A novel bifunctional GH51 exo-α-l-arabinofuranosidase/endo-xylanase from Alicyclobacillus sp. A4 with significant biomass-degrading capacity

Background Improving the hydrolytic performance of xylanolytic enzymes on arabinoxylan is of importance in the ethanol fermentation industry. Supplementation of debranching (arabinofuranosidase) and depolymerizing (xylanase) enzymes is a way to address the problem. In the present study, we identified a bifunctional α-l-arabinofuranosidase/endo-xylanase (Ac-Abf51A) of glycoside hydrolase family 51 in Alicyclobacillus sp. strain A4. Its biochemical stability and great hydrolysis efficiency against complex biomass make it a potential candidate for the production of biofuels. Results The gene encoding Ac-Abf51A was cloned. The comparison of its sequence with reference proteins having resolved 3D-structures revealed nine key residues involved in catalysis and substrate-binding interaction. Recombinant Ac-Abf51A produced in Escherichia coli showed optimal activity at pH 6.0 and 60 °C with 4-nitrophenyl-α-l-arabinofuranoside as the substrate. The enzyme exhibited an exo-type mode of action on polyarabinosides by catalyzing the cleavage of α-1,2- and α-1,3-linked arabinofuranose side chains in sugar beet arabinan and water-soluble wheat arabinoxylan and α-1,5-linked arabinofuranosidic bonds in debranched sugar beet arabinan. Surprisingly, it had capacity to release xylobiose and xylotriose from wheat arabinoxylan and was active on xylooligosaccharides (xylohexaose 1.2/mM/min, xylopentaose 6.9/mM/min, and xylotetraose 19.7/mM/min), however a lower level of activity. Moreover, Ac-Abf51A showed greater synergistic effect in combination with xylanase (2.92-fold) on wheat arabinoxylan degradation than other reported enzymes, for the amounts of arabinose, xylose, and xylobiose were all increased in comparison to that by the enzymes acting individually. Conclusions This study for the first time reports a GH51 enzyme with both exo-α-l-arabinofuranosidase and endo-xylanase activities. It was stable over a broad pH range and at high temperature, and showed greater synergistic effect with xylanase on the degradation of wheat arabinoxylan than other counterparts. The distinguished synergy might be ascribed to its bifunctional α-l-arabinofuranosidase/xylanase activity, which may represent a possible way to degrade biomass at lower enzyme loadings. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0366-0) contains supplementary material, which is available to authorized users.


Background
Hemicellulose, the second most abundant polysaccharide in plants, combines with cellulose and lignin to compose lignocellulose of plant cell walls, and accounts for about 20-35 % of lignocellulosic biomass [1,2]. It mainly consists of xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan, and has attracted much attention for their industrial importance in bioconversion of plant biomass to biofuel, improvement of animal feedstock digestibility, and organic synthesis [3,4]. Heterogeneous xylans are the major constituents of hemicellulose. Among them, arabinoxylans such as those found in wheat straw [5] consist of a backbone of β-1,4-linked d-xylopyranose residues that are extensively decorated at C-2 and/or C-3 positions with arabinofuranose side chains [6,7]. Arabinan, a component of pectin, contains a backbone of α-1,5-linked l-arabinofuranosyl residues as well as α-1,2-and α-1,3-linked side chains [8]. Thus, due to the structural complexity of xylans, efficient hydrolysis of wheat arabinoxylan to achieve high xylose yields by complete xylan monomerization requires supplementing xylanases with α-l-arabinofuranosidases and other accessory enzymes [9].
Until now, bacterial GH51 Abfs from Anoxybacillus kestanbolensis [17], Clostridium stercorarium [18], Geobacillus stearothermophilus [19], Bacillus pumilus [20], Bifidobacterium longum [21], and Streptomyces spp. [22][23][24] have been characterized, and the structures of six bacterial Abfs have been resolved, which all perform hydrolysis via a retaining mechanism [25]. Previous studies indicate that GH51 Abfs have various modes of action on different substrates. This broad specificity against distinct branching modifications represents a great advantage for biotechnological processing of complex and branched polysaccharides [26]. In this study, we report the characterization of a novel Alicyclobacillus sp. A4 GH51 α-l-arabinofuranosidase (Ac-Abf51A) with both exo-α-l-arabinofuranosidase and endo-xylanase activities, which is significantly different from previously reported GH51 arabinofuranosidases in substrate specificity and would play an important role in biomass hydrolysis.

Gene cloning and sequence analysis
The full-length Abf gene, Ac-abf51A (GenBank accession no.KT781102), contains 1509 bp and encodes a 502-residue polypeptide with a calculated molecular mass of 56.7 kDa. The deduced amino acid sequence of Ac-Abf51A is most similar to a putative Abf of Alicyclobacillus hesperidum (99 % identity; WP_006446014.1), and 68 % identical with the crystal structure-resolved Abf from G. stearothermophilus T6 (1PZ3). Using the Accelrys Discovery Studio software with 1PZ3 as the template, modeled Ac-Abf51A folds into two modules: the N-terminal catalytic module of the frequently encountered (α/β) 8 barrel (TIM barrel) of GH51 and the C-terminal module of 12-stranded β-sandwich with a jelly-roll topology (Additional file 1). The structure of the (α/β) 8 barrel domain places Ac-Abf51A into the superfamily of clan GH-A, in which two conserved glutamates (Glu175 and Glu294) were found on strands β-4 (acid/base) and β-7 (nucleophile), respectively ( Fig. 1). Based on homology analysis of amino acid sequences with the characterized GH51 Abf with crystal structure [17], nine key residues including Glu29, Arg69, Asn74, Asn174, Glu175, His244, Tyr246, Glu294, and Gln351 are conserved and probably involved in catalysis and substrate-binding interaction (Fig. 1).

Expression and purification of recombinant Ac-Abf51A
After induction with IPTG at 30 °C for 6 h, the whole E. coli cell lysates showed an Abf activity of 2.6 U/mL. The recombinant Ac-Abf51A was purified by one-step immobilized metal-affinity chromatography (IMAC). The specific activity of purified Ac-Abf51A was 18.2 U/mg with (See figure on next page.) Fig. 1 Amino acid sequence alignment of Ac-Abf51A from Alicyclobacillus sp. A4 with CelA4 from Alicyclobacillus sp. A4 (ADI82825.1) and other GH51 Abfs from Bifidobacterium longum B667 (PDB: 2Y2 W), Geobacillus stearothermophilus T6 (PDB: 1PZ3), Clostridium thermocellum ATCC 27405 (PDB: 2C7F), Thermobacillus xylanilyticus D3 (PDB: 2VRK), Thermotoga maritima MSB8 (PDB: 3UG3), and Thermotoga petrophila RKU-1 (PDB: 3S2C), using the ClustalW program. Identical and similar amino acids are indicated by black and gray shades, respectively. The catalytic glutamate residues are indicated by asterisk, and the conserved residues are indicated by hash 4-nitrophenyl-α-l-arabinofuranoside as the substrate. The purified enzyme migrated as a single band with an apparent molecular mass of 57.0 kDa, as determined by SDS-PAGE analysis (Fig. 2).

Biochemical characterization
With 4-nitrophenyl-α-l-arabinofuranoside as the substrate, Ac-Abf51A exhibited the highest Abf activity at pH 6.0 (Fig. 3a). The enzyme retained more than 80 % of the initial activity after incubation at pH 4.0-11.0, 37 °C for 1 h (Fig. 3b). When assayed at pH 6.0, Ac-Abf51A was most active at 60 °C (Fig. 3c). The enzyme retained full activity after 12-h incubation at 60 °C and 65 °C, more than 80 % of the initial activity at 70 °C for 1 h, and more than 30 % of the activity at 80 °C for 30 min (Fig. 3d).
Most metal ions and chemicals had little or no effect on Ac-Abf51A (Table 1). However, the presence of K + , Co 2+ , Ag + , and SDS strongly inhibited the enzymatic activity of Ac-Abf51A.

Substrate specificity and kinetic parameters
Ac-Abf51A had broad substrate specificity. The capacity of purified Ac-Abf51A to hydrolyze a range of different substrates was evaluated. For 4-nitrophenyl-glycosides and polysaccharides with reaction time within 10 min, it acted solely on 4-nitrophenyl-α-l-arabinofuranoside. When the incubation period was lengthened to 5 h, Ac-Abf51A showed weak activity against 4-nitrophenylβ-d-xylopyranoside (0.026 % activity relative to that on 4-nitrophenyl-α-l-arabinofuranoside). The K m , V max , and k cat values of Ac-Abf51A towards 4-nitrophenyl-αl-arabinofuranoside were determined to be 0.46 mM, 24.4 μmol/min/mg, and 23.0/s, respectively. The k cat /K m value was 50/mM/s. For longer incubation (up to 12 h), it was active on sugar beet arabinan and debranched sugar beet arabinan, releasing arabinose as the final product (0.57 and 0.30 mg/mL, respectively) (Additional file 2). It also released arabinose, xylobiose, xylotriose, and xylotetraose from water-soluble wheat arabinoxylan.
Further time course analysis of the water-soluble wheat arabinoxylan digestion by Ac-Abf51A indicated that arabinose was yielded as the sole product at the beginning, and xylobiose, xylotriose, and xylotetraose were also released after a prolonged incubation (Fig. 4). When using xylooligosaccharides as the substrates, Ac-Abf51A exhibited an endo-mode of action on xylohexaose, xylopentaose and xylotetraose, releasing products of lower degree of polymerization. However, it showed barely detectable activity on xylotriose and xylobiose. The k cat /K m values of Ac-Abf51A on xylotetraose, xylopentaose, and xylohexaose were 1.2/mM/min, 6.9/mM/min, and 19.7/mM/min, respectively (Additional file 3).

Characterization of four mutants of Ac-Abf51A
Four Ac-Abf51A mutants containing the (α/β) 8 barrel module alone (Ac-Abf51An), the jelly-roll module alone (Ac-Abf51Ac), and the two catalytic glutamic acid residues substituted by glutamine (E175Q and E194Q), respectively, were successfully expressed in E. coli BL21 (DE3) and purified to homogeneity by Ni-NTA columns individually. Except for Ac-Abf51An that has both exoα-l-arabinofuranosidase and endo-xylanase activities, other three mutant proteins were completely inactive towards 4-nitrophenyl-α-l-arabinofuranoside, water-soluble wheat arabinoxylan, and xylotetraose, xylopentaose, and xylohexaose. The results showed that Ac-Abf51A catalyzes the two types of substrate at the same active sites within a single catalytic domain.

Synergetic effects
The product composition of water-soluble wheat arabinoxylan hydrolysis by Ac-Abf51A and endo-xylanase XynBE18 individually or in combination were determined by HPAEC-PAD. XynBE18 released only xylose and xylobiose, and Ac-Abf51A liberated arabinose and trace amounts of xylobiose and xylotriose. In comparison with the hydrolysis products by XynBE18 or Ac-Abf51A alone, all enzyme combinations had significant synergistic effects on wheat arabinoxylan degradation, producing Lanes: M, the standard protein molecular weight markers; 1, the recombinant Ac-Abf51A purified by Ni 2+ -NTA affinity chromatography substantially more arabinose, xylobiose, and xylose. The greatest synergy showed no difference, when simultaneous incubation of wheat arabinoxylan with Ac-Abf51A and XynBE18 (2.92-fold increase, Table 2). First addition of Ac-Abf51A followed by XynBE18 had no difference with XynBE18 alone on the production of arabinose and xylose. However, the amount of xylobiose was increased by 1.4-fold (1.26-fold increase in total, Table 2). When XynBE18 was added followed by Ac-Abf51A, more arabinose, xylobiose, and xylotriose were released (2.12-fold increase, Table 2).

Discussion
To our knowledge, this is the first report on the gene cloning, expression, and characterization of a GH51 α-larabinofuranosidase (Ac-Abf51A) from the genus Alicyclobacillus. This genus contains unusual ω-alicyclic fatty acids in the cell membrane and is thus highly thermoacidophilic [27]. It produces a spectrum of GHs, including GH9 and GH51 cellulases [28][29][30], GH10 xylanase [31], GH52 xylosidase [32], and GH113 mannanase [33]. Analysis of the sequence alignment and structure modeling indicated that Ac-Abf51A consists of two modules: the catalytic module of the frequently encountered (β/α) 8 barrel fold and a C-terminal jelly-roll module. The overall structure of Ac-Abf51A is very similar to that of the six previously published Abfs from bacteria. Furthermore, among the nine conserved residues within the GH51 arabinofuranosidases, the catalytic acid/base Glu175 is involved in the formation of a hydrogen bond with the conserved His244, and the Arg69 residue appears to keep the catalytic nucleophile Glu294 deprotonated [34]. All these data strongly support the conclusion that Ac-Abf51A is a putative GH51 Abf.
On the other hand, Ac-Afb51A yielded arabinose as the sole hydrolysis product from branched and debranched sugar beet arabinan. These results showed Ac-Afb51A was an exo-acting enzyme that had hydrolytic activity against α-1,2-, α-1,3-, or α-1,5-linked non-reducing terminal l-arabinofuranose residues [37,38]. Moreover, it exhibited higher activity against branched (1.9-fold) than debranched sugar beet arabinan, suggesting it preference for the α-1,2-and α-1,3-rather than α-1,5-glycosidic bonds. Surprisingly, Ac-Abf51A exhibited activity against water-soluble wheat arabinoxylan, releasing arabinose, xylobiose, xylotriose, and xylotetraose after a prolonged The time course analysis of wheat arabinoxylan hydrolysis revealed that Ac-Abf51A preferentially removes α-1,2-and α-1,3-linked arabinofuranose side chains from arabinoxylan, and hydrolyzes internal β-1,4-linkages at a much lower rate. Furthermore, Ac-Abf51A also had ability to hydrolyze xylohexaose, xylopentaose, and xylotetraose, but not xylotriose and xylobiose, to shorter xylooligosaccharides. The catalytic efficiency of Ac-Abf51A improved slightly with increasing polymerization degree of xylooligosaccharides up to six (the catalytic efficiency ratio of xylotetraose, xylopentaose, and xylohexaose was 1:5.8:16.4). These results suggested that Ac-Abf51A exhibits an endo-mode of action against xylan. The replacement of Glu175 or Glu294 with Gln resulted in a complete loss of both α-l-arabinofuranosidase and endo-xylanase activities, indicating that Ac-Abf51A catalyzes the hydrolysis of these substrates at the same active center. Thus, this is the first report on a novel GH51 Abf having endo-xylanase activity. Synergy of hydrolytic enzymes has been extensively studied in prior studies [39][40][41], which is a useful way to improve sugar yield at low cost. When Ac-Abf51A was incubated in combination with endo-xylanase XynBE18 sequentially or simultaneously to degrade wheat arabinoxylan, more reducing sugars were released. There was no significant synergy increase in sequential reaction of Ac-Abf51A followed by XynBE18. But when Ac-Abf51A was added after XynBE18, the amounts of arabinose, xylobiose, and xylotriose released were higher than that by the two enzymes acting separately. The results suggest that efficient degradation of wheat arabinoxylan is achieved by the first cleavage of the main chains with XynBE18 followed by branch removal with Ac-Abf51A. Moreover, the synergy degree of the simultaneous reactions was higher than that of the sequential reactions. As results, Ac-Abf51A plays a role in the release of both arabinose and xylose moieties from arabinose-containing xylooligosaccharides generated by xylanase [42]. Discovery of this bifunctional Abf/xylanase is seminal to the structural analysis of the catalytic mechanisms of GH51 Abfs. Considering the distinct substrate specificity and biochemical stability, Ac-Abf51A has great application potentials for biomass degradation and refining.

Conclusions
Thermoacidophilic Alicyclobacillus sp. A4 is an excellent producer of CAZymes, especially of hemicellulases and cellulases. This study firstly reported the gene cloning, expression and characterization of an auxiliary α-l-arabinofuranosidase of GH51. Neutral Ac-Abf51A remained stable over a broad pH range (pH 4.0-11.0) and high temperature (70 °C), and exhibited an exo-type mode of action towards polyarabinosides. It had ability to release arabinose, xylobiose, and xylotriose from wheat arabinoxylan, and exhibited endo-type action towards xylooligosaccharides with a lower level of activity. This is the first report of a GH51 Abf that has endo-xylanase activity. The broad substrate specificity, biochemical stability, and great synergistic effect with xylanase make Ac-Abf51A a potentially interesting enzyme for application in industrial biomass degradation.

Strains and culture medium
Alicyclobacillus sp. A4 CGMCC 3147 (whole genome sequenced, unpublished) was maintained in our laboratory [31]. Escherichia coli strains Trans I-T1 and BL21 (DE3) (TransGen) were maintained in Luria-Bertani (LB) broth or on agar plates at 37 °C for gene cloning and expression, respectively. Substrates were purchased from Sigma or Megazyme. All other chemicals were of analytical grade and commercially available.

Table 2 Simultaneous and sequential reactions of Ac-Abf51A and XynBE18 against water-soluble wheat arabinoxylan
The amounts of xylooligosaccharides and arabinose were determined by HPAEC -, not detected a Degree of synergy is defined as the ratio of the amounts of saccharide released from simultaneous or sequential enzyme combinations to the sum of that released by the individual enzymes

Gene cloning and sequence analysis
The gene fragment of Ac-abf51A (no signal peptide coding sequence based on SignalP 4.0 prediction) was amplified from the genome of strain A4 by PCR using the expression primers (abf51F: 5′-CGCCATATGTCCAA-GTCGATTGCACGTATG-3′ and abf51R: 5′-GACGCG-GCCGCCACGCGCAATCGAATGACG-3′, NdeI and NotI sites underlined, respectively). The PCR products were gel-purified with Agarose Gel DNA Purification kit (TaKaRa), ligated into a pGEMT easy vector (Promega), and transformed into E. coli Trans I-T1 for sequencing. The nucleotide sequence was assembled and analyzed using Vector NTI Advance 11.5 software (Invitrogen). BlastN and BlastP programs (http://www.ncbi.nlm.nih. gov/BLAST/) were used to analyze the nucleotide and deduced amino acid sequences, respectively.

Protein expression and purification
Ac-abf51A was obtained by digesting the recombinant plasmid pGEMT-Ac-abf51A with restriction endonucleases NdeI and NotI (TaKaRa), and was cloned into the NdeI and NotI sites of pET-30a(+) vector (Novagen). The recombinant plasmid pET-Ac-abf51A was then transformed into the E. coli BL21 (DE3) competent cells. The positive transformants harboring pET-Ac-abf51A were identified by PCR amplification, and further cultured in LB medium supplemented with 100 μg/mL kanamycin at 37 °C to an A 600 of approximately 0.6. Gene expression was induced at 30 °C for 6 h by isopropyl-β-d-1thiogalactopyranoside (IPTG) with a final concentration of 0.6 mM. The induced cultures were centrifuged at 12,000×g for 5 min at 4 °C to harvest cells. The pellet (5 g) re-suspended in 25 ml of lysis buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.6) was sonicated with an Ultrasonic Cell Disruptor (Scientz) on ice with 100 short bursts at 150 W of 6 s followed by intervals of 15 s for cooling. After removal of cell debris by centrifugation, the supernatant was subjected to Ni 2+ -NTA chromatography with a linear 20-300 mM imidazole gradient in 50 mM Tris-HCl, 500 mM NaCl, pH 7.6. The fractions exhibiting enzyme activities were pooled, concentrated, and assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Bradford protein assay was employed to determine protein concentration at 595 nm with bovine serum albumin as the standard.

Enzyme assay
Abf activity was assayed as described previously [21]. The standard reaction system consisted of 250 μL of appropriately diluted enzyme and 250 μL of McIlvaine buffer (pH 6.0) containing 2 mM 4-nitrophenyl-α-larabinofuranoside. After incubation at 60 °C for 10 min, 1.5 mL of 1.0 M Na 2 CO 3 was added into the system to terminate the reaction. The amount of p-nitrophenol released was determined spectrophotometrically by reading the absorbance at 405 nm. One unit of Abf activity was defined as the amount of enzyme that released 1 μmol of 4-nitrophenol per min under the assay conditions. The dinitrosalicylic acid (DNSA) method [43] was used to assay the xylanase activity. The reaction system consisted of 100 µL of appropriately diluted enzyme solution and 900 µL of 1 % (w/v) xylan in McIlvaine buffer (pH 6.0). The reaction mixture was incubated at 60 °C for 10 min, then 1.5 mL of DNSA reagent was added to terminate the reaction. The mixture was boiled for exactly 5 min and cooled down to room temperature. The color formation was monitored at 540 nm and quantified by comparison with a standard curve of d-xylose. One unit of xylanase activity was defined as the amount of enzyme releasing 1 μM of reducing sugars equivalent to d-xylose per minute under assay conditions. Each reaction was performed in triplicate, as was each experiment.

Biochemical characterization
The optimal pH for the Abf activity of purified recombinant Ac-Abf51A (3 μg/mL) was determined at 37 °C for 10 min in 100 mM McIlvaine buffer (pH 3.0-8.0), 100 mM Tris-HCl (pH 8.0-9.0) and 100 mM glycine-NaOH (pH 9.0-10.0). The stability of Ac-Abf51A under different pH conditions was assessed by measuring the residual activities under standard conditions after incubation of the enzyme (60 μg/mL) in the buffers as described above at 37 °C for 1 h. The optimal temperature for Ac-Abf51A activity (3 μg/mL) was carried out at various temperatures from 20 to 80 °C in 100 mM McIlvaine buffer (pH 6.0). Thermal stability was determined by measuring the residual activities under standard conditions [100 mM McIlvaine buffer (pH 6.0), 60 °C for 10 min] after incubating the enzyme (60 μg/mL) at 60 °C, 65 °C, 70 °C, and 80 °C for different times without substrate.
Enzyme kinetic assays were performed at pH 6.0, 60 °C for 5 min with 4-nitrophenyl-α-l-arabinofuranoside (1-10 mM) as the substrate. The K m and V max values of Ac-Abf51A were plotted based on Lineweaver-Burk method. The catalytic efficiency (k cat /K m ) of Ac-Abf51A towards xylooligosaccharides was determined following the Matsui equation as previously described by Cervera-Tison et al. [44].

Mutant construction and characterization
To further verify whether the α-l-arabinofuranosidase and endo-xylanase activities of Ac-Abf51A were derived from the same module and the active center, truncation mutants of single module (Ac-Abf51An and Ac-Abf51Ac) and substitution mutants (Glu175 and Glu294) were performed using the Quik-Change method (Stratagene). All mutations were generated in the wild-type Ac-Abf51A DNA in the expression plasmid. The resulting mutant plasmids were transformed into E. coli BL21 (DE3) cells and confirmed by DNA sequencing. Expression, purification, and characterization of the mutant enzymes followed the same procedure as for the Ac-Abf51A.

Synergetic effects of Ac-Abf51A and xylanase XynBE18
Endo-xylanase XynBE18 from Paenibacillus sp. E18 [41,45] is an excellent xylooligosaccharide producer and was selected for enzyme combination. The reaction mixtures were composed of 900 μL of 0.5 % (w/v) water-soluble wheat arabinoxylan and 100 μl of enzyme(s) (0.5 U each of XynBE18 and/or Ac-Abf51A). All reactions were carried out at 37 °C in 100 mM McIlvaine buffer (pH 6.0). After 12-h incubation, the reaction mixtures were immersed in boiling water for 10 min to stop the reactions. Blank controls containing substrate alone were treated under the same conditions. For the sequential reactions, the second enzyme solutions were added after heat denaturation. The hydrolysates were assessed using HPAEC as described above. The degree of synergy was defined as the ratio of reducing sugar equivalents released when enzymes were incubated simultaneously or sequentially to the sum of the reducing sugar equivalents released by each enzyme alone.