Gene Expression and Molecular Characterization of a Xylanase from Chicken Cecum Metagenome

A xylanase gene xynAMG1 with a 1,116-bp open reading frame, encoding an endo-β-1,4-xylanase, was cloned from a chicken cecum metagenome. The translated XynAMG1 protein consisted of 372 amino acids including a putative signal peptide of 23 amino acids. The calculated molecular mass of the mature XynAMG1 was 40,013 Da, with a theoretical pI value of 5.76. The amino acid sequence of XynAMG1 showed 59% identity to endo-β-1,4-xylanase from Prevotella bryantii and Prevotella ruminicola and 58% identity to that from Prevotella copri. XynAMG1 has two conserved motifs, DVVNE and TEXD, containing two active site glutamates and an invariant asparagine, characteristic of GH10 family xylanase. The xynAMG1 gene without signal peptide sequence was cloned and fused with thioredoxin protein (Trx.Tag) in pET-32a plasmid and overexpressed in Escherichia coli Tuner™(DE3)pLysS. The purified mature XynAMG1 was highly salt-tolerant and stable and displayed higher than 96% of its catalytic activity in the reaction containing 1 to 4 M NaCl. It was only slightly affected by common organic solvents added in aqueous solution to up to 5 M. This chicken cecum metagenome-derived xylanase has potential applications in animal feed additives and industrial enzymatic processes requiring exposure to high concentrations of salt and organic solvents.


Introduction
Microbial enzymes have been recognized as a major source of various types of biocatalysts which can be successfully applied in different industrial processes [1]. To be practically useful and economically competitive, industrial enzymes must display high activity and stability under harsh conditions to help reduce the production costs [2]. Carbohydrases, enzymes that degrade polymeric carbohydrates, are currently employed in various industries including food and beverages, detergent, biofuel production, textile, paper and pulp, leather industries, and animal feed. However, there are only two enzymes, xylanase and glucanase that dominate the global enzyme market by more than 80% [1,3].
Generally, intestinal microorganisms of plant-eating animals are known to be excellent sources of various hydrolytic enzymes. Considering that poultry feeds consist mainly of cereal grains which are rich in nonstarch polysaccharides including xylans and arabinoxylans, microorganisms 2 International Journal of Microbiology producing nonstarch polysaccharide hydrolases including xylanase should be abundant in chicken intestine. Chicken cecal microbiota has been shown, in a culture-based study, predominated by the phyla Bacteroidetes and Firmicutes [10]. However, majority of gut microorganisms are unculturable. Therefore, metagenomic strategy is used to recover the genes of desired enzymes [11]. Recently, a metagenomic study of chicken cecal microbiome showed that up to 1.5% of the sequences represented glycosyl hydrolase (GH) domains with over two hundred different sequences of nonstarch polysaccharide-degrading enzymes found [12].
In this paper, we report the gene cloning, sequence and phylogenetic analyses, structural prediction, heterologous expression, and molecular and catalytic characterizations of a new GH10 family endo-xylanase derived from a chicken cecum metagenome.

Bioinformatic Analysis of DNA and Amino Acid Sequences.
Nucleotide sequence recognized as xylanase gene was translated into amino acids. Similar sequences were retrieved from the GenBank database using the BLAST search. Sequence alignment and phylogenetic analysis were done using the CLC Main Workbench 7.7 sequence analysis software package (CLC bio). To determine the family of the xylanase, the position of glutamate residues of the active site, and the highly conserved motifs of GH10, the ExPASy-PROSITE (http:/www.expasy.org/prosite) was used. The signal peptide was predicted by using SignalP 4.1 server (http:// www.cbs.dtu.dk/services/SignalP). The theoretical pI and molecular weight were predicted using an online prediction tool (http://www.expasy.org/tools/pi_tool.html). The structure of XynA MG1 protein was predicted using the SWISSMODEL (https://swissmodel.expasy.org/) and Int-FOLD (http://www.reading.ac.uk/bioinf/IntFOLD/) servers. Images were generated using PyMOL software (http://www .pymol.org/). 1 Plasmid, Gene Expression, and Purification of XynA 1 Xylanase. The xylanase gene xynA MG1 was amplified by PCR from the pCC1FOS fosmid clones, using the Xyl524GH10CH-Fw: 5 -ATGAGC-TCGCTGACACCC-3 forward and Xyl524GH10CH-Rv: 5 -CTAAGCTTGTCACTGCTTGAAC-3 reverse primers. The primer pair targeted the truncated gene, excluding the leader peptide encoding sequences, and introduced SacI and HindIII restriction sites at 5 -and 3 -end of the gene. The amplified PCR product was digested with SacI and HindIII and ligated, using T4 DNA polymerase, into pET-32a(+) vector previously linearized with the same restriction enzymes and dephosphorylated with Antarctic Phosphatase (New England BioLabs). The expression plasmid (pET32a-xynA MG1 ), having the xynA MG1 gene under the control of the T7 promoter, was used to transform chemically competent [13] E. coli Tuner (DH3)pLysS cells to give E. coli Tuner (pET32a-xynA MG1 ) expression strain. Positive clones were proven by colony PCR and the presence of xylanase activity in the cell lysate. Selected E. coli Tuner (pET32a-xynA MG1 ) clone was grown in LB broth containing 34 g/mL and 100 g/mL of chloramphenicol and ampicillin, respectively, incubated at 37 ∘ C with 200 rpm shaking until the culture reached an OD 600 nm of 0.6. Then the xynA MG1 gene expression was induced by adding IPTG to a final concentration of 0.4 mM and the culture was further incubated for 5 h at 37 ∘ C with 200 rpm shaking. The cells were harvested by centrifugation, resuspended in nine volumes of ice-cold phosphate buffer (50 mM sodium phosphate, 1.2 M NaCl, 10 mM imidazole) pH 7.2, and subjected to ultrasonic cell disruption while keeping in an ice-bath. Unbroken cells and insoluble cell debris were removed by centrifugation at 12,000 for 25 min yielding the clear soluble cell lysate. The cell lysates were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, 12% separating gel) to determine the expression profile of the XynA MG1 xylanase. The recombinant XynA MG1 was purified from cell lysate by Immobilized Metal Affinity Chromatography (IMAC) using the TALON Metal Affinity Resin (Clontech Lab, Inc.) charged with cobalt. The IMAC column (Tricon 10 × 100 mm) was operated using anÄKTA purifier FPLC system (GE Healthcare Bio-Sciences) at the 0.2 mL/min flow rate. The bound proteins were eluted with 10 bed volumes of linear gradient of 1-150 mM imidazole in phosphate buffer (50 mM sodium phosphate, 1.2 M NaCl) pH 7.2, at 0.5 mL/min flow rate. Fractions containing xylanase activity were pooled and concentrated by the Amicon5 30 kDa cut off centrifugal filter device (EMD Millipore) at 4,000 rpm, 4 ∘ C. The concentrated XynA MG1 was repurified using the IMAC and then desalted as described above. The pET-32a derived Trx-His•tag which fused to the N-terminus of the expressed XynA MG1 was removed by digestion with enterokinase (New England BioLabs) for 16 h at 23 ∘ C. The digestion mixture was then loaded onto the IMAC column as above, and the International Journal of Microbiology 3 recombinant XynA MG1 was retrieved from the flow-through, while the Trx-His•tag and the uncleaved Trx-His-XynA MG1 remained bound to the TALON resin. Protein concentration was determined by the Bradford method using bovine serum albumin (BSA) as the standard. The purity of XynA MG1 was analyzed on SDS-PAGE. The zymogram analysis was done on 12% gel native PAGE containing 0.2% oat-spelt xylan as the substrate [14].

Effect of pH and Temperature on XynA 1 Activity and Stability.
To determine the optimum pH for XynA MG1 activity, the enzyme was allowed to function in buffers ranging from pH 3.0 to 11.0 with 1 pH unit interval. The buffers used were 50 mM citrate buffer for pH range of 3-6, 50 mM sodium phosphate buffer for pH range of 6-8, and 50 mM glycine NaOH buffer for pH range of 8-11. XynA MG1 activities were determined using the standard enzyme assay, and a pH versus enzyme activity profile was plotted [19]. The enzyme stability at different pH was determined by preincubating XynA MG1 in different buffers from pH 3.0 to 11.0 (with 1 pH unit interval) for 30 min and 60 min at 25 ∘ C. Then, the remaining activities of the enzyme were measured under standard condition [20].
The optimum temperature for xylanase activity was determined by assaying the enzyme activity at different temperatures from 20 to 100 ∘ C with step increment of 5 ∘ C. Thermal stability was determined by incubating the enzyme in pH 5.5 buffer in the absence of substrate at the temperature range of 45 to 70 ∘ C. Then, the residual activities were measured under standard conditions [21].
To investigate the impact of sodium chloride on XynA MG1 activity, the purified XynA MG1 was incubated with 0-4 M NaCl for 0 and 2 h and then assayed under standard conditions in the reaction containing NaCl at the same concentration as previously incubated [23].
XynA MG1 was incubated in aqueous solution of different solvents (acetone, methanol, ethanol, and propanol) at different concentrations (0-5 M) for 30 min. Residual activity was determined by using assay system under optimal conditions [22].
The nucleotide sequence of the xynA MG1 gene reported in this paper has been deposited in the GenBank database with an accession number of KX347434.

In Silico Sequence and Structural Analyses of XynA 1
Xylanase. An E. coli clone harboring DNA from chicken cecum metagenome was found to contain an open reading frame of 1,116 bp encoding a protein of 372 amino acids, sequence of which was related to xylanase family GH10 and it was named the XynA MG1 xylanase. The first 23 N-terminus amino acids were predicted to be the signal peptide which guides secretion of the 349 amino acids' mature enzyme having a molecular mass and a pI value of 40,013 Da and 5.76, respectively.
Multiple sequence alignment of the XynA MG1 protein (including the signal peptide) with similar proteins, BLASTed and retrieved from the GenBank, revealed the highest identity at 59% to those of endo-1,4-beta-xylanases from Prevotella bryantii and Prevotella ruminicola and 58% identity to that from Prevotella copri. XynA MG1 has two conserved motifs, DVVNE and TEXD, of the GH10 family xylanase which contained the two glutamate residues (E157 and E 262) predicted to be the catalytic sites, and an invariant asparagine (N156) preceding the glutamate in the first motif serving as an acid/base catalyst ( Figure 1) [24][25][26].
Phylogenetic analysis of XynA MG1 placed it in the cluster within Prevotella xylanases clade with high bootstrap value support ( Figure 2). Among these, the highest amino acid sequence identity was only 59% which was the xylanase from Prevotella bryantii and Prevotella ruminicola. This indicated that XynA MG1 is a distinctly new endo-1,4 -xylanase member of the family GH10, probably from a bacterium in the genus Prevotella.
Structure of XynA MG1 was modeled [27] using the protein crystal structure 2cnc.1.A of a closely related thermostable GH10 xylanase from Cellvibrio mixtus as a template. The amino acid sequence stretches in the XynA MG1 molecule predicted to form different secondary structures (alphahelices, beta-sheet, and loops) are shown in Figure 3(a). Despite the low degrees of amino acid sequence homology of the mature forms of the two enzymes (40.2% identity; 58.6% similarity), the secondary structure profiles at corresponding positions along the amino acid sequences of the two xylanases are highly similar (Figure 3(a)). Three-dimensional structure   modeling of XynA MG1 revealed the typical ( / ) 8 TIM-barrel fold resembling the shape of a salad bowl (Figures 3(b) and 3(c)) which is similar to other known GH10 family xylanases [24].

Cloning, Expression, and Purification of XynA 1
Xylanase. XynA MG1 without the signal peptide was expressed in E. coli with thioredoxin A peptide and hexa-histidine tags fused to its N-terminus to enhance soluble protein folding and facilitate protein purification, respectively. The purified fusion protein was digested with enterokinase to remove the peptide tags and subjected to second round of purification to yield the purified mature XynA MG1 enzyme for characterization experiments. The Trx-His-XynA MG1 fusion enzyme was catalytically active; however, its activity was around half of that of the mature XynA MG1 (data not shown). Figure 4 shows the SDS-PAGE analysis of different forms of the enzyme through successive purification steps. The molecular mass of the purified XynA MG1 after removal of the tags was determined to be around 41.0 kDa which was close to the theoretical value of 40.013 kDa calculated based on the amino acid sequence. Xylanase zymogram analysis displayed a clear band of xylan digestion corresponding with the position of the XynA MG1 protein band (Figure 4).

Substrate Specificity.
The purified XynA MG1 was found to exert its highest hydrolytic activity towards the beechwood xylan while the activity towards oat-spelt xylan was 94% relative to that of the beechwood xylan. No activity could be measured with -cellulose, carboxymethyl cellulose, starch, and -glucan as substrates. Upon testing with synthetic chromogenic substrates, XynA MG1 could hydrolyze only the 4nitrophenyl--D-xylopyranoside while 4-nitrophenyl--Dcellobioside or 4-nitrophenyl--D-galactopyranoside could not serve as substrates ( Table 1). The relatively specific xylanase property of XynA MG1 is in contrast with most xylanases of family 10 which have both xylanase and cellulase activities. However, a number of xylanases of family 10 showed only xylanase activity such as xylanase Xyn10N18 derived from a bovine rumen metagenomic library [28] and Xyn10J from a compost metagenomic library which showed negligible hydrolytic activity against carboxymethyl cellulose [18]. Added to the latter group is XynA MG1 isolated in this study and the possession of the cellulase-free xylanase activity makes it potentially very useful for biobleaching of pulps [18]. TLC analysis of the reaction mixture of XynA MG1 catalyzed hydrolysis of beechwood xylan, compared with the results reported for the novel family 10 xylanase Xyn10J from a compost metagenomic library [18], suggested that xylobiose (X 2 ) and xylotetraose (X 4 ) were two major end products, and xylose (X 1 ) was produced as a minor product ( Figure 5(a)). XynA MG1 showed high hydrolytic activity on 4-nitrophenyl--D-xylopyranoside (pNP-X), a synthetic chromogenic substrate, releasing equimolar of two products 4-nitrophenol and xylose. The extent of the reaction is determined by adding 1 mL of 1 M Na 2 CO 3 to stop the reaction and turn the 4nitrophenol to yellow color which was quantified by measuring the absorbance at 400 nm [17]. The present xylose as a reaction product was shown by TLC ( Figure 5(b)). XynA MG1 effectively digested the synthetic substrate 4-nitrophenyl--D-xylopyranoside releasing large amount of xylose. However, upon digestion of beechwood xylan, a natural substrate, the major end product was not xylose (X 1 ) but the xylotetraose (X 4 ) and xylobiose (X 2 ) suggested that XynA MG1 could catalyze transglycosylation reactions. Xylanases with transglycosylation activity have been previously reported in the GH10 family, such as Xyn10J from compost metagenome and XynB from Thermotoga maritima and several GH11 xylanases such as Xyn1 from Paenibacillus sp. W-61, TfxA from Thermomonospora fusca, AnxA from Aspergillus niger, and XynA from Paecilomyces thermophila [18]. With the transglycosylation activity, XynA MG1 can be useful in the application for the synthesis of alkyl oligoglycosides [18].

Effects of pH and Temperature on Enzyme Activity and
Stability. The purified XynA MG1 showed the typical bellshaped pH profile with an optimal pH of 6. It retained 32% of its initial activity at pH 9 and completely lost all of its activity at pH 10 ( Figure 6(a)). This is consistent with xylanases previously reported [26,29] and with the physiological function of xylanases in the cecum of broiler chickens since the pH values in cecum broiler chicken are generally 5.5-6 [30]. The xylanase XynA MG1 enzyme was stable between pH 5.0 and 8.0 for 60 min, retaining around 70% of its activity at pH 8.0 (Figure 6(b)). It was fairly stable at high pH similar to other bacterial xylanases [26,31].
10 were known to have optimum temperature of 40-80 ∘ C [32]. XynA MG1 retained 72% of its activity after incubating at 60 ∘ C for 45 min (Figure 6(d)). However, when the temperature was 70 ∘ C, the enzyme completely lost the activity. Interestingly, it had around 30% activity even at 100 ∘ C in the presence of the substrate (Figure 6(c)). Many enzymes were known to be more stable in the presence of their substrates [33,34].

Effect of Metal Ions and Chemicals on the Xyn 1 Activity.
As shown in Table 2, the XynA MG1 activity was slightly deactivated by metal ions Co 2+ , Zn 2+ , Mn 2+ , and Mg 2+ at 2 mM and 10 mM in a concentration-dependent manner. Moderate inhibitions were found with Ca 2+ and strong inhibitions were found with Cu 2+ . The inhibitions of xylanase activity by Cu 2+ ion were commonly reported [35,36].
Reducing agents such as -mercaptoethanol and DTT slightly affected the XynA MG1 activity suggesting that xylanase does not seem to need disulfide bonds to achieve the hydrolysis reaction [37].
XynA MG1 enzyme retained more than 87% of its activity after incubation for 60 min with 10 mM of EDTA. The resistance of XynA MG1 to the chelating reagent suggested that it is not a metalloenzyme and no metal ion is essential for XynA MG1 activity. Its stability against the chelating agent, one of the indispensable ingredient in detergent formulations, is of great importance and worth further investigation on its application.  The anionic surfactant SDS which is well-known to cause protein denaturation severely deactivated XynA MG1 . Many xylanases were known to be strongly affected by SDS. These include xylanase rMxyl from compost-soil metagenome, xylanase from Burkholderia sp. DMAX, Aspergillus awamori VTCC-F312, and Aspergillus giganteus [9,[38][39][40]. This indicated that hydrophobic interactions are important in maintaining the structure of XynA MG1 .
Nonionic surfactants like Triton X-100 and Tween 80 at 2 mM showed mild inhibitory effects. However at 10 mM, International Journal of Microbiology they slightly enhanced the XynA MG1 activity which are in good agreement with other recombinant xylanases [41].

Effects of Sodium Chloride.
Purified XynA MG1 showed excellent salt tolerance. While NaCl was not required for its xylanase activity, XynA MG1 could function at more than 96% of its activity in the presence of 1 to 4 M NaCl for at least 2 h (Figure 7). High salt-tolerant and halophilic xylanases have been reported. Recently, a xylanase from Massilia sp. RBM26 isolated from feces of Rhinopithecus bieti was reported to maintain around 86% activity in 5 M NaCl for 1 h [26]. Xylanase from Aspergillus giganteus was shown to tolerate to up to 20% (3.4 M) of NaCl [38]. A cold-active xylanase from Glaciecola mesophila KMM 241 exhibited its highest activity at 0.5 M NaCl and retained 90% of the activity in 2.5 M NaCl at its optimal temperature of 30 ∘ C [35]. A xylanase from a marine bacterium Bacillus subtilis cho40 was reported to be greatly activated to 140% when preincubated with 0.5 M NaCl for 4 h [19]. A cold-active and halo-tolerant Xyn10A xylanase from Bacillus sp. SN5 was reported to exhibit the highest activity (134%) in 0.5 M NaCl and retain 90% activity in 2.5 M NaCl [42]. In comparison to the above-mentioned xylanases, our XynA MG1 reported here has the highest salt-tolerance property. ( Figure 8). At the highest concentration tested (5 M), acetone and methanol showed nearly no effect on XynA MG1 activity which is similar to the xylanase from Streptomyces rameus L2001 [43]. Comparing among alcohols, 1-propanol and ethanol which have lower polarity index values (and thus more hydrophobic) than methanol slightly inhibited XynA MG1 by 10% and 14%, respectively. These were similar to those reported for a xylanase (XynA) from Clostridium cellulovorans [44]. Hydrophobic interactions with the dissolved organic solvent molecules seem to be the major factor affecting the protein stability and the enzyme activity in this case [45]. In addition to microbial xylanases mentioned in the above discussions, biochemical properties of bacterial GH10 xylanases especially those from animal intestines are listed in Table 3 for comparison with the XynA MG1 xylanase described in this study. XynA MG1 is shown to be better tolerance to organic solvent and high salt (NaCl) concentration than other xylanases compared.

Conclusion
XynA MG1 derived from a chicken cecum metagenome is a new member of the GH10 family xylanase related to the xylanases from Prevotella sp. It is resistant to metal ions, reducing agents, and certain detergents, and highly tolerant to high concentrations of salt and water-miscible organic solvents. These make XynA MG1 a potential candidate for applications in the enzymatic processes operated at high concentration of salt and organic solvent such as in food and biofuels industries. The salt-tolerant xylanase property of XynA MG1 can be useful in the processing of sea food and food with a high salt content which contain 0.5 to 2.5 M NaCl, such as marine algae, pickles, and sauce [35]. Furthermore, food materials washing, food processing, and fermentation under high salt condition could reduce cost because sterilization is not required [26]. In bakery industry, salt-tolerant XynA MG1 xylanase has potential applications in increasing the strength of the dough and adding flavor to baked goods [52].
Modern biofuel (bioethanol) production from lignocellulose biomass employs consolidated bioprocess where both the saccharification (enzymatic hydrolysis of cellulose and hemicellulose components to simple sugar) and fermentation steps take place within the same bioreactor, which makes the process more economical [44]. Tolerance to the carried over biomass pretreatment agents (acid, alkali, and inhibitors) and ethanol tolerance have been identified as two key elements for the enzymes in this consolidated process. The biomass saccharification enzymes, including xylanase, must be able to withstand and function well in the presence of 23 to 63 g/L (0.5-1.4 M) ethanol produced from the fermentation step. XynA MG1 can be a potential candidate for this application as it could tolerate up to 5 M ethanol.
XynA MG1 functions optimally at the temperature and the pH range of the chicken intestine, its native environment where its gene was retrieved by metagenomic cloning. This xylanase has potential utility in animal feed to improve nutrient digestibility and growth performance for animals, especially broiler chickens and hens [53].

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this article.