Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum

Background Arabinan is an important plant polysaccharide degraded mainly by two hydrolytic enzymes, endo-arabinanase and α-L-arabinofuranosidase. In this study, the characterization and application in arabinan degradation of an endo-arabinanase from Thermotoga thermarum were investigated. Results The recombinant endo-arabinanase was expressed in Escherichia coli BL21 (DE3) and purified by heat treatment followed by purification on a nickel affinity column chromatography. The purified endo-arabinanase exhibited optimal activity at pH 6.5 and 75°C and its residual activity retained more than 80% of its initial activity after being incubated at 80°C for 2 h. The results showed that the endo-arabinanase was very effective for arabinan degradation at higher temperature. When linear arabinan was used as the substrate, the apparent Km and Vmax values were determined to be 12.3 ± 0.15 mg ml−1 and 1,052.1 ± 12.7 μmol ml−1 min−1, respectively (at pH 6.5, 75°C), and the calculated kcat value was 349.3 ± 4.2 s−1. Conclusions This work provides a useful endo-arabinanase with high thermostability andcatalytic efficiency, and these characteristics exhibit a great potential for enzymatic conversion of arabinan.

A few reports about the endo-arabinanases from bacteria and fungi have been published so far [5,6,8,[10][11][12][13][14][15][16][17][18][19]. Among all the studies above, only a novel hyperthermophilic endo-arabinanase from Thermotoga petrophila was biophysically characterized and the three-dimensional (3D) structure was released [1,17]. Although the genome of hyperthermophilic Thermotoga thermarum DSM 5069 has been sequenced, no information is currently available on the endo-arabinanase. In this study, a putative GH43 β-xylosidase from Thermotoga thermarum, Tth Abn, was cloned and expressed. Its biochemical characterization and application in arabinan degradation were also investigated. Upon the evaluation of substrate specificity, it was interesting to find that the recombinant enzyme was an endo-arabinanase, which has not yet been reported.

Amino acid sequence of Tth Abn
The Tth abn gene isolated from the T. thermarum genome is 1,437 bp in length coding for 479 amino acids. There is a signal peptide sequence (32 amino acids, 96 bp) in Tth Abn when analyzed by SignalP 4.0 (http://www.cbs. dtu.dk/services/SignalP/). Therefore, only the DNA fragment of 1,341 bp was amplified. Amino acid sequence of Tth Abn in GenBank is described as a putative β-xylosidase. However, it contains putative domains of α-L-arabinofuranosidase (EC 3.2.1.55), endo-arabinanase (EC 3.2.1.99) and β-xylosidase (EC 3.2.1.37) through the BLAST. In this study, the provisional β-xylosidase was finally confirmed as an endo-arabinanase through the biochemical analysis. Tth Abn exhibited 64% identity to the β-xylosidase from Thermotoga sp. RQ2 (GenBank accession number YP_001738698) and Thermotoga petrophila RKU-1 (GenBank accession number YP_001244233), 59% identity to putative β-xylosidase Dictyoglomus thermophilum H-6-12 (GenBank accession number YP_002251442), and 50% identity to the endo-arabinanase from B. subtilis Abn2 H318A mutant (GenBank accession number YP_002251442 2X8T). To gain insights into the evolutionary relationships among endo-arabinanases, the phylogenetic trees with 13 candidate sequences were constructed using the NJ and MP methods, respectively, which both supported the same topological structures as shown in Figure 1. Phylogenetic analysis indicated that Tth Abn from T. thermarum had a close relationship with an endo-arabinanase from T. petrophila and a putative βxylosidase from the same genus T. sp.
Homology modeling revealed that Tth Abn had a similar 5-fold β-propeller structure as the B. subtilis endo-arabinanase Abn2 H318A mutant [11] (Figure 2). Both sequence comparison and homology modeling indicated that Asp 146 and Glu 199 residues were the catalytic nucleophile and proton donor, respectively [11]. Furthermore, there are other conserved amino acid residues adjacent to the catalytic center, His 9 , Asp 10 , Pro 11 , Trp 38 , Ala 73 and Asn 143 , which may play some roles in catalytic activity.

Expression and purification of Tth Abn
Mature protein coding 447 amino acids was successfully expressed in the cytoplasmatic fraction of E. coli BL21 (DE3). Then, the protein in the cell-free extract was purified by more than 95% homogeneity after heat treatment and affinity chromatography (Additional file 1: Table S1). Finally, the purified fusion enzyme showed a single band on a SDS-PAGE gel with an estimated molecular weight of 36 kDa ( Figure 3). To examine the oligomerization state of the enzyme, size exclusion

Biochemical properties of Tth Abn
Enzymatic characteristics of purified recombinant Tth Abn were determined and summarized in Tables 1, 2 and 3. Substrate specificity was assayed with different substrates (Table 1). It was interesting to find that Tth Abn was active towards linear arabinan, debranched arabinan and sugar beet arabinan, but there was no activity towards 1,4-β-D-mannan, galactan, p-Nitrophenyl-β-D-xylopyranoside (pNPX), and p-nitrophenyl-α-L-arabinofuranoside (pNPAF). These results indicated that the enzyme only exhibited arabinanase activity.
Effect of pH on Tth Abn activity was determined in 50 mM imidazole-potassium buffer ranging from pH 5.5 to 8.5 (Figure 4a). Tth Abn was found to exhibit an optimum activity at pH 6.5 and was able to retain more than 95% of its initial activity at 70°C for 1 h (Figure 4b).
As a function of temperature, the enzyme displayed the highest activity at 75°C and retained more than 90% of its maximum activity even at 85°C after 10 min incubation ( Figure 4c). Incubations at different temperatures to determine the enzyme's thermal stability were also carried out. As shown in the result, the enzyme kept nearly its initial activity at 75°C after 2 h incubation, and could still retain more than 80% of its original activity at 80°C for 2 h (Figure 4d).
Effects of metal ions and chemicals on Tth Abn activity were shown in Table 2. In various assays, the enzyme activity was apparently stimulated by 1 mM Mn 2+ , Ca 2+ , Ba 2+ , or chemical reagents 0.05% Tween 60 and 0.1% SDS. On the contrast, Al 3+ , Cu 2+ and Zn 2+ could inhibit the enzyme activity evidently. Kinetic studies in the presence of three arabinans as the substrates at optimum temperature and pH allowed the determination of the Michaelis-Menten parameters as shown in Table 3.

Degradation of arabinan by Tth Abn
Catalytic ability of Tth Abn on arabinan was investigated by analyzing the digestion products of linear , debranched and sugar beet arabinans (Figure 5a,b,c). Clearly from the result, the end products for hydrolysis of linear and debranched arabinans were arabinose, arabinobiose and arabinotriose after degradation for 3 h. However, it displayed very weak catalytic ability towards the hydrolysis of sugar beet arabinan.
We reported an endo-arabinanase (Tth Abn) from the hyperthermophilic bacterium T. thermarum here which was originally predicted as a β-xylosidase yet possessed the capability of arabinan depolymerization. Similar results have been published that endo-arabinanase from Thermotoga petrophila RKU-1 and B. subtilis were confirmed as endo-arabinanases through biophysical characterizations although once were predicted as β-xylosidases [1,20]. It is well known that hydrolases from GH43 family utilize a single displacement mechanism, which results in an inverted anomeric configuration for hydrolysis products [8]. Amino acid sequence alignment and homology modeling indicated that Asp 146 and Glu 199 in Tth Abn were the catalytic nucleophile (base) and proton donor, respectively. The result is also supported by the fact that aspartic acid and glutamic acid residues are the catalytic nucleophile and proton donor in all members of GH43 family (http://www.cazy.org). Residue Asp 10 may act as a pKa modulator and maintain the correct alignment of the general acid residue relative to the substrate [21]. Residues Asp 10 , Asp 146 and Glu 199 located in the deep cavity at the centre of the β-propeller are also found in all members of GH32, 43, 62 and 68 [22]. As the consequence of the flexibility reduction of the polypeptide chain, residue Pro 11 may contribute to the high thermal stability of the enzyme [23].
Phylogenetic analysis and enzymatic properties showed that Tth Abn was distant with the endo-arabinanases from Bacillus subtilis, Bacillus licheniformis and Aspergillus aculeatus [4,5,19]. However, it revealed a close relationship with T. thermarum and T. petrophila, indicating that they share the similar properties [1,17]. Other endoarabinanases from genus Thermotoga including T. thermarum endo-arabinanase have not yet been studied. Since there is 64% amino acid sequences similarity between endo-arabinanases T. thermarum and T. petrophila and is also inferred by the experimental data shown in Table 3, it is confirmed that Tth Abn could be an endo-arabinanase with some specific properties.    Tth Abn exhibited good thermostability when incubated at 75°for 2 h. Endo-arabinanase from Gram-negative bacterium T. petrophila RKU-1 has been reported to have a pH optimum at 6.5 and retained 95% of initial activity at 90°C for 10 h [1]. While Gram-positive bacteria B. subtilis and B. licheniformis endo-arabinanases show optimum activity at near-neutral pH at 60°C and 35°C, respectively [4]. Endo-arabinanase Aspergillus aculeatus from fungus displays optimum activity at pH 5.5 and 50°C [16]. In general, endo-arabinanases from bacteria exhibit the nearneutral pH optima, while those from fungi display the acid pH optima. The enzyme activities regarding pH from bacteria and fungi are also consistent with their inherent properties.
Cations and chemical reagents exhibited different effects on the activity of the endo-arabinanase Tth Abn. With the evidence that one Ca 2+ ion was observed in the catalytic cavity of Bacillus subtilis about 5 Å below the catalytic carboxylates and the presence of ions in an equivalent location in other arabinanases [22].
Compared to other endo-arabinanases, Tth Abn demonstrated the highest V max value among all the known  Effects of pH and temperature on the activity and stability of the recombinant Tth Abn endo-arabinanase. a. Optimal pH of the Tth Abn. b. pH stability of the Tth Abn. c. Effect of temperature on Tth Abn activity. d. The thermostability of the Tth Abn. The residual activity was monitored, and the maximum activity was defined as 100% (a, c) or initial activity was defined as 100% (b, d). Values shown were the mean of triplicate experiments, and the variation about the mean was below 5%. endo-arabinanases from thermophile and hyperthermophile when linear arabinan was used as the substrate. The catalytic efficiency (k cat /K m ) of endo-arabinanase Tth Abn on debranched arabinanwas approximately 6-fold and 18fold higher than B. licheniformis and Caldicellulosiruptor saccharolyticus, respectively (Table 3). In addition, catalytic efficiency for linear, debranched and sugar beet arabinans existed obvious differences (linear > debranched > sugar beet). This may be caused by substrate's composition and structure. As we know, a typical sugar composition of linear arabinan is: arabinose: galactose: rhamnose: galacturonic acid = 97.5: 0.4: 0.1: 2.0. For debranched and sugar beet arabinans, the contents of arabinose are lower than linear arabinan, and sugar beet arabinan has more substitutions attached to backbone while 1,2-and 1,3-α-L-arabinofuranosyl branch units of debranched arabinan have been all removed.
The fact that Tth Abn could hydrolyze linear and debranched arabinan into oligomers arabinose, arabinobiose and arabinotriose implied that the Tth Abn had endoarabinanase activity but no exo activity. Only a few endoarabinanases especially that from hyperthermophile have been reported to possess the function in generating arabino-oligosaccharides [1,17]. Although it was insensitive to the branched arabinan (eg. sugar beet arabinan), Tth Abn could be used together with other arabinandegrading enzymes in synergistic reaction for the hydrolysis of branched ones.

Conclusions
In this study, a useful endo-arabinanase (Tth Abn) from T. thermarum DSM 5069 was expressed in E. coli with desirable features, such as high thermostability, catalytic efficiency, and hydrolytic reaction at high temperature. This is easily envisioned that Tth Abn endo-arabinanase exhibits a great potential for enzymatic conversion of arabinan through synergetic action with other arabinandegrading enzymes.
Bacterial strains and growth conditions E. coli Top10 (Novagen, San Diego, USA) was used for routine molecular cloning work and E. coli BL21 (DE3) (Novagen) was employed as the host for the expression of the recombinant Tth Abn. E. coli strains, both were grown in Luria-Bertani (LB) medium. Ampicillin (100 μg ml −1 , Shanghai, China) and isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mM, Dalian, China) were added when necessary.

Construction of plasmids and strains
DNA manipulations were carried out according to standard methods [24]. Restriction enzymes and high-fidelity Ex-Taq DNA polymerase were purchased from Takara Biotechnology Co. Ltd. (Dalian, China) and used according to the manufacturer's instructions. DNA was extracted from agarose gels with BIOMIGA Gel Extraction Kit (Shanghai, China). Sequencing was performed using ABI 3730 DNA analyzer (Applied Biosystems, Foster City, USA). PCR amplifications were conducted using highfidelity Ex-Taq DNA polymerase, and the resulting products were purified with BIOMIGA PCR Purification Kit (Shanghai, China).
The coding sequence of Tth abn gene was amplified from T. thermarum genomic DNA using primers 5'-GGAATTCCATATGGTATTCAACTGGGCAACTGTA-CAC-3' (forward) and 5'-CCGCTCGAGATCTTCGA TCCGAACTCCCCAG-3' (reverse). The primers contained restrictions sites NdeI and XhoI (underlined) for forward and reverse primers, respectively. The amplified DNA fragment was digested with NdeI and XhoI, and inserted into the corresponding sites in plasmid pET-20b (Novagen) to produce pET-20b-Tth abn. The correctness of the insert was confirmed by DNA sequencing.

Expression and purification of recombinant Tth Abn
E. coli BL21 (DE3) cells harboring recombinant plasmids were grown (200 rpm, 37°C) in LB medium (200 ml) with appropriate antibiotic selection. When the OD 600 reached 0.6~0.8, the expression of Tth Abn was induced by the addition of 0.5 mM IPTG and the culture was further incubated (150 rpm, 25°C, 12 h) . Cells were harvested by centrifugation (10000 rpm, 4°C, 5 min). The pellet was washed twice with 20 mM Tris-HCl buffer (pH 8.0), and re-suspended in 5 ml of 5 mM imidazole, 0.5 mM NaCl, and 20 mM Tris-HCl buffer (pH 7.9). All steps were carried out at 4°C. The cell extracts after sonication were heat treated at 70°C for 30 min, and subsequently cooled in an ice bath. After centrifugation (15000 g, 4°C, 20 min), the resulting supernatant was loaded onto a 1 ml nickle affinity column (Novagen) and the bounded proteins were eluted by discontinuous imidazole gradient (30-1000 mM). The fractions that contained Tth Abn were dialyzed overnight against storage buffer (20 mM Na-phosphate, 50 mM NaCl, 10% glycerol, pH 7.0) and then were kept at −80°C until further use. The analysis of production and purity of the enzymes were determined by a 12% SDS-PAGE gel [25] using broad range molecular weight markers (MBI Fermemtas, Burlington, Canada) as standards. The protein concentration was determined by Bradford method using bovine serum albumin (BSA) as a standard [26]. Oligomerization state of Tth Abn was analyzed by size exclusion chromatography on a AKTAFPLC™ (GE Healthcare Life Sciences, New Jersey, USA) system with a Superdex 200 10/30 GL column as described by Zhang et al. [27].