Characterization of a glucose-tolerant β-glucosidase from Anoxybacillus sp. DT3-1

Background In general, biofuel production involves biomass pretreatment and enzymatic saccharification, followed by the subsequent sugar conversion to biofuel via fermentation. The crucial step in the production of biofuel from biomass is the enzymatic saccharification. Many of the commercial cellulase enzyme cocktails, such as Spezyme® CP (Genencor), Acellerase™ 1000 (Genencor), and Celluclast® 1.5L (Novozymes), are ineffectively to release free glucose from the pretreated biomass without additional β-glucosidase. Results In this study, for the first time, a β-glucosidase DT-Bgl gene (1359 bp) was identified in the genome of Anoxybacillus sp. DT3-1, and cloned and heterologously expressed in Escherichia coli BL21. Phylogenetic analysis indicated that DT-Bgl belonged to glycosyl hydrolase (GH) family 1. The recombinant DT-Bgl was highly active on cello-oligosaccharides and p-nitrophenyl-β-d-glucopyranoside (pNPG). The DT-Bgl was purified using an Ni–NTA column, with molecular mass of 53 kDa using an SDS-PAGE analysis. It exhibited optimum activity at 70 °C and pH 8.5, and did not require any tested co-factors for activation. The Km and Vmax values for DT-Bgl were 0.22 mM and 923.7 U/mg, respectively, with pNPG as substrate. The DT-Bgl displayed high glucose tolerance, and retained 93 % activity in the presence of 10 M glucose. Conclusions Anoxybacillus DT-Bgl is a novel thermostable β-glucosidase with low glucose inhibition, and converts long-chain cellodextrins to cellobiose, and further hydrolyse cellobiose to glucose. Results suggest that DT-Bgl could be useful in the development of a bioprocess for the efficient saccharification of lignocellulosic biomass.

Cellulose from forestry and agricultural wastes can be degraded to cellodextrin and cellobiose or glucose. This last step in cellulose hydrolysis involves the use of β-glucosidase (BGL) [11]. BGL is known as a bottleneck factor in the cellulose hydrolysis process. Most commercial fungal BGLs have low stability at higher temperatures (>50 °C). In addition, glucose inhibition is a common problem for several BGL enzymes [12]. To maximize the overall biofuel production efficiency, harnessing thermostable and active BGL with high glucose tolerance is needed [13].
Anoxybacillus sp. DT3-1 (DSM 28778) is a thermophilic bacterium which was isolated from a Malaysian hot spring of a temperature of 75 °C [18], where its genome has been sequenced. A putative BGL (GH1) identified (UniProtKB M5QUM2) in Anoxybacillus sp. DT3-1 as well as its homologues annotated in other genomes of Anoxybacillus spp. have not been biochemically investigated. The purpose of this work was to characterize DT-Bgl from Anoxybacillus sp. DT3-1 and to evaluate its potential use in the conversion of cello-oligosaccharides to glucose.

Protein sequence analysis
The draft genome of Anoxybacillus sp. DT3-1 was sequenced as previously reported [19]. An open reading frame for BGL (1362 bp) was identified in the genome sequence of Anoxybacillus sp. DT3-1. The gene for DT-Bgl encodes a protein of 453 amino acids, and is expressed intracellularly inside Anoxybacillus sp. DT3-1 due to the lack of a signal peptide. Multiple sequence alignments of DT-Bgl with six GH1 BGL enzymes revealed highly conserved sequence elements and its active sites (Fig. 1). DT-Bgl contains the characteristic NEPW (amino acid 163-166) and TENG (353-356) motifs of the GH1 BGL. The two conserved carboxylic acid catalytic residues (Glu164 and Glu354), as shown by highlighted in yellow ( Fig. 1), are located at β-strands 4 and 10, and act as the catalytic acid-base and nucleophile, respectively ( Fig. 1) [20]. The overall three-dimensional predicted structure of DT-Bgl resembles the typical (β/α) 8 -barrel structure observed in other structurally characterized GH1 family member, with the active site pocket located at the barrel (Fig. 2).
The protein sequence showed 92-96 % identity to homologous sequences present in the genomes of A. ayderensis [21], A. thermarum [22], A. flavithermus [23], and other Anoxybacillus spp. None of these homologous proteins have been characterized so far. The phylogenetic tree shown in Fig. 3 was constructed by comparing the protein sequence of DT-Bgl with other BGL sequences present in database. β-glucosidases from Anoxybacillus spp. clustered together and formed Clade II with BGL from Jeotgalibacillus malaysiensis, Bacillus, Tumebacillus, Alicyclobacillus, Thermoanaerobacterium, and Thermoanaerobacter spp. The similarity between DT-Bgl and the neighboring taxa other than Anoxybacillus spp. was in the range of 51-56 %. A previous whole-genome comparison of four Anoxybacillus strains and several Geobacillus spp. elucidated that proteins from these two genera are closely related [19]. Nevertheless, DT-Bgl exhibited low-protein sequence identity of 42 % to a BGL from Geobacillus kaustophilus strain HTA426. Therefore, it is believed that DT-Bgl is a BGL uniquely affiliated to Anoxybacillus.
Clade II has been previously described as a group of enzymes constituted of GH1 thermostable β-glucosidases [12]. It is clear from Fig. 3 that Clade II is probably not restricted to enzymes obtained from thermophilic genera, since Tumebacillus [24] and Jeotgalibacillus [25] are mesophiles. Clade II is phylogenetically closer to Clade III, which is well represented by Streptomyces spp. and other mesophilic bacteria, such as Paenibacillus panacisoli. Members in Clade I are GH1 β-glucosidases from fungi. Clade IV and Clade V are constituted of enzymes classified in the GH3 family, sourced from fungi and bacteria, respectively.

Purification of the recombinant DT-Bgl
DT-Bgl was overexpressed in E. coli using the expression vector pET28a. Under current experimental conditions, the recombinant DT-Bgl was produced as a soluble protein in large quantity. In contrast to this, some β-glucosidases from other thermophiles, for instance, Tt-BGL from Thermotoga thermarum DSM 5069 T formed inclusion body in E. coli host [26].
The purified fractions of DT-Bgl eluted from an Ni-NTA column were pooled and analyzed by SDS-PAGE. A single band corresponding to a molecular mass of approximately 53 kDa was observed ( Fig. 4a) with a specific activity of 1158 U/mg using pNPG as the substrate.

Effects of temperature and pH optimum and stability on DT-Bgl
The biochemical properties of DT-Bgl were determined using the purified recombinant DT-Bgl. When using pNPG as substrate, the purified DT-Bgl exhibited maximum activity at 70 °C and pH 8.5, while its relative activity was higher than 70 % of the maximum activity at the temperature range from 60 to 70 °C and pH range from 6.5 to 9.0 (Fig. 4b, c). The optimum temperature of DT-Bgl is comparable or higher than BGLs from Nasutitermes takasagoensis, Thermoanaerobacterium aotearoense, and T. thermosaccharolyticum DSM 571 (Table 1), but DT-Bgl exhibits a much higher optimum pH value. DT-Bgl was stable at pH 6-11, and a negligible reduction in activity was observed when it was incubated at temperatures lower than 60 °C (Fig. 4b, c). The activity halflife of DT-Bgl at 60 °C was 24 h (Fig. 4d). The hydrolysis of cellulose is usually performed under mild conditions at 45-50 °C [27]. Due to the feature of stability at elevated temperature and pH, DT-Bgl is a potential β-glucosidase candidate for wide industrial saccharification processes, including textiles, pulp and paper, and waste treatment for cellulose-degrading function [28].
Despite the optimum temperature of DT-Bgl was 70 °C, its stability was drastically reduced over time. DT-Bgl lost its activity completely after pre-incubated in the absence of pNPG substrate for 30 min at 70 °C (Fig. 4b, d). This indicated that pNPG substrate may stabilize DT-Bgl at high temperatures. Therefore, for subsequent analyses, the reaction was conducted at 60 °C instead of 70 °C, since the enzyme is more stable at 60 °C.

Effect of metal ions and reagents on purified DT-Bgl
The influence of various metal ions (2 or 5 mM) on DT-Bgl activity was investigated ( Table 2). None of the tested metal ions significantly elevated the activity of DT-Bgl, suggesting that the enzyme does not require any of these metals as co-factors. Similar observation was noticed for BGL of T. thermarum DSM 5069 T [26]. Besides, it has been reported that Ca 2+ improves the activity of multiple GH enzymes, including BGL from T. thermarum DSM 5069 T and T. thermosaccharolyticum DSM 571 [12,26]. However, CaCl 2 did not influence the catalytic efficiency of DT-Bgl. Salts, such as NaCl and KCl, showed little effect on its activity; however, DT-Bgl was sensitive to Fe 3+ , Ni 2+ , Co 2+ , Zn 2+ , and Mn 2+ ions (Table 2). Nevertheless, Mn 2+ was known to be an activator for BGL from T. thermosaccharolyticum DSM 571 [12]. The addition of 2 or 5 mM urea had no significant effect on its activity. DT-Bgl retained approximately 90 % of its original activity in the same molarity of EDTA indicating that DT-Bgl is not a metalloprotein. Furthermore, DT-Bgl tolerated a variety of chemical reagents, including 1 % (v/v) Tween-20, 1 % (v/v) Tween-40, DMSO, or Triton-100; however, it lost its activity in 5 % (v/v) Tween-20, 5 % (v/v) Tween-40, or 5 % (v/v) Tween-80.

Kinetic parameters
Using pNPG as substrate, DT-Bgl showed K m value of 0.22 mM, V max of 923.7 U/mg, and a k cat value of 812.7 s −1 using Michaelis-Menten function (Fig. 5). The comparison of these values to closest taxa shown in Fig. 3 could not be performed because of lack of information on these BGLs and the homologues. K m of DT-Bgl was found to be lower than the K m values reported for the BGLs of other microbial species, including T. thermosaccharolyticum and T. thermarum (Table 1).

Substrate specificity
DT-Bgl showed broad substrate-cleaving activity ( Table 3). Despite cellobiose is the main substrate for β-glucosidase, DT-Bgl was unable to hydrolyse completely the dimeric compound to glucose monomer using 1 U of purified DT-Bgl at 60 °C for 15 min (Table 3). Using  (Table 3). This emphasizes the importance of DT-Bgl in the hydrolysis of biomass by containing the activities of both laminaribiase and cellobiase. It also showed low hydrolysing activity towards β-gentiobiose (21 %) and lactose (33 %). Negligible hydrolysis was detected when maltose and sucrose were used as substrates. Interestingly, DT-Bgl also showed high hydrolysis efficiencies on cellotriose, cellotetraose, cellopentaose, and cellohexaose (Table 3). These data suggested that DT-Bgl was able to convert these cellodextrins to glucose efficiently, where this finding is in contrast to the hydrolysis preference of BGL obtained from Aspergillus niger [33], Pichia pastoris [31], and Trichoderma koningiopasis [34], where the enzyme efficiency decreased as the length of cellooligosaccharides increased [31]. It is not clear the maximum length of substrate (degree of polymerisation) for DT-Bgl, since we have not reacted the enzyme with any   ability of DT-Bgl, the enzyme can be assumed to belong to the first group "broad range β-glucosidase".

Effect of glucose on DT-Bgl activity
DT-Bgl activity was not affected by glucose up to 5 M when pNPG was used as a substrate. The enzyme retained 93 and 43 % relative activity in 10 and 15 M glucose, respectively (Fig. 6). Glucose inhibition in DT-Bgl is lower than β-glucosidase from T. thermosaccharolyticum DSM 571, which was known to be one of the glucose-tolerant β-glucosidases [12]. When cellobiose was used as substrate, DT-Bgl retained 76 and 54 % relative activity in 0.2 and 0.4 M glucose, respectively. DT-Bgl was fully inhibited in 3 M glucose (Fig. 6).

Conclusions
To our knowledge, this is the first study of the heterologous expression of the GH1 β-glucosidase gene from Anoxybacillus genus. The DT-Bgl was found to be stable and active in a broad range of pH and temperatures, and it did not require co-factors for activation. DT-Bgl efficiently hydrolyses cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, and was defined as a "broad range β-glucosidase". Notably, DT-Bgl has high resistant to glucose up to 5 M. Thus, this study provides a useful novel BGL, which may be used to improve the enzymatic conversion of cellulosic biomass to glucose.

Bioinformatic analyses
Multiple amino acid sequence alignment of DT-Bgl and other selected GH1 BGL enzymes was carried out using the Clustal Omega (Fig. 1) Putative catalytic residues for DT-Bgl were inferred from the sequence alignment. The position of secondary structures of DT-Bgl was predicted with ESPript [35]. Phylogenetic tree among DT-Bgl and other β-glucosidases was constructed using the neighbor-joining  [38].

Cloning and expression of DT-Bgl
Stock culture of Anoxybacillus sp. DT3-1 was grown on solid (15 g/L agar) or liquid media containing 4.0 g/L of peptone, 4.0 g/L of tryptone, 4.0 g/L of yeast extract, 2.0 g/L of NaCl, and 1.0 g/L of MgSO 4 , as reported previously [18]. Genomic DNA was purified using the Wizard Genomic DNA Purification Kit (Promega, Madison, USA). The DT-Bgl ORF was PCR-amplified using F5′-ACG CCC GGA TCC ATG TTT CAG TTT CC-3′) and R5′-CAC CTC GAG TTT GTC GTT GTC TAC TTG C-3′) containing the BamHI and XhoI (underlined sequences) restriction sites, respectively. The gene was cloned into pET-28a, and transformed into Escherichia coli BL21 (DE3) for its expression. The recombinant E. coli was cultured overnight in LB medium supplemented with 50 μg/mL kanamycin at 37 °C with shaking (200 rpm). Two milliliters of culture were inoculated into 200 mL of fresh LB/kanamycin and incubated at 37 °C with shaking at 200 rpm. When OD 600 reached to 0.6, protein expression was induced by adding filtered sterilised 0.4 mM isopropyl-β-d-thiogalactopyranoside, and the culture was incubated at 37 °C under shaking condition (200 rpm) for 3 h.

Purification of recombinant DT-Bgl
The induced E. coli BL21 (DE3) cells were lysed using the B-PER ® Bacterial Protein Extraction Reagent (Thermo  Scientific, Waltham, USA). The crude enzyme was dialysed against sodium phosphate buffer (20 mM, pH 7.4) overnight at 4 °C and then loaded onto an Ni-NTA Superflow column (Qiagen, Venlo, Netherlands) using an ÄKTA start system (GE Healthcare, Uppsala, Sweden). The column was equilibrated with binding buffer (20 mM sodium phosphate buffer, pH 7.4, containing 500 mM NaCl and 60 mM imidazole). The resin was washed with binding buffer, and bound protein was eluted with elution buffer (20 mM sodium phosphate buffer, pH 7.4, containing 400 mM NaCl and 350 mM imidazole). The purified enzyme was dialysed against glycine-NaOH buffer (100 mM, pH 8.5) overnight at 4 °C.

Enzyme assay
β-glucosidase activity was determined by measuring the hydrolysis of pNPG (Calbiochem, Darmstadt, Germany). The reaction mixture contained 1.0 mL of 20 mM pNPG in 100 mM glycine-NaOH (pH 8.5) and 100 μL DT-Bgl. After incubation at 60 °C for 15 min, the reaction was stopped by adding 1.0 mL ice-cold 1 M Na 2 CO 3 . The released p-nitrophenol was measured using a spectrophotometer at 405 nm. The enzyme assays were performed in triplicates. Enzyme-and substrate-free controls were also incubated and measured under the same conditions. One unit (U) of DT-Bgl activity was defined as the amount of enzyme liberating 1 µmol of p-nitrophenol per min per mL under the assay conditions.

Effect of temperature and pH on DT-Bgl
The optimum temperature and pH of DT-Bgl were determined by reacting the enzyme with pNPG for 15 min in the ranges of 30-100 °C and pH 4.0-11.0, respectively. The effects of pH on β-glucosidase activity and stability were tested using 100 mM sodium acetate buffer (pH 4.0-5.5), sodium phosphate buffer (pH 6.0-8.0), or glycine-NaOH buffer (pH 8.5-11.0) at 60 °C. The enzyme activity obtained at the optimum temperature or pH was used to calculate the relative percentage of enzyme activity at varying temperature and pH values. The temperature stability was determined by incubating the enzyme for 30 min without pNPG at 60 °C and measuring the residual activity.

Determination of kinetic parameters
The apparent Michaelis-Menten constant (K m ), maximum velocity (V max ), and turnover number (k cat ) for the purified DT-Bgl were assessed by measuring the rate of hydrolysis of pNPG at various concentrations (0.01-3.0 mM) at 60 °C for 15 min in glycine-NaOH buffer (100 mM, pH 8.5). The enzymatic kinetic parameters were determined from the Michaelis-Menten function using the SigmaPlot software version 12.0 (Systat Software, Chicago, USA).

Substrates hydrolysis by DT-Bgl
To test the ability of DT-Bgl to degrade various substrates (Table 3), 100 μL 1 % (w/v) of these compounds was reacted with 10 μL 1 U of purified DT-Bgl. All reactions were carried out in glycine-NaOH buffer (100 mM, pH 8.5), at 60 °C for 15 min, unless specified. Reactions were stopped by boiling for 5 min, and the samples were analyzed on the HPLC 1260 system (Agilent Technologies, Santa Clara, USA), using a 7.8 × 300 mm Rezex RPM-Monosaccharide Pb +2 column (Phenomenex Inc, Torrance, USA) fitted with a 7.8 × 50 mm LC guard column filled with the same stationary phase. An Agilent 385-Evaporative Light Scattering Detector was used, and the nebulizer and evaporator were kept at 30 °C with a nitrogen flow rate of 1.6 slm (standard liters per min). Nanopure water, filtered through a nylon membrane filter (0.45 µm pore size, Millipore, Darmstadt, Germany) and degassed, was used as the mobile phase. All the insoluble particles were filtered through a polytetrafluoroethylene syringe filter membrane (0.45 µm pore size) prior to injection into the column. All substrates and standards used were of high purity grade.

Effect of glucose on DT-Bgl
The effect of glucose on purified DT-Bgl was evaluated by reacting pNPG or cellobiose in the presence of glucose at various concentrations using the standard assay procedure and HPLC analysis, respectively. The relative activity (%) was defined as the relative value to the activity of a control without glucose.
Authors' contributions CSC, LLS, and FAM performed the cloning, expression, purification, and characterization of the enzyme. CSC, MSS, and KMG conducted the bioinformatic analyses. CSC, KGC, RKS, and KMG designed the experiments, wrote, and reviewed the manuscript. All authors read and approved the final manuscript.