Microorganism and culture media
The ascomycete T. amestolkiae is deposited in the Collection of the Institute Jaime Ferrán of Microbiology (IJFM) at the Centro de Investigaciones Biológicas, with the access number A795. Fungal strains were maintained in tubes with PDA (Potato dextrose agar) medium, stored at 4 °C, and periodically reseeded in PDA plates, incubated at 28 °C.
To obtain spore suspensions from this culture, agar pieces (1 cm2) were cut and added to a tube containing 5 mL of a solution of 1% NaCl and 0.1% Tween 80. The mixture was used to inoculate 250 mL flasks with 50 mL of CSS medium (40 g/L glucose, 0.4 g/L FeSO4·7H2O, 9 g/L (NH4)2SO4, 4 g/L K2HPO4, 26.3 g/L corn steep solid, 7 g/L CaCO3, and 2.8 ml/L soybean oil), incubating at 28 °C and 250 rpm for 5 days. These cultures were used as pre-inoculum.
Production and purification of BGL-3
For BGL-3 production, 2 mL from the CSS cultures of T. amestolkiae was inoculated in 250-mL Erlenmeyer flasks containing 50 mL of Mandels medium [26] with 1% of glucose as carbon source, and incubated in an Innova 4330 orbital shaker (New Brunswick Scientifics) at 28 °C and 250 rpm. All culture media were prepared with autoclaved distilled water.
When maximal β-glucosidase activity was detected in the supernatants (7 days), the cultures were cropped and centrifuged at 10,000×g for 30 min to separate mycelium and supernatant, which was vacuum-filtered through filter paper and nitrocellulose membrane discs (Millipore) of 0.8, 0.45, and 0.22 µm to complete clarification. This treated supernatant was further concentrated using a tangential flow filtration system (7518-02 Masterflex, from Millipore) equipped with a 10 kDa polysulfone membrane (Membrane Cassette, Filtron) and an ultrafiltration cell (Amicon, Millipore) with a 10 kDa cutoff polysulfone membrane (Millipore). Protein purifications were performed using an ÄKTA Purifier HPLC system (GE Healthcare Life Sciences). The crude extract was first dialyzed in 10 mM sodium phosphate, pH 6.0, and applied onto a Capto Adhere HiTrap cartridge (GE Healthcare Life Sciences) equilibrated with the same buffer at 2 mL/min. Retained proteins were eluted by using a linear gradient of NaCl in the same buffer (0–0.25 M of NaCl in 30 min) and 100% NaCl (15 min). Finally, the cartridge was equilibrated with the initial buffer.
The purified BGL-3 was dialyzed against acetate buffer, pH 4.0, and its homogeneity confirmed by SDS-PAGE using 10% gels stained with Coomassie brilliant blue R-250.
bgl-3 gene sequencing and real-time quantitative qRT-PCR analysis
In order to identify bgl-3 sequence, a BLASTP against predicted proteins of T. amestolkiae was carried out. The gene sequences of the best hits were used as queries to run a local BLASTN against the assembled genome. An alignment between the gene and the best hits of the BLAST search was done to identify possible introns in the sequences of other BGLs. With the predicted coding sequence, the presence of a possible signal peptide was analyzed with the SignalP server, and the putative mature gene of BGL-3, without introns and signal peptide, was translated to protein by using the ExPASy Bioinformatics resource portal (ProtParam tool), in order to obtain the theoretical molecular mass and isoelectric point of BGL-3.
Primers for qRT-PCR were designed based on bgl-3 sequence (BGL-3FWQPCR (TTCGTATCATGTCTGCATTC) and BGL-3RVQPCR (ATTCTTGAGGAGAACAATGC)). 18S rRNA was chosen for normalization of expression across all treatments [27] (primers 18sFW (ATTGGAGGGCAAGTCTGGTG) and 18sRV (CCAGTGAAGGCCATGGGATT)).
RNA was extracted from T. amestolkiae cultures growing in 1% of glucose using TRIzol reagent [28]. One-step qRT-PCR was performed using total RNA preparations treated with a Turbo DNA-free kit (Ambion). Brilliant III Ultra-Fast SYBR® Green qRT-PCR Master Mix, from Agilent, was used for qRT-PCR reactions. Each reaction was performed according to the manufacturer’s instructions, adding 5 ng of the respective RNA.
Reactions were done in a LightCycler® 96 detection system, and analyzed with LightCycler® 96 SW. The running method consisted of several steps: 50 °C for 10 min, 95 °C for 3 min, 40 cycles of 95 °C for 10 s, and 60 °C for 20 s. All reactions were performed six times. The amplification efficiency for each primer pair was determined with serial dilutions from an RNA sample (100 ng RNA/µL) with at least five dilution points. The relative quantification of PCR products was calculated by the comparative 2−ΔΔCT (cycle threshold) method.
Cloning and expression of bgl-3 in P. pastoris
RNA was isolated from fungal cultures by using TRIzol reagent, as explained before. The isolated transcripts were converted to cDNA using the Superscript II Reverse Transcriptase RT-PCR kit (Invitrogen) using 50 µM random hexamers. PCR amplifications were performed in a thermocycler Mastercycler pro S (Eppendorf) using genomic DNA as template. Primers were designed based on the nucleotide sequence of the bgl3 gene from T. amestolkiae genome (GenBank accession no. MIKG00000000), but excluding the region corresponding to the signal peptide. Restriction sites for XhoI and NotI were, respectively, added to the forward and reverse primers (BG3FWXHOI: 5′-ATCTCGAGAAAAGATACTCTCCTCCAGCTTACCCT-3′, and BG3 RV NOTI: 5′-ATGCGGCCGCATGCCCAATCTTCAAAGCCAA-3′). Reaction mixtures were initially subjected to denaturation at 95 °C for 5 min, followed by 36 cycles of amplification consisting of denaturation at 95 °C for 45 s, primer annealing at 55 °C for 45 s, and elongation at 72 °C for 3 min, followed by a final extension step at 72 °C for 10 min. The PCR product was ligated to the yeast expression vector pPICzα (Invitrogen), and it was used for transforming P. pastoris X-33 after linearization with SacI (New England Biolabs). Transformed colonies were grown on YPD medium plates (10 g/L Yeast extract, 20 g/L peptone, 20 g/L glucose, and 10 g/L of agar) with 100 μg/mL of zeocin as selection marker. Since it is considered that the better the zeocin resistance, the better the protein production, the scored transformants were re-screened for resistance to a zeocin concentration of 1 mg/mL, selecting the clones with the highest tolerance for protein production.
Production and purification of recombinant BGL-3
To prepare a fresh inoculum, the selected clones were grown overnight in 250 mL flasks with 50 mL of YEPS medium at 28 °C and 250 rpm. Then, recombinant protein production was carried out in 2-L flasks with 400 mL of YEPS medium (20 g/L peptone, 10 g/L yeast extract, 10 g/L sorbitol). Cultures were incubated at 28 °C and 250 rpm for 7 days with daily addition of 5 g/L methanol. Samples were periodically taken to measure β-glucosidase activity.
For BGL-3* purification, 7-day-old cultures were harvested and centrifuged at 10,000×g and 4 °C for 20 min. The supernatant was first concentrated by tangential filtration and finally concentrated and dialyzed against 10 mM phosphate buffer (pH 6.0) using a 50-kDa cutoff membrane (Merck-Millipore). BGL-3* was purified after two chromatographic steps. First, a QFF Hi Trap cartridge (GE Healthcare) equilibrated with phosphate buffer pH 6.0 was used. Elution of the bound proteins was carried out by applying a linear gradient from 0 to 0.25 M of NaCl in 25 min, at 2 mL/min. The column was then washed with 10 mL of 1 M NaCl and re-equilibrated using 10 mL of the starting buffer. Fractions with β-glucosidase activity were collected, dialyzed, and concentrated. To complete the purification of BGL-3*, the sample from the previous stage was analyzed by size exclusion chromatography on Superose 12 column (GE Healthcare Life Sciences). To avoid unspecific interactions, the same buffer (plus 100 mM NaCl) was used for column equilibration and proteins elution, at a flow of 0.5 mL/min.
Protein quantification, enzyme assays, and substrate specificity
Total protein was estimated by the BCA method using bovine serum albumin as standard, measuring the absorbance of the sample at 280 nm in a Nanodrop (Thermo Fisher Scientific). The β-glucosidase standard reaction was performed using 0.1% (w/v) p-nitrophenyl-β-d-glucopyranoside (pNPG, Sigma), at 70 °C, in sodium acetate buffer 100 mM, pH 4.0. Other nitrophenyl derivatives, as pNPX (p-nitrophenyl-β-d-xylopyranoside), β-pNPgal (p-nitrophenyl-β-d-galactopyranoside), α-pNPG, α-pNPgal (p-nitrophenyl-α-d-galactopyranoside), p-nitrophenyl-α-L-rhamnopyranoside, and p-nitrophenyl-β-d-fucopyranoside were assayed analyzing pNP release. The reactions were stopped after 10 min by adding 2% (w/v) Na2CO3, and the pNP released was spectrophotometrically measured at 410 nm. One BGL activity unit was defined as the amount of enzyme capable of releasing 1 micromole of pNP per minute (the molar extinction coefficient of pNP is 15,200 M−1 cm−1).
β-glucosidase activity on cellobiose, gentiobiose, laminaribiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, maltose, sucrose, and lactose, was quantified by measuring the glucose released from these compounds after enzyme hydrolysis, using the Glucose-TR commercial kit (Spinreact), according to the manufacturer’s instructions. Reactions were performed in sodium acetate 100 mM, pH 4.0, incubating in a heating block for 10 min at 1200 rpm. Then, the reactions were stopped by heating at 100 °C for 5 min.
The activity of BGL-3 was also determined against different polysaccharides, all of them prepared in 50 mM sodium acetate buffer, pH 4.0: 1.25% Avicel (microcrystalline cellulose), 3% carboxymethyl cellulose (CMC), 1% laminarin from L. digitata and L. hyperborea, and 3% beechwood xylan. The substrates were incubated with BGL-3 in a heating block at 60 °C and 1200 rpm for 10 min. The released reducing sugars were determined by the Somogyi–Nelson method [29]. A degree of polymerization of 25 units has been previously described for laminarin [30].
The kinetic constants of the purified BGL-3 were determined against pNPG (over a range of concentrations from 10 μM to 5 mM), oNPG (40 μM to 20 mM), cellobiose (80 μM to 40 mM), gentiobiose (80 μM to 40 mM), laminaribiose (80 μM to 40 mM), cellotriose (80 μM to 40 mM), cellotetraose (80 μM to 40 mM), cellopentaose (40 μM to 20 mM), and cellohexaose (20 μM to 10 mM). The values of Km and Vmax were determined using the program SigmaPlot, based on the Michaelis–Menten model.
Ki for BGL-3 was calculated using pNPG as substrate, in the presence of different concentrations of glucose (0, 2.5, 5, and 10 mM).
All enzymatic assays were performed including 0.1% BSA, a protein which does not affect the catalytic activity of the BGL-3, to prevent the activity loss when working with low enzyme concentrations [15].
Physicochemical properties
To obtain the peptide mass fingerprint of the protein, the sample was run in a SDS-PAGE gel as explained before, excising the BGL-3 band. After tryptic digestion [31], the peptides’ mixture was analyzed in a MALDI-TOF/TOF Autoflex III (Bruker Daltonics) equipped with a laser and a Smartbeam LIFT-MS/MS device. The data from MS and MS/MS experiments were combined using the 3.0 BioTools (Bruker Daltonics) software and searched against the NCBInr database using 2.3 MASCOT as the search engine (Matrix Science). Relevant search parameters were trypsin as enzyme, carbamidomethylation of cysteines as fixed modification, methionine oxidation as variable modification, 1 missed cleavage allowed, peptide tolerance of 50 ppm, and MS/MS tolerance of 0.5 Da. Protein scores greater than 75 were considered significant.
The molecular mass of the native BGL-3 was determined both by size exclusion chromatography using a Superose 12 column (GE Healthcare), and by MALDI-TOF in the instrument described above. Isoelectric point (pI) was determined by isoelectrofocusing (IEF) in 5% (w/v) polyacrylamide gels, prepared with Pharmalyte (pH 3.0–10.0) as carrier ampholytes (GE Healthcare), using a Mini Protean III Cell system (Bio-Rad). 1 M H3PO4 and 1 M NaOH were the anode and cathode buffers, respectively. The pH gradient was directly measured on the gel using a contact electrode (Crison). The activity of BGL-3 was tested in zymograms after IEF, incubating the gel with 2 mM p-methylumbelliferyl-β-d-glucopyranoside (Sigma-Aldrich) for 10 min, and observing the gel under UV light with a Gel Doc XR + system (Bio-Rad) to detect free 4-methylumbelliferone.
The optimal values of pH and temperature and the stability of BGL-3 were evaluated with pNPG as model substrate, measuring the residual activity after the treatments in standard conditions. The buffer Britton–Robinson (100 mM) was used to study the effect of pH on BGL-3 activity, adjusting different aliquots to pH values from 2.0 to 10.0. BGL-3 was incubated at 4 °C and different pH values for 3 days. After this time, a standard BGL reaction was performed to determine its optimal pH. Temperature assays were done between 30 and 80 °C using solutions of BGL-3 in acetate pH 4.0. Its thermostability was analyzed in the same temperature range for 72 h, taking aliquots at different incubation times to measure the residual activity.
Saccharification of wheat straw slurry and laminarin
Enzymatic saccharification was tested in samples of wheat straw slurry from steam explosion (kindly provided by Abengoa). For saccharification, 100 mg of wheat straw slurry was treated with 2 U/mL of BGL activity in 100 mM sodium acetate buffer, pH 4 (final volume of 2 mL), incubating in a heat block at 50 °C and 1200 rpm, for 120 h. The sources of BGL activity tested were Celluclast 1.5L (Novozymes), a basal cocktail for biomass degradation with low BGL activity, NS-50010 (Novozymes), which is a β-glucosidase-rich cocktail, and the purified BGL-3. The control sample contained Celluclast 1.5L as the unique source of BGL activity. To compare the efficiencies of NS-50010 and BGL-3, 1 U/mL of BGL activity from Celluclast 1.5L was supplemented with 1 U/mL of either NS-50010 or the purified BGL-3. The glucose released was measured in sample supernatants at different time intervals, using the Glucose-TR commercial kit (Spinreact), according to the manufacturer’s instructions.
Similarly, the release of glucose from laminarin, from Laminaria digitata (Sigma-Aldrich) and Laminaria hyperborea (Koch-light laboratories), was evaluated. The reaction mixtures contained 100 mg of laminarin in 10 mL of 100 mM sodium acetate buffer, pH 4.0 and 3 U/mL of laminarinase activity. This was provided by either the purified BGL-3 or a commercial β-1,3-glucanase from Helix pomatia (Sigma-Aldrich). Reactions were performed in a heat block at 50 °C and 1200 rpm for 24 h, measuring the glucose released at different times as detailed above.