Cloning of NpaBGS cDNA
The cDNA of NpaBGS was amplified by PCR using the Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) with the gene-specific primers flanked by restriction site sequences, EcoRI/F1-1 (AATTCATGAAGTTCTCATCTGTTTTATCTACTG), EcoRI/F1-2 (CATGAAGTTCTCATCTGTTTTATCTACTG), XhoI/R1-1 (GTTAGTAAAGTTTGTAAGCTCTCTTC), XhoI/R1-2 (TCGAGTTAGTAAA GTT TGTAAGCTCTCTTC), and PCR products were cloned into the pGEM-T vector (Promega Corp. Madison, WI, USA). The plasmid DNA from a positive clone was digested with XhoI and EcoRI and subcloned in frame with the P. pastoris pPICZ A expression vector. After sequencing confirmation of the NpaBGS cDNA, the recombinant plasmid, pPA-NpaBGS, was used to transform the P. pastoris GS115 (his4) strain (Invitrogen).
Construction ofPichia pastorisrecombinant strains expressing NpaBGS
Ten μg of the pPICZA-NpaBGS DNA was linearized with BglII and transformed into the yeast P. pastoris GS115 strain by electroporation. A 200 μL aliquot was spread on YPD plates containing 100 μg/mL zeocin and incubated at 30°C. Another aliquot of electroporated cells was spread onto YPD plates containing 1,000 μg/mL zeocin to screen for colonies with high copy insertion. One transformant confirmed for the Mut+ phenotype was scored and grown in 10 mL of BMGY medium. The yeast colony was cultured at 30°C with orbital shaking at 250 rpm for about 20 h until the density reached OD600 of 5.0. The yeast culture was harvested by centrifugation at 2,000 x g for 5 min at room temperature. To induce expression via the AOX1 promoter, the pellet was resuspended in 50 mL of BMMY medium and grown at 30°C with 250 rpm shaking for 5 days, during which methanol was added to the concentration of 0.5 % at 24-hr intervals to maintain induction, and the activity of the culture was examined simultaneously. In order to increase the level of induction, various concentrations of methanol (from 0.5 to 3 %) were also tested.
Purification of recombinant NpaBGS
The yeast broth (4 L) was percolated through filter paper (Toyo Roshi Kaisha, Japan) and concentrated with a stirred ultrafiltration cell (model 8400; Millipore Corp.) equipped with a PM 10 membrane (Millipore Corp., USA) under the nitrogen pressure of 4.0 kg. f/cm2 and dialyzed against 20 mM sodium acetate buffer (pH 5.0). The extracted enzyme was condensated by precipitation at increasing concentrations of ammonium sulfate (0-30 %, 30-50 %, and 50-70 %) at 5°C. The fraction contained better activity and amount of enzyme was found at 50-70 % ammonium sulfate precipitation. The resulting precipitates were collected by cold centrifugation, dissolved in distilled water and dialyzed (0.1 M phosphate buffer, pH 6.0, 48 h, 5°C) to remove excessive salt. The protein (30 mL) was then loaded onto a Toyopearl DEAE-650 S (Tosoh, Japan) column (2.0× 20 cm) and eluted with a step gradient of 0, 200, 300, 400, and 500 mM of NaCl in a volume of 1,000 mL. The fractions showing cellulolytic activity were pooled and concentrated by ultrafiltration, then dialyzed against 50 mM sodium acetate (pH 5.0) containing 0.15 M NaCl. The dialyzed sample (4 mL) was applied to a Sephacryl 300-S HR (GE Healthcare Bio-Sciences AB) column (1.6 × 60 cm) and eluted with the same buffer at a flow rate of 0.5 ml/min. The active fractions were concentrated by ultrafiltration and dialyzed against 20 mM Tris–HCl buffer (pH 8.0) containing 1.5 M (NH4)2SO4. The dialyzed enzyme solution (2.0 mL) was then loaded onto a Resource PHE (Amersham Biosciences, USA) column (1.0 × 1.0 mL) equilibrated with the same buffer containing 1.5 M (NH4)2SO4. The active fractions were eluted with a decreasing linear gradient from 1.5 to 0 M of (NH4)2SO4 in the buffer at a flow rate of 1.0 mL/min. The fractions containing the β-glucosidase activity were further concentrated with an Ultrafree-0.5 centrifugal filter (Millipore Corp., USA), and the purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples were electrophoresed on 12 % (w/v) SDS–PAGE gels and visualized by staining with Coomassie Brilliant Blue R-250 . The molecular weight markers were obtained from Fermentas (USA). Protein concentration was determined using a protein assay kit (Bio-Rad Laboratories Inc., Hercules, California, USA). Native PAGE electrophoresis was carried out similarly by exclusion of SDS from all solutions. Zymogram after native PAGE was performed as described by Feng et al.  and Duan et al. .
Characterization of recombinant NpaBGS enzymatic properties
After optimizing the culture conditions of the P. pastoris recombinant strain for producing NpaBGS, the β-glucosidase activity in the culture medium supernatant was measured. β-glucosidase activity was assayed by adding 10 μl of culture supernatant to 90 μL of 2 % (w ⁄ v) cellobiose (low viscosity), and the amount of glucose assay was carried out according to the manual of Glucose (HK) assay kit (Sigma-Aldrich, USA). One unit (U) of β-glucosidase activity was defined as the amount of 1 μmole glucose released per minute at pH 7.0 under the assay conditions described below. Furthermore, other natural substrates, such as amygdalin, arbutin, larminarin, phenyl-β-D-glucoside, and β-gentiobiose, and substrates, such as 4-methylumbel-liferyl-β-D-cellobioside (MUC), 4-mehtylumbelliferyl-β-D-galactopyronoside (MUG) and 4-methylumbel-liferyl-β-D- glucopyranoside (MUD) were also assayed at in the same condition.
The temperature profile and the optimum temperature of NpaBGS were determined by the activity on cellobiose at 30, 40, 50, and 60°C. The optimum pH was determined using 100 mM buffers: sodium citrate (pH 2.0 and 3.0), sodium acetate (from pH 4.0 to pH 6.0) and Tris–HCl (form pH 6 to pH 9). The thermostability of NpaBGS and the commercial enzyme Novo™ 188 (Novozymes, Bagsvaerd, Denmark) were compared by incubation at 40°C in 100 mM in Tris–HCl buffer (pH 6.0) during a time course and then measured their β-glucosidase activity against cellobiose.
To evaluate the effect on the β-glucosidase activity of metal cations, such as Al3+, Ca2+, Cu2+, Fe3+, Mg2+, Mn2+, and Zn2+, and reducing agents, such as DTT, and β-mercaptoenthanol, these elements were added separately to the standard assay in 100 mM Tris–HCl buffer, pH 6.0, to a final concentration of 1 and 10 mM, respectively.
Efficiency of β-glucosidase in SSF application
S. cerevisiae BY4741 and Kluyveromyces marxianus KY3  were used in SSF experiments to study the effect of different culture temperatures. Both yeasts could grow at 30°C on the solid YPAD medium (1 % yeast extract, 2 % peptone, 24 mg/L adenine hemisulfate, 2 % glucose, and 2 % agar), while separate cultures of K. marxianus KY3 was also examined at 37°C and 40°C for its thermotolerance. First, the two microbes were pre-cultured overnight in YPAD medium and inoculated to initial O.D.600nm 0.1 in 10 ml of fresh YPA medium containing either 2 % glucose or 2 % cellobiose under an aerobic condition. The experimental groups were the YPA medium with 2 % cellobiose and supplemented with an equal unit (2 units) of NpaBGS or Novo™ 188 β-glucosidase in the culture. The growth curve, glucose generation and ethanol production of these cultures were determined at 0, 12, 16, 20, and 24 hrs. The cell density was measured at 600 nm by spectrophotometer (Ultrospec 2100 pro, GE Healthcare Bio-Sciences AB). In a parallel experiment, the SSF experiment was conducted using 2 % dry napiergrass as the sole carbon source, and adding 2 ml of commercial Celluclast 1.5 L in a 50 ml yeast culture at 40°C. In addition, K. marxianus KY3 and KY3-NpaBGS, in which KY3 was transformed with the NpaBGS gene via a commercial expression system (K. lactis Protein Expression Kit, New England Biolabs) , were used in the SSF experiment. Glucose and ethanol assays were performed using Glucose (HK) assay kits and Ethanol Enzymatic BioAnalysis kits (Roche Molecular Biochemicals, Germany) following manufacturer’s procedures.