and Andreotti medium with 5% glucose (MAG). Subsequently complete medium lactose (CML) was used for the overexpression of the transformants. The medium protocols of MAG and CML were performed following published protocol . For large-scale fermentation, the positive transformants spore stocks were streaked on potato dextrose agar plates and allowed to grow 2 to 3 days. Then the spore was extracted from the plate and deposited into 1.0 L of MAG medium in a 2.8 L shake flask. The culture was grown at 28 °C with at 225 RPM for 24 h, after which the entire 1.0 L was transferred to 7.0 L of the same medium in a bioreactor.
Then the culture broth were extracted for further analysis by SDS-PAGE and activity assay. Culture broths were clarified via centrifugation and transferred to microcentrifuge tubes. Broths were diluted 3:1 in 4×LDS sample buffer (Life Technologies Corp., Carlsbad, CA, USA) with 50 μL/mL β-mercaptoethanol as a reducing agent. Samples were incubated at 95 °C for 5 min prior to loading onto NuPAGE SDS gels with MOPS buffer, electrophoresed at 200 V constant for approximately 40 min.
Fermentation broths were harvested and sequentially vacuum-filtered. This filtered broth was then concentrated by tangential ultrafiltration with a 10 kDa MWCO. The broths were roughly concentrated to 100 mL. The final concentrated volume was exchanged with at least 2.0 L of 20 mM Bis-Tris pH 6.5 to remove residual peptides and other low molecular weight debris. The following purification steps were then performed according to previous publications . The crude enzyme was purified through hydrophobic interaction chromatography (HIC) using a 26/10 Phenyl Sepharose Fast Flow column (GE Healthcare, Chicago, USA). Then the protein went through anion exchange chromatography using a 10/100 anion exchange column packed with Source 15Q (GE Healthcare, Chicago, USA) followed by HIC using a Source15 iso 10/100 column (GE Healthcare, Chicago, USA) and SEC using a 26/60 Superdex 75 column (GE Healthcare, Chicago, USA) and 20 mM acetate buffer pH 5.0, 100 mM NaCl as mobile phase.
SDS-PAGE were performed to assess purity of the protein TlSWO. Electrophoretically separated on a 12% SDS-PAGE and visualized by Coomasie Blue staining. Protein concentration was measured with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific Inc., Rockford, USA) and the Bradford protein assay kit (Bio-Rad).
TlSWO activity assays
The activity of TlSWO was measured using the 3,5-dinitrosalicylic acid (DNS) assay (Miller, 1959). Enzyme activity was assayed in a final volume of 1.5 mL, with 1% (w/v) barley β-glucan (Megazyme Co., Bray, Ireland), lichenan (Megazyme Co., Bray, Ireland), laminarin (Megazyme, Wicklow, Ireland) and carboxymethyl cellulose sodium (CMC-Na) (Sigma-Aldrich, St. Louis, MO) as the substrates, and 10 μg/mL of enzyme at optimal conditions for 10 min. The effect of pH was studied using 100 mM citric acid-Na2HPO4 (pH 3.0–7.0) buffers at 50 °C and the effect of temperature was evaluated by incubation at pH 4.0, between 30 °C and 90 °C.
Effect of pH and temperature on TlSWO activity
Further, the effects of pH and temperature on the activities of TlSWO were measured and compared. To determine the optimum pH of TlSWO, the activities were assayed with 1% lichenan (w/v) in buffer of different pH, 100 mM glycine-HCl (pH 1.0–3.0), McIlvaine buffer (pH 3.0–8.0) and glycine–NaOH (pH 9.0–12.0). For pH stability, TlSWO was preincubated at 37 °C for 1 h in buffers of different pH (1.0–12.0) and subjected to the residual activity assay. For the optimum temperature of TlSWO was determined at optimal pH over the temperature range from 30 °C to 80 °C. The thermostability assay of TlSWO (100 μg/mL) was carried out by preincubating at 37 °C, 50 °C, 60 °C or 70 °C for 0–60 min, and aliquots of 100 μL were withdrawn at different time points for residual activity assay.
Light and scanning electron microscopic analyses
Avicel PH-101was used as a solid cellulosic substrate. Ten milligrams of Avicel were incubated with different amount of purified TlSWO in 100 mM citric acid-Na2HPO4 buffer (pH 4.0). The experiment was carried out on a rotary shaker at 40 °C for different time intervals. Control experiments without TlSWO were also performed under the same conditions. The physical structure of Avicel fibers was initially observed using light microscopy (Olympus TH4-200, Japan). Subsequently, photomicrographs of the samples were captured using a scanning electron microscope (Hitachi SU8010, Tokyo, Japan) at a voltage of 15 kV.
Polysaccharide depolymerization analysis
Hydrolysis reactions on 1% barley β-glucan, 1% CMC-Na, 1% laminarin and 0.5% lichenan, were carried out overnight at pH 4.0, 37 °C using the enzyme in a final concentration of 100 μg/mL. High-performance anion-exchange chromatography (HPAEC) (Thermo Fisher Scientific, Sunnyvale, CA, USA) equipped with a Carbo-Pac PA200 column (3×250 mm) was used to determine the reaction products released from the polysaccharide.
Synergism between TlSWO and cellulases
The substrate used in this work were NREL dilute acid pre-treated corn stover (PCS) P120927, cellulose nanocrystals (CNCs), phosphoric acid swollen cellulose (PASC) and each substrate equivalent to 8.5 mg of glucan. For CNC preparations from Avicel, about 2 g of Avicel were added to pre-heated HCl at 80 °C. Then run the acid hydrolysis for 4 hours, stirring every 15 minutes with a glass or Teflon rod followed by centrifugation several times, 1600×g for 10 minutes. Decant supernatant and resuspend pellet in DI water until the pH reached 5.0. The CNCs were then suspended in the supernatant after centrifugation. Collect the translucent supernatant and resuspend pellet in DI water and shake vigorously to break up clumps until the supernatant is no longer translucent. The reaction mixtures were carried out in triplicate vials at 40 °C and each substrate was suspended in 20 mM sodium acetate buffer, pH 5.0. The enzyme cocktail comprised endoglucanase I from Trichoderma longibrachiatum (Megazyme Co., Bray, Ireland), cellobiohydrolases Cel7A from Penicillium funiculosum and β-glucosidase from Aspergillus niger (Megazyme Co., Bray, Ireland) at a concentration (mg protein/g of glucan) of 2, 13 and 1, respectively. The reaction was followed for 120 h, with sampling every 24 h. 100 μL samples containing both solids and liquid are removed from the mixtures and diluted for sugar analysis by high performance liquid chromatography (HPLC) with a BioRad HPX-87H column. Control experiments using BSA were also performed under the same conditions as mentioned above.