Molecular Characterization of a Thermophilic and Salt- and Alkaline-Tolerant Xylanase from Planococcus sp. SL4, a Strain Isolated from the Sediment of a Soda Lake

Abstract

To enrich the genetic resource of microbial xylanases with high activity and stability under alkaline conditions, a xylanase gene (xynSL4) was cloned from Planococcus sp. SL4, an alkaline xylanase-producing strain isolated from the sediment of soda lake Dabusu. Deduced XynSL4 consists of a putative signal peptide of 29 residues and a catalytic domain (30-380 residues) of glycosyl hydrolase family 10, and shares the highest identity of 77% with a hypothetical protein from Planomicrobium glaciei CHR43. Phylogenetic analysis indicated that deduced XynSL4 is closely related with thermophilic and alkaline xylanases from Geobacillus and Bacillus species. The gene xynSL4 was expressed heterologously in Escherichia coli and the recombinant enzyme showed some superior properties. Purified recombinant XynSL4 (rXynSL4) was highly active and stable over the neutral and alkaline pH range from 6 to 11, with maximum activity at pH 7 and more than 60% activity at pH 11. It had an apparent temperature optimum of 70℃ and retained stable at this temperature in the presence of substrate. rXynSL4 was highly halotolerant, retaining more than 55% activity with 0.25-3.0 M NaCl and was stable at the concentration of NaCl up to 4M. The enzyme activity was significantly enhanced by β-mercaptoethanol and Ca²⁺ but strongly inhibited by heavy-metal ions and SDS. This thermophilic and alkaline- and salt-tolerant enzyme has great potential for basic research and industrial applications.

Introduction

Xylan is composed mainly of backbones of β-1,4-linked xylopyranose units with substituted side chains at different positions and represents approximately one-third of all renewable organic carbon on Earth [4,33]. Complete hydrolysis of xylan requires a large variety of cooperatively acting enzymes, including endo-1,4-β-D-xylanase, β-D-xylosidase, α-D-glucuronidase, α-L-arabinofuranosidase, acetyl xylanesterase, and arylesterase [11]. Endo-1,4-β-D-xylanase (E.C. 3.2.1.8) is the crucial enzyme that catalyzes the random hydrolysis of β-1,4-xylosidic linkages. Based on sequence similarities of the catalytic domain, xylanases have been classified into glycosyl hydrolase (GH) families (http://www.cazy.org/fam/acc_GH.html; [8]) 5, 7, 8, 10, 11, and 43 [10]. The majority of xylanases fall into the GH 10 and 11, which have distinct substrate specificities, three-dimensional structures, and mechanisms of action [6,18].

Xylanases are widely distributed in diverse organisms, including bacteria, algae, fungi, protozoa, gastropods, and anthropods [10]. Microbial xylanases have been attracting particular attention for their great application in food, animal feed, pulp and paper, and fuel alcohol industries [5,22]. Of these, the application of xylanases in the pulp and paper industry is of great interests owing to its facilitation of the release of lignin from the pulp and reduction of chlorine [41]. Industrial pulping is usually carried out under high temperature and alkaline conditions, and hence thermostable and alkaline-tolerant xylanases are the favorite [5,41]. Moreover, bioconversion processes and the detergent industry also require thermostable and alkalinetolerant xylanases [20,29,34]. Thus, mining of genetic resources of favorite xylanases is becoming important to meet the requirements of industrial applications.

Many alkaline xylanases and their coding genes have been reported from strains derived from environments like soil [15,36], alkaline wastewater [9,40,45,46], soda lakes [26], compost [39], and insect termite [31]. Those from extreme environments are the research focus [10,38]. Soda lake is one of the most stable naturally occurring alkaline environments, which usually has high alkalinity, generally pH 9.0 to 11.0, and moderate to extremely high salinity [2,19]. Despite the extreme conditions, soda lakes harbor abundant and diverse microorganisms that are excellent alkaline enzyme producers [17]. In this study, a bacterial strain, Planococcus sp. SL4, was isolated from the sediment of soda lake Dabusu, and a novel GH 10 xylanase gene was cloned from it. The gene was expressed in Escherichia coli, and the purified recombinant enzyme was characterized to be thermophilic and alkaline- and salt-tolerant.

 

Materials and Methods

Strains, Vectors, and Chemicals

Escherichia coli DH5α and the pMD 18-T vector (TaKaRa, Otsu, Japan) were used for gene cloning and sequencing. Vector pET-22b(+) (Novagen, San Diego, CA, USA) and E. coli BL21 (DE3) (TaKaRa) were used for gene expression. Nickel-NTA agarose (Qiagen, Valencia, CA, USA) was used to purify the His6-tagged protein. Kits for genomic DNA isolation, DNA purification, and plasmid isolation were purchased from Omega (Norcross, GA, USA). Restriction endonucleases, T4 DNA ligase, DNA polymerase, and dNTPs were purchased from New England Biolabs (Ipswich, MA, USA). The substrates beechwood xylan, oat spelt xylan, birchwood xylan, carboxymethyl cellulose-sodium (CMC-Na), lichenan, barley β-glucan, 4-nitrophenyl β-D-cellobioside (pNPC), and p-nitrophenyl β-D-xyloside (pNPX) were purchased from Sigma (St. Louis, MO, USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was purchased from Amresco (Solon, OH, USA). All other chemicals were of analytical grade and commercially available.

Microorganism Isolation

Soda lake Dabusu is located in the southwest of Qian’an County, Jilin Province, China. It has a salinity of 62.34 to 347.34 g/l and a pH of 10 to 11 [35]. One gram of sediment was suspended in sterilized lake water and spread onto screening agar plates containing 0.5 % (w/v) beechwood xylan, 0.1 % (w/v) peptone, and 0.1 % (w/v) NaCl. The pure culture of strain SL4 was obtained through repeated streaking on the screening medium. The taxon of the strain was identified by the 16S rDNA sequence PCR-amplified using primers 27F and 1492R (Table 1).

Table 1.aRestriction sites are bold and underlined.

Gene Cloning of the Full-Length Xylanase Gene (xynSL4)

Genomic DNA from strain SL4 was extracted using the Omega genomic DNA isolation kit following the manufacturer’s instructions. To obtain the xylanase gene fragment, the purified genomic DNA as template and a degenerate primer set specific for GH 10 xylanases (X10-F: 5’-CTACGACTGGGAYGTNIBSAAYGA-3’; X10-R:5’-GTGACTCTGGAWRCCIABNCCRT-3’) were used for PCR amplification [42]. The PCR products were excised, purified, and ligated into vector pMD 18-T, transformed into E. coli DH5α, and sequenced by Invitrogen (Carlsbad, CA, USA). The flanking regions of the gene fragment were obtained by using thermal asymmetric interlaced-PCR (TAIL-PCR) procedure [25] with three nested specific primers (Table 1). PCR products of the expected size that appeared between the second and third rounds of amplification were purified, cloned into the pMD 18-T vectors, sequenced, and then assembled with the known fragment sequence. The full-length xylanase gene was designated as xynSL4.

Sequence and Phylogenetic Analysis

Assembly of the xylanase gene sequences and identification of the open reading frame (ORF) were performed using the programs of the Vector NTI 10.3 (InforMax, Gaithersburg, MD, USA). The signal peptide sequence was predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/) [32]. The DNA and protein sequence similarities were assessed by using the BLASTn and BLASTp programs (http://www.ncbi.nlm.nih.gov/BLAST/), respectively. Multiple sequence alignments were performed with Clustal W (http://www.ebi.ac.uk/Tools/clustalw2/). A phylogenetic tree, including XynSL4 and its closest homologs, was constructed using the neighbor-joining algorithm in MEGA 4.0 [37]. Confidence for the tree topology was estimated using the bootstrap values based on 1,000 replicates.

Expression and Purification of XynSL4 in E. coli

The coding sequence of mature XynSL4 without the predicted signal peptide was amplified by PCR using primers xynSL4-m-F and xynSL4-m-R (Table 1), and cloned into the BamHI-NotI site of pET-22b(+). The recombinant plasmid, pET-xynSL4, was transformed into E. coli BL21 (DE3) competent cells. Positive transformants harboring the recombinant plasmid (pET-xynSL4) were identified by PCR and further confirmed by DNA sequencing. The cells were grown in LB medium containing 100 µg/ml of ampicillin at 37℃ to an A600 of 0.6. Protein expression was induced by addition of IPTG at a final concentration of 1 mM at 30℃ for 12 h. Xylanase activities of the cell pellet and culture supernatant were assayed as described below.

To purify the His-tagged recombinant protein (rXynSL4), culture supernatant was collected after centrifugation (12,000 ×g, 4℃ for 15 min) and further concentrated using an ultrafiltration membrane (PES5000; Sartorius Stedim Biotech, Göettingen, Germany). The concentrated supernatant was loaded onto a Ni2+-NTA agarose gel column with a linear imidazole gradient of 20– 200 mM in Tris-HCl buffer (20 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH 7.6).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the purity and apparent molecular mass of rXynSL4. The protein concentration was determined by the Bradford method [7], using bovine serum albumin as a standard. The identity of the purified enzyme was verified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Enzyme Assay

Xylanase activity was determined by measuring the release of reducing sugar from substrates using the 3,5-dinitrosalicylic acid (DNS) method [30]. The standard reaction contained 0.1 ml of appropriately diluted enzyme and 0.9 ml of McIlvaine buffer (pH 7.0) containing 1% (w/v) beechwood xylan as substrate. After incubation at 70℃ for 10 min, the reaction was stopped with 1.5 ml of DNS reagent and boiled for 5 min. The absorption at 540 nm was measured when the aforementioned mixture cooled to room temperature. Using a standard curve generated with D -xylose, the absorbance was converted into moles of reducing sugars produced. One unit (U) of xylanase activity was defined as the amount of enzyme that released 1 µmol of reducing sugar equivalent to xylose per minute. The enzyme activity was assayed by following this standard procedure unless otherwise noted. All reactions were performed in triplicate.

Biochemical Characterization

The substrate specificity of purified rXynSL4 was assayed by incubating the enzyme solution with 1% (w/v) of oat spelt xylan, birchwood xylan, beechwood xylan, CMC-Na, lichenan, or barley β-glucan under standard conditions (pH 7.0, 70℃, 10 min). Enzymatic activities against pNPC and pNPX were examined under standard conditions at a final concentration of 5 mM.

The optimal pH for xylanase activity of the purified rXynSL4 was determined at 37℃ in buffers with pH ranging from 4 to 11. The buffers used were McIlvaine buffer (0.2 M Na2HPO4/0.1 M citric acid) for pH 4–7, 0.1 M Tris-HCl for pH 7–9, and 0.1 M glycine-NaOH for pH 9–11. The stability of purified rXynSL4 at different pH values was estimated by incubating the enzyme solution (0.02 mg) at 60℃ in various buffers over a pH range of 7-11 for 5, 10, 20, 30, or 60 min, with or without 1% (w/v) of beechwood xylan. The remaining activity was measured in Tris-HCl buffer (pH 7) at 70℃ for 10 min. The initial activity of rXynSL4 was set as 100%.

The optimal temperature for purified rXynSL4 activity was determined over the range of 40–90℃ in Tris-HCl buffer (pH 7). The thermostability of rXynSL4 was determined by measuring the residual activities after pre-incubation of the enzyme in Tris-HCl buffer (pH 7 or 9) at 55℃, 60℃, 65℃, and 70℃, with or without 1% (w/v) of beechwood xylan for various periods.

To investigate the effects of different metal ions and chemical reagents on the purified rXynSL4 activity, activities were measured at 37℃ in McIlvaine buffer (pH 7) containing 5 mM (final concentration) of KCl, CaCl2, CoCl2, NiSO4, CuSO4, MgSO4, FeSO4, FeCl3, MnSO4, ZnSO4, Pb(CH3COO)2, AgCl, HgCl2, EDTA, SDS, or β-mercaptoethanol.

The Km, Vmax, and kcat values for rXynSL4 were determined in Tris-HCl buffer (pH 7) containing 1–10 mg/ml beechwood xylan at 70℃, respectively. Km and Vmax were determined from a Lineweaver-Burk plot using the nonlinear regression computer program GraFit (Erithacus, Horley, Surrey, UK).

The effect of NaCl on the purified rXynSL4 was determined at 70℃ in Tris-HCl buffer (pH 7) containing 0.25–4.5 M NaCl. To examine its resistance to salt, rXynSL4 was incubated with 3 or 4 M of NaCl at 37℃ for 1 h, and the residual enzyme activities were measured.

Nucleotide Sequence Accession Numbers

The nucleotide sequences of the Planococcus sp. SL4 16S rDNA and GH 10 xylanase gene (xynSL4) were deposited into the GenBank database under accession numbers KM360097 and KM360098, respectively.

 

Results

Strain Identification

Based on the BLASTn analysis, the 16S rDNA sequence from strain SL4 (1,508 bp) showed a nucleotide identity of 99.6% with Planococcus sp. L4 (DQ435614), 98.9% with Planomicrobium alkanoclasticum HWG-A6 (JQ684229), and 98.8% with Planomicrobium sp. SS7.14 (KC160645). Thus, strain SL4 was classified into the genus Planococcus. The distance tree created by the neighbor-joining method also revealed the same classification (data not shown).

Gene Cloning and Sequence Analysis of xynSL4

A gene fragment of xynSL4 (256 bp) was amplified by using the CODEHOP primers X10-F and X10-R [42]. DNA fragments amplified by TAIL-PCR were assembled with the core region, and an ORF of 1,143 bp starting with ATG and terminating with TGA was identified. The full-length gene (xynSL4) encoded a polypeptide of 380 amino acid residues, including a putative signal peptide of 29 residues and a catalytic domain belonging to family 10. Its calculated molecular mass and theoretical isoelectric point were estimated to be 43.3 kDa and 4.82, respectively.

The deduced amino acid sequence of xynSL4 showed the highest identities (74-77%) with hypothetical xylanases from Planomicrobium glaciei CHR43 (ETP70492), Bacillus sp. J37 (WP_026562290), Bacillus sp. NSP22.2 (WP_026570660), Sediminibacillus halophilus (WP_026771505), Bacillus kribbensis (WP_026692539), Bacillus nealsonii (WP_016201570), and Bacillus sp. UNC41MFS5 (WP_026564811). Furthermore, XynSL4 showed 63-65% identity with the functionally characterized thermo-alkali-stable xylanase of Geobacillus thermoleovorans (AEW07375) [40], the xylanase Xt6 from Geobacillus stearothermophilus (ABI49951) [21], the alkaline thermostable endoxylanase from Bacillus sp. NG-27 (AAB70918) [15], the Xyn10A from Bacillus firmus (AAQ83581) [9], the alkaline active endoxylanase from Bacillus halodurans S7 (AAV98623) [26], the thermostable xylanase from thermophilic Geobacillus sp. TC-W7 (ACX42569) [23], and the thermostable xylanase from alkaliphilic Bacillus sp. N16-5 (ADI24221) [44].

A phylogenetic tree was constructed based on the amino acid sequences of XynSL4 and its closest homologs derived from GenBank Database. High bootstrap values separated these xylanases into four major groups (Fig. 1). XynSL4 was closely related to the putative xylanases from P. glaciei CHR43 and B. nealsonii (WP 016201570), and grouped in a large cluster together with thermostable alkaline xylanases from Geobacillus and Bacillus. Based on the multiple sequence alignment of XynSL4 and seven characterized GH 10 xylanases, two putative catalytic residues (Glu179 and Glu285) were identified in XynSL4 (Fig. 2).

Fig. 1.Phylogenetic tree of the amino acid sequences of XynSL4 and its close homologs with accession numbers in parentheses. The tree was constructed using the neighbor-joining method (MEGA 4.0). Bootstrap values (n = 1,000 replicates) are reported as percentages. The scale bar represents the number of changes per amino acid position.

Fig. 2.Multiple alignment of XynSL4 and seven functionally characterized GH 10 xylanases. Identical and similar amino acids are highlighted in solid black and grey, respectively. The two conserved catalytic residues (Glu) are marked with asterisks. Xylanase names, microbial sources, and GenBank accession numbers are given as follows: Xyn_GT: Geobacillus thermoleovorans (AEW07375); Xyn_TC-W7: Geobacillus sp. TC-W7 (ACX42569); Xyn_XT6: Geobacillus stearothermophilus (ABI49951); Xyn_NG-27: Bacillus sp. NG-27 (AAB70918); Xyn_BF: Bacillus firmus (AAQ83581); Xyn_N16-5: Bacillus sp. N16-5 (ADI24221); Xyn_BH: Bacillus halodurans S7 (AAV98623); and XynSL4: Planococcus sp. SL4 (HM156498).

Expression and Purification of rXynSL4

The gene fragment coding for the mature protein was expressed in E. coli BL21 (DE3). After induction with 1 mM IPTG at 30℃ for 12 h, significant xylanase activity (about 2.8 U/ml) was detected in the culture supernatant. No xylanase activity was detected in the cultures of uninduced transformant. The crude enzyme was purified to electrophoretic homogeneity by ultrafiltration and Ni-affinity chromatography (Fig. 3). The purified rXynSL4 migrated as a single band at about 44 kDa on SDS-PAGE, which was identical to the calculated value (43.4 kDa). Three internal peptides obtained from LC–ESI-MS/MS (HHYNSIVAENVMK, ESQWYQLTGTDYIK, and PAYPTYDAIPEER) matched the deduced amino acid sequence of XynSL4 (Fig. 2), confirming that the purified enzyme was indeed XynSL4.

Fig. 3.SDS-PAGE analysis of purified rXynSL4. Lanes: M, the low-molecular-weight protein marker; 1, culture supernatant of an uninduced transformant harboring pET-xynSL4; 2, culture supernatant of an induced transformant harboring pET-xynSL4; 3, purified rXynSL4 after Ni-affinity chromatography.

Enzyme Characterization

When assayed at 37℃, purified rXynSL4 showed apparent optimal xylanase activity at pH 7, and retained greater than 95% of the maximum activity between pH 7 and 9 and greater than 60% even at pH 11.0 (Fig. 4A). The thermal activity of purified rXynSL4 was apparently optimal at 70℃ when assayed at pH 7, and retained greater than 50% of the maximum activity when assayed at 50–75℃ (Fig. 4B). Without substrate, the purified rXynSL4 exhibited more than 50% of the initial activity after incubation in buffers ranging from pH 7 to 9 at 60℃ for 1 h and was not stable at pH 10 and pH 11 (Fig. 4C). However, in the presence of 1% beechwood xylan, rXynSL4 retained 92% and 58% activity after incubation at pH 10.0 and 11.0, respectively (Fig. 5A). Without substrate, the enzyme was stable at 55℃ for more than 60 min, whereas at 60℃ and 70℃, the half-lives of the enzyme were approximately 15 min and 2 min (Fig. 4D), respectively. In the presence of 1% beechwood xylan, rXynSL4 retained 76 % activity after incubation at pH 7.0 at 70℃ for 30 min (Fig. 5B).

Fig. 4.Enzymatic properties of purified rXynSL4. (A) Effect of pH on XynSL4 activity. Activities at various pHs were assayed at 37℃ for 10 min. (B) Effect of temperature on XynSL4 activity in McIlvaine buffer (pH 7.0). (C) pH stability of XynSL4. Residual activities after incubation of the purified enzyme at various pHs for different periods of time at 60℃ were assayed at pH 7.0 and 70℃ for 10 min. (D) Thermostability of XynSL4. Residual activity was assayed at pH 7.0 and 70℃ for 10 min after pre-incubation at 55℃, 60℃, 65℃, or 70℃ for different periods of time. The error bars represent the means ± SD (n = 3).

Fig. 5.Stability of rXynSL4. (A) pH stability of purified rXynSL4 at 60℃ and pH values of 10.0 and 11.0 in the absence or presence of 1% beechwood. (B) Thermostability of purified rXynSL4 at 70℃ in the absence or presence of 1% beechwood xylan. The error bars represent the means ± SD (n = 3).

At pH 7.0 and 70℃, rXynSL4 had high specific activities towards various xylans, including oat spelt xylan (236.2 U/mg), birchwood xylan (219.6 U/mg), and beechwood xylan (244.7 U/mg). It also had weak activity to synthetic substrate pNPC (5.6 U/mg), but no activity against p-nitrophenyl xyloside, CMC-Na, lichenan, and barley β-glucan.

Using beechwood xylan as the substrate, the Km, Vmax, and kcat values were 1.45 ± 0.08 mg/ml, 362.74 ± 8.55 µmol mg-1min-1, and 262.62 ± 6.19 s-1, respectively, based on a Lineweaver-Burk plot.

The chemical effect on rXynSL4 activity was also investigated (Table 2). Ca2+ and β-mercaptoethanol of 5 mM enhanced the activity by 1.34-fold and 1.86-fold, respectively. K+, Cr3+, Li+, and Na+ had little or no effect on rXynSL4 activity. Other chemicals strongly inhibited the activity, and 5 mM of Ag+, Cu2+, Hg2+, Pb2+, Zn2+, Fe3+, and Cr3+ resulted in an almost complete loss of activity.

Table 2.aValues represent the means ± SD (n = 3) relative to the untreated control samples.

Purified rXynSL4 retained greater than 55% xylanase activity in the presence of 0.25–3.0 M NaCl, and 33% at 4.5 M NaCl. Purified rXynSL4 showed strong tolerance to high concentrations of NaCl, retaining more than 90% xylanase activity after 1 h incubation with 3 or 4 M NaCl at 37℃ and pH 7 (Fig. 6).

Fig. 6.Effect of NaCl on rXynSL4 activity and stability. (A) Effect of different concentrations of NaCl on the activity of rXynSL4. (B) Effect of 3 and 4 M of NaCl on the stability of rXynSL4. The error bars represent the mean ± SD (n = 3).

 

Discussion

Soda lakes are one of the most stable alkaline ecosystems that occur naturally on Earth, thus representing an ideal niche for alkaliphilic microorganisms [19]. A lot of alkaliphilic bacteria have been isolated from soda lakes, which produce alkaline enzymes capable of functioning at high pH and possibly high temperature and salt concentration, with application value in various industries [13,17]. In this study, a Planococcus strain was isolated from the sediment of Lake Dabusu, and a xylanase gene was cloned from it. Based on phylogenetic analysis (Fig. 1), XynSL4 is distant from functionally characterized GH 10 xylanases, suggesting its novelty. To our best knowledge, this is the first study on the gene cloning, heterologous expression, and biochemical characterization of a xylanase from Planococcus.

Enzymes from alkaliphilic bacteria are usually capable of functioning at high pH and possibly high temperature and salt concentration [17]. The rXynSL4 had a pH optimum of 7, but it retained more than 95% activity at pH 9.0 and retained more than 60% activity even at pH 11, which is similar to the alkaline xylanases from Bacillus firmus [9] and Bacillus sp. NG-27 [15]. The change of the number of surface charged amino acids might be the main factor that makes alkaline active enzymes to adapt to high pH conditions. The crystal structure analysis indicated that alkaline xylanases from B. halodurans S7 [27] and Bacillus sp. NG-27 [28] have more surface-accessible, negatively charged residues when compared with non-alkaline xylanases. A homology-based model of XynSL4 with xylanase from Bacillus sp. NG-27 as the template showed that XynSL4 has a high percentage of acidic amino acids on the surface, similar to alkaline xylanases from B. firmus, B. halodurans S7, and Bacillus sp. NG-27. It is the possible reason why rXynSL4 had high activity and stability at alkaline pHs.

rXynSL4 was thermophilic with an apparent temperature optimum at 70℃. To our surprise, its thermostability at this temperature was worse, with a half-life of approximately 2.5 min, far less than its closest characterized homologs. By mining the literature database, several xylanases that have worse thermostability at their apparent temperature optima have been reported [1,43]. A possible explanation is that the substrate changes the structure of the enzyme or is involved in hydrogen bonding in the active site in a pHdependent manner [43]. To determine whether the presence of substrate can improve the enzyme thermostability, the thermostability of rXynSL4 with or without substrate at the same reaction conditions was assayed (Fig. 5). As a result, rXynSL4 was far more thermostable at 70℃ in the presence of beechwood xylan, which confirmed that the substrate can protect the enzyme against heat.

Salt-tolerant xylanases have potential applications in the processing of marine foods, which are usually isolated from microorganisms of marine or saline environments [3,14,16,24,47]. Because Lake Dabusu has a high salinity, we also investigated the effect of NaCl on the activity and stability of rXynSL4. The purified rXynSL4 has great tolerance to different concentrations of NaCl, and it was very stable at high concentrations of NaCl up to 4 M (Fig. 6). Previous studies showed that the salt-tolerant proteins usually have an excess of acidic amino acids, which have a high water binding capacity, and could form a salvation shell on the surface of the proteins to keep them hydrated, facilitating their adaptation to the environmental pressure that represents the high salt concentration [12,24,38]. Amino acid sequence analysis found that more acidic amino acids were present on the surface of XynSL4 when compared with other salt-tolerant xylanases [24], which might one of the reasons that enable XynSL4 to maintain a good stability of the protein in a high salt environment.

Hundreds of xylanases have been characterized so far (http://www.cazy.org/). Most of them are mesophilic and active under acidic or neutral conditions, and only a few are thermophilic and alkaline [9,15,21,26,39,40]. In comparison with the thermophilic alkaline xylanases from B. halodurans S7 [27] and G. thermoleovorans [40], XynSL4 had a higher Vmax value (362.74 µmol · mg-1 min-1 vs. 252 and 42.5 µmol · mg-1 min-1). The Km value of rXynSL4 was lower than that of these two counterparts, indicating its higher affinity to substrate. Furthermore, rXynSL4 had no activity towards barley β-glucan, CMC-Na, and Avicel. Thus, rXynSL4 represents a novel thermophilic alkaline xylanase with application potential in the biobleaching of paper pulp.

In conclusion, a novel GH 10 xylanase gene was cloned from Planococcus sp. SL4, a strain isolated from the sediment of a soda lake with high alkalinity and salinity. rXynSL4 produced in E. coli showed high activity and stability at alkaline pH and was thermophilic and highly salt-tolerant. These properties indicate that XynSL4 has great potential for basic research and industrial applications.