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Molecular and Biochemical Characterization of a Novel Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti S
Molecular and Biochemical Characterization of a Novel Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti S
Journal of Microbiology and Biotechnology. 2016. Feb, 26(1): 9-19
Copyright © 2016, The Korean Society For Microbiology And Biotechnology
  • Received : April 08, 2015
  • Accepted : September 17, 2015
  • Published : February 28, 2016
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About the Authors
Bo Xu
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
Liming Dai
School of Life Science, Yunnan Normal University, Kunming 650500, P.R. China
Junjun Li
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
Meng Deng
School of Life Science, Yunnan Normal University, Kunming 650500, P.R. China
Huabiao Miao
School of Life Science, Yunnan Normal University, Kunming 650500, P.R. China
Junpei Zhou
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
Yuelin Mu
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
Qian Wu
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
Xianghua Tang
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
Yunjuan Yang
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
Junmei Ding
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
Nanyu Han
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
Zunxi Huang
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China
huangzunxi@163.com

Abstract
Xylanases sourced from different bacteria have significantly different enzymatic properties. Therefore, studying xylanases from different bacteria is important to their applications in different fields. A potential xylanase degradation gene in Massilia was recently discovered through genomic sequencing. However, its xylanase activity remains unexplored. This paper is the first to report a xylanase (XynRBM26) belonging to the glycosyl hydrolase family (GH10) from the genus Massilia . The gene encodes a 383-residue polypeptide (XynRBM26) with the highest identity of 62% with the endoxylanase from uncultured bacterium BLR13. The XynRBM26 expressed in Escherichia coli BL21 is a monomer with a molecular mass of 45.0 kDa. According to enzymatic characteristic analysis, pH 5.5 is the most appropriate for XynRBM26, which could maintain more than 90% activity between pH 5.0 and 8.0. Moreover, XynRBM26 is stable at 37℃ and could maintain at least 96% activity after being placed at 37℃ for 1 h. This paper is the first to report that GH10 xylanase in an animal gastrointestinal tract (GIT) has salt tolerance, which could maintain 86% activity in 5 M NaCl. Under the optimum conditions, K m , V max , and k cat of XynRBM26 to beechwood xylan are 9.49 mg/ml, 65.79 μmol/min/mg, and 47.34 /sec, respectively. Considering that XynRBM26 comes from an animal GIT, this xylanase has potential application in feedstuff. Moreover, XynRBM26 is applicable to high-salt food and seafood processing, as well as other high-salt environmental biotechnological fields, because of its high catalytic activity in high-concentration NaCl.
Keywords
Introduction
Xylan is a main component of hemicelluloses in plant cell walls. The complete hydrolysis of xylan needs the synergistic effect of multiple enzymes such as xylanase and xylosidase. Xylanases mainly include endo-1,4-β-D-xylanase (E.C. 3.2.1.8), β-D-xylosidase (E.C. 3.2.1.37), and α-L-arabinofuranosidase (E.C. 3.2.1.55) [4] . Endo-1,4-β-D-xylanase could hydrolyze the β-1,4 glucosidic bonds of the xylan main chain. This xylanase is the most important enzyme in the degradation of xylan. Considering its potential to degrade plant xylan, xylanase is widely used in various industrial fields, such as food, feedstuff, wine, papermaking, linen degumming, and fuel production.
The gastrointestinal tract (GIT) of herbivorous animals has rich enzymes related with the degradation of lignocelluloses. However, different animals have different enzymes [5 , 14 , 29] . Several researchers have recently acquired various xylanases from the GIT of longicorn beetle, cow rumen, and goat rumen through isolated culture of microbes and metagenomics [38 , 33 , 32] . As a typical herbivorous primate, Rhinopithecus bieti is fed on lichen, wild fruit, tender leaves of needle-leaved tree, overwintering cataphyll, and sunglo [19 , 22] . Given the different feeding types and species, the GIT of R. bieti may contain new microorganism xylanase gene resources different from other animals. No report on enzymes in the GIT of R. bieti has been reported yet.
Xylanase has extensive sources, mainly including bacteria, fungus, terrestrial plant tissues, and animal digestive juice. Among microorganisms, xylanase mainly comes from Bacillus, Aspergillus, Trichoderma, Ruminococci , and Fibrobacteres [4] . Potential xylan degrading genes in Massilia were recently discovered through genomic sequencing [9] . However, no research on the activity of xylanase has been reported yet. A number of previous research demonstrated that xylanase from different bacterial sources have significantly different enzymatic properties [10 , 18] . Therefore, studying xylanase from different bacterial sources is significant to their applications in different fields.
Salt, playing a key role in food safety and preservation by retarding the growth of spoilage microorganisms, is the world’s oldest food additive. Xylanases that are magically active and stable at high salt concentrations could be used in harsh industrial processes, such as food processing and washing [24] . Furthermore, fermentation and materials processing under high salt condition could reduce cost because sterilization is unnecessary [24] . In recent years, salt-tolerant xylanases have become an interesting research topic. Previous research studied reported that xylanases with salt resistance have been acquired from extreme environments (seawater and high-salt soil). Occasionally, the properties of the enzymes are different from the source environments, such as a halotolerant cellulase isolated from non extreme soil [31] . In this study, a GH10 salt-tolerant endoxylanase was revealed from Massilia harbored in an animal GIT.
Genomic sequencing could comprehensively reveal genetic information on microbial genomics. With the rapid development of sequencing techniques, genomes of numerous microorganisms have been sequenced. Based on genomic sequencing, xylanase genes from different microorganisms have been cloned, heterologously expressed, and characterized. Zhou et al. [37] cloned and heterologous expressed a xylanase gene with multiple structural domains from Arthrobacter sp. GN16, an isolate in feces of Grus nigricollis . Bhalla et al. [2] gained a heat-resistant GH10 xylanase from Geobacillus sp. WSUCF1 and made heterologous expression. In the present paper, a GH10 xylanase gene was cloned from Massilia for the first time through genomic sequencing. Sequence analysis, phylogenetic analysis, and heterologous expression of this xylanase were conducted. Enzymatic characteristics of its recombinase were analyzed. The results showed that XynRBM26 has catalytic activity in high-concentration NaCl and is applicable to high-salt food and seafood processing and other high-salt environmental biotechnological fields. Moreover, this xylanase has application potential in feedstuff materials because XynRBM26 comes from an animal GIT.
Materials and Methods
- Main Reagents and Vectors
The DNA polymerase and dNTP were bought from TaKaRa (Otsu, Japan). Beechwood xylan and p -nitrophenyl-β-D-xylopyranoside were bought from Sigma (St. Louis, MO, USA). Escherichia coli BL21 and the expression vector pEasy -E2 were bought from TransGen (Beijing, China). Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was purchased from Amresco (Solon, OH, USA). The Genomic DNA Clean and Concentration Kit was bought from Zymo Research (Orange, CA, USA). The Tureseq DNA Sample Preparation Kit was bought from Illumima (San Diego, CA, USA). Nickel-NTA resin was bought from Qiagen (Valencia, CA, USA). All other chemicals were of analytic grade.
- Microorganism Isolation and Identification
Massilia sp. RBM26 was extracted from fecal microorganism of R. bieti . Feces samples were collected from the National Nature Reserve in Baima Snow Mountain in Weixi County, Yunnan Province, People’s Republic of China. Two grams of the feces was suspended in 0.7% (w/v) NaCl and spread onto screening agar plates containing 0.2% (w/v) carboxymethyl cellulose sodium salt, 0.1% (w/v) peptone, 0.1% (w/v) yeast extract, and 0.02% (w/v) Congo red. The pure culture of strain RBM26 was obtained through repeated streaking on the screening agar plates at 30℃. Strains were identified on the basis of their 16S rDNA. Two universal primers of bacteria, namely, 27F (AGAGTTTGATCC TGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT), were used for 16S rDNA amplification. Amplification conditions included 94℃ for 5 min, 30 cycles at 94℃ for 30 sec, 52℃ for 30 sec, and 72℃ for 2 min; and then, 72℃ for 10 min.
- Genomic Sequencing
We accomplished the genomic sequencing of Massilia sp. RBM26 in the authors’ laboratory. Library construction, sequencing, and data analysis were similar to those reported by Zhou et al. [37] .
Library preparation. Genomic DNA of RBM26 was extracted using the Tiangen Genomic DNA Isolation Kit (Beijing, China), assessed using NanoDrop-2000 (Thermo Scientific, Waltham, MA, USA), quantified using the Qubit DNA Quantification Kit (Invitrogen, Carlsbad, CA, USA), randomly fragmented using the Bioruptor sonicator (Diagenode, Liège, Belgium), and purified using the Zymo Genomic DNA Clean & Concentration Kit (Orange, CA, USA). Then, the DNA library was prepared usingthe Illumina TruSeq DNA Sample Preparation Kit according to the manufacturer’s instruction (San Diego, CA, USA). After adapter ligation, a library fragment size of 400–600 bp was chosen and PCR enriched. The library quantity and quality were confirmed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA).
Sequencing. The MiSeq Reagent Kit V2 provided reagents for the cluster amplification and sequencing on a Miseq sequencer (Illumina).
Data analysis. Real-time image analysis and base calling were performed using the compatible sequencing software RTA (Illumina).
- Sequence Analysis and Phylogenetic Analysis
The open reading frame (ORF) was predicted using GeneMark.hmm (Ver. 2.4; http://exon.gatech.edu/GeneMark/gmhmm2_prok.cgi ). DNA comparison and protein comparison were conducted using BLASTn and BLASTp programs ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). The signal peptide was predicted by SignalP ( http://www.cbs.dtu.dk/services/SignalP/ ). Glucoside hydrolases were classified by InterPro ( http://www.ebi.ac.uk/interpro/ ).
Sequences were compared by ClustalX. A phylogenetic tree was built by the neighbor-joining algorithm and Poission correction matrix in the MEGA 6 . 0 software p ackage. The bootstrap value was 1,000.
- Heterologous Expression ofXynRBM26
PCR amplification was implemented using XynRBM26F (ATG ACATCTCGACGCGATAC) and XynRBM26R (GGCTTTACG CATCGGCATCG) as primers and genome DNA of Massilia sp. RBM26 as template. Touchdown-PCR was performed as follows to amplify the xylanase gene: 94℃ for 5 min, and then 20 touchdown cycles of 94℃ for 30 sec, 72℃ for 30 sec (decreasing by 1℃ each cycle), and 72℃ for 1.5 min; followed by 10 cycles of 94℃ for 30 sec, 5 2℃ for 30 s ec, a nd 7 2℃ for 1. 5 min, a nd o ne f inal extension a t 72℃ for 7 min. The PCR product was gel purified, ligated to pEasy -E2 vector, transformed into E. coli BL21, and sequenced by Beijing Genomics Institute (Guangzhou, China).
- Purification of Recombinant XynRBM26
The transformed strains were grown in LB medium (100 μg/ml Amp) u ntil t he OD600 reached 0 . 6–1. 0. Protein e xpression w as induced by adding IPTG to 0.05 mM, and the culture was shaken for 20 h at 20℃. Cells were harvested by centrifugation at 12,000 × g f or 5 m in. After b eing s uspended b y an a ppropriate amount of pH 7.0 McIlvaine buffer solution, the collected cells were disrupted by ultrasonic wave in a low-temperature water bath. The processed intracellular concentrated initial enzyme solution was centrifuged for 12 min at 12,000 ×g. The supernatant was extracted and purified into target protein by Nickel-NTA resin (XynRBM26; His6-tagged at C terminal). The purified protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% SDS-PAGE) and the protein concentration was determined by the Bradford method using bovine serum albumin as the standard. The protein within the gel was identified using matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF/MS) performed by Tianjin Biochip (Tianjin, China).
- Enzyme Assay and Substrate Specificity
The activity of XynRBM26 was determined by measuring the release of reducing sugar or p -nitrophenol from the substrate. The reaction system contained 100 μl of enzyme solution (concentration of XynRBM26: 0 . 02 mg/ml) and 9 00 μl of McIlvaine buffer (pH 5.5) containing 0.5% (w/v) beechwood xylan, oat spelts xylan, carboxymethyl cellulose sodium salt, laminarin and β-gulcan, microcrystalline cellulose, or 2 mmol/l p -nitrophenyl-β-Dxylopyranoside. The substrate was pre-heated for 5 min at 45℃ and added with enzyme solution for another 10 min reaction. Then 1.5 ml of DNS was added to end the reaction and boiled for 5 min, or the reaction was stopped with 1.5 ml of 1 mol/l Na 2 CO 3 . The absorption at 540 nm or 405 nm was measured when the above mixture was cooled down to room temperature. One unit of enzyme activity (U) was defined as the amount of enzyme that produced 1 μmol of xylose or p -nitrophenol equivalent per minute.
- Analysis of Enzymatic Characteristics
Characterization of the purified XynRBM26 activity was determined using beechwood xylan as the substrate. The buffer solutions used were 0.1 mol/l McIlvaine buffer (pH 3.0–8.0), 0.1 mol/l Tris-HCl (pH 8.0–9.0), and 0.1 mol/l glycine-NaOH (pH 9.0–12.0).
The optimal pH for the purified XynRBM26 was determined at 37℃ in buffers with pH ranging from 3.0 to 12.0. The enzyme stability at different pH values was estimated by measuring the residual enzyme activity after incubating the enzyme solution at 37℃ for 60 min, with the untreated enzyme defining 100% activity.
The optimal temperature for purified XynRBM26 activity was determined over the range of 0–70℃ in McIlvaine buffer (pH 5.5). The thermostability of purified XynRBM26 was determined after pre-incubation of the enzyme in McIlvaine buffer (pH 7.0) at different temperatures (37℃, 45℃, 50℃, and 55℃) without substrate for various periods, with the untreated enzyme defining 100% activity.
The NaCl resistance test of XynRBM26 was determined by enzymatic reaction in the presence of 0.5–5 M NaCl at pH 5.5 and 45℃; without adding NaCl was set as the control group. For the NaCl stability test of XynRBM26, purified enzyme solution was placed in 0.5–5 M NaCl for 1 h at 37℃, and the residual enzyme activity was determined. The enzyme solution without added NaCl was kept at 37℃ for 1 h, set as the control group.
To examine its resistance to proteinases, the purified XynRBM26 was incubated at 37℃ for 60 min with proteinase K (30 U/mg) or trypsin (250 U/mg) at a ratio of 1 : 0.1 (proteinase : xylanase (w/w)), and the residual enzyme activity was measured in McIlvaine buffer (pH 5.5) at 45℃. A similar experiment without proteinase K and trypsin was done as a control experiment.
To investigate the effects of different metal ions and chemical reagents on the purified XynRBM26 activity measured in McIlvaine buffer (pH 5.5) at 45℃, 1 or 10 mM (final concentration) K + , Ca 2+ , Co + , Ni 2+ , Cu 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Mn 2+ , Zn 2+ , Pb 2+ , Ag + , Hg 2+ , EDTA, Tween-80, Triton-100, β-mercaptoethanol, or SDS was individually added to the reaction solution. The reaction without metal ions or chemical reagents was used as a control.
For the kinetic parameter test of XynRBM26, using 0.05%–2% (w/v) beechwood xylan as the substrate, K m , V max , and k cat were determined by the Lineweaver-Burk method at pH 5.5 and 45℃.
- Hydrolysis Products
For the hydrolysis product analysis, 0.5% (w/v) beechwood xylan was incubated at 45℃, pH 5.5, for 10–60 min with 1 U of the purified XynRBM26 in a reaction volume of 1 ml. Enzyme inactivated at 90℃ was used as the control group. Products were analyzed through thin-layer chromatography (TLC) [37] . An aliquot (4 μl) of each sample was spotted on the TLC plates and developed at room temperature with the solvent system of n -butanol/water/acetic acid (2:1:1 (v/v/v)). Oligosaccharides and monosaccharides were located using aniline-diphenylamine-phosphoric acid-acetone reagent [37] . Xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose were used as standards.
- Nucleotide Sequence Accession Numbers
The nucleotide sequences of the 16S rDNA and endoxylanase gene ( XynRBM26 ) were deposited in GenBank under the accession numbers KP677390 and KP677391, respectively.
Results
- Identification of Strains
Based on the BLASTn analysis, 16S rDNA (1, 398 bp) of RBM26 was consistent with those of Massilia aurea strain AP13 (NR_042502), Massilia oculi strain CCUG 43427A (NR_117180), and Massilia timonae strain UR/MT95 (NR_026014) at 99%, 97%, and 97%, respectively. The distance tree created by the neighbor-joining method also revealed the closest phylogenetic position of RBM26 with Massilia strains (Fig. S1). Thus, strain RBM26 was classified into the genus Massilia .
- Gene Clone and Sequence Analysis
XynRBM26 was obtained on the basis of ORF prediction and BLAST comparison of the genomic sequencing data. The full-length XynRBM26 (1,152 bp) gene starts with the putative codon ATG, ends with TGA, and encodes a 383-residue polypeptide with a calculated mass of 43.17 kDa. No signal peptide was discovered. A BLASTp analysis showed that XynRBM26 was most similar to a number of hypothetical proteins and putative endo-1,4-beta-xylanase. XynRBM26 is highly consistent (96%) with the putative protein (WP005666746) from M. timonae in GenBank. The second most consistent protein with XynRBM26 is the endo-1,4-beta-xylanase A precursor (ACN58881) of uncultured bacterium BLR13 from soil microorganism, showing a consistency of 62%. However, the enzymatic properties of this gene are unidentified.
According to multi-alignment of the XynRBM26 protein sequence with others in GenBank ( Fig. 1 ), XynRBM26 has reported conserved regions of GH10 xylanase (DVVNE and TEXD) [3 , 26] . E169 and E288 are predicted to be the catalytic sites.
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Multi-alignment analysis of the XynRBM26 protein sequence.

Identical residues are shaded in black and conserved residues are shaded in gray. The asterisks show the following putative catalytic residues. AAB70918, alkaline thermostable endoxylanase from sp. NG-27; ABN52146, endo-1, 4-beta-xylanase from ATCC 27405; AEE64767, Xyn10A from 8; this study, XynRBM26 protein sequence (KP677391).

- Phylogenetic Analysis
In recent years, several researchers have gained various xylanases from GIT microorganisms of longicorn beetle, cow, goat, and human beings through isolated culture of microbes and metagenomics. Considering the different feeding types and species, xylanase from different animal GITs may differ from one another. A phylogenetic tree was built with XynRBM26 from Massilia sp. RBM26 of R. bieti and GH10 xylanase from GIT of other animals in GenBank ( Fig. 2 ). The results showed that XynRBM26 is closer to xylanase from cow GIT gained through metagenomic technology [33] and could cluster with xylanase from Ampullaria gigas [6] , but has far genetic relationship with xylanase from other GITs.
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Phylogenetic tree of XynRBM26 with GH10 family proteins using the neighbor-joining method.

The bootstrap values ( = 1,000 replicates) are reported as percentages. The scale bar represents the number of changes per amino acid position. Accession numbers are given at the end of each species name. Based on annotation of the sequences in public databases, the sources from which the strains were isolated are indicated in parentheses.

- Heterologous Expression and Purification of XynRBM26
The xylanase gene gained from PCR amplification was ligated with the expression vector pEasy -E2. Thus, the recombinant expression vector containing XynRBM26 was acquired. Positive clone was proven by sequencing, and the activity of xylanase in ultrasonic disruption enchylema was detected. After being purified by nickel-NTA resin, the purified XynRBM26 migrated as a single band on SDS-PAGE with a molecular mass of 45.0 kDa ( Fig. 3 ), which is close to the calculated value (43.17 kDa). The deduced molecular mass of three internal peptides from XynRBM26 (SFRDPAYR, SKPVAHAEAILR, and MGAVEQIEFAFR) matched three peaks of MALDI-TOF/MS (Fig. S2), confirming that the purified enzyme was indeed XynRBM26.
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SDS-PAGE analysis of XynRBM26.

Lanes: M, marker from 35–116 kDa; 1, cell lysate of harboring empty -E2 following IPTG induction; 2, cell lysate from induced transformant harboring -XynRBM26; 3, purified protein.

- Substrate Specificity
Determined at pH 5.5 and 45℃, specific activities of the purified XynRBM26 towards substrates of 0.5% (w/v) oat spelt xylan and beechwood xylan were 20.14 ± 0.4 U/mg and 20.42 ± 0.9 U/mg, respectively. However, no activity of XynRBM26 was detected towards substrates of 0.5% (w/v) barley β-glucan, carboxymethyl cellulose sodium salt, laminarin, microcrystalline cellulose, or 2 mmol/l p -nitrophenyl-β-D-xylopyranoside.
- Enzyme Characterization
When assayed at 37℃, the purified XynRBM26 showed apparent optimal xylanase activity at pH 5.5, and more than 90% of the maximum activity between pH 5.0 and 8.0 ( Fig. 4 A). The enzyme retained more than 80% of the initial activity after incubation in buffers ranging from pH 5.0 to 10.0 at 37℃ for 60 min ( Fig. 4 B). The purified XynRBM26 activity was optimal at 45℃, retaining more than 62% of the maximum activity when assayed at 30℃–50℃, 27% at 20℃, and 10.7% at 10℃. ( Fig. 4 C). After being processed at 37℃, 45℃, and 50℃ for 60 min, XynRBM26 maintained 95.6%, 81.2%, and 59.7% activity, respectively ( Fig. 4 D). Purified XynRBM26 exhibited good salt tolerance, retaining greater than 96% xylanase activity in the presence of 0.5–3 M NaCl ( Fig. 4 E), and more than 100% xylanase activity after 60 min incubation with 0.5–3.5 M NaCl at 45℃ and pH 5.5 ( Fig. 4 F).
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Characterization of purified XynRBM26.

() Effect of pH on xylanase activity. The enzyme activity was determined at 37℃ from pH 3.0–12.0. () pH stability assay. After pre-incubation of the enzyme at pH 3.0–12.0 at 37℃ for 1 h, the enzyme activity was determined in McIlvaine buffer (pH 5.5) at 37℃. () Effect of temperature on XynRBM26 activity measured in McIlvaine buffer (pH 5.5) at 0–70℃. () Thermostability assay. Purified XynRBM26 was pre-incubated in McIlvaine buffer (pH 5.5) at 37℃, 45℃, 50℃, or 55℃, and aliquots were removed at specific time points for the measurement of residual activity at 45℃. () Effect of NaCl on XynRBM26 activity (pH 5.5, 45℃). The activity of the enzyme was measured in the presence of 0–5 M NaCl. () Stability in NaCl. The enzyme was incubated at 37℃ for 1 h with 0–5 M NaCl, and the residual enzyme activity was determined in McIlvaine buffer (pH 5.5) at 45℃. The error bars represent the mean ± SD ( = 3).

XynRBM26 was resistant to trypsin, as it did not lose xylanase activity after incubation with trypsin at 37℃ for 60 min. However, XynRBM26 was moderately resistant to proteinase K, losing 82.4% of xylanase activity at the same treatment condition.
In the presence of different metal ions or chemical reagents ( Table 1 ), the activity of XynRBM26 was completely inhibited by 1 or 10 mM Ag + , SDS, and Hg 2+ . At a concentration of 10 mM, Pb 2+ inhibited XynRBM26 activity completely; activity was strongly inhibited (retaining less than 60% activity) by 10 mM Mn 2+ and Fe 2+ , and partially inhibited (retaining 71.9–75.3% activity) by 10 mM Co 2+ , and Fe 3+ . β-Mercaptoethanol enhanced the activity. The rest of the metal ions or chemical agents exerted no or small influences on XynRBM26 activity.
Effects of metal ions and chemical reagents on the xylanase activity of purified XynRBM26.
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XynRBM26 activity was measured in McIlvaine buffer (pH 5.5) at 45℃. aFinal concentration.
XynRBM26 was quantified by the Bradford method, showing a protein concentration of 0.9786 mg/ml. Based on a Lineweaver-Burk plot, the K m , V max , and k cat values were 9.49 mg/ml, 65.79 μmol/min/mg, and 47.34 /sec, respectively.
- Hydrolysis Products
Hydrolysis products of 0.5% (w/v) beechwood xylan by XynRBM26 were analyzed by TLC ( Fig. 5 ). Xylobiose, xylopentaose, and xylose were released after 10 min and their amounts increased with increasing time of incubation. The result revealed that the purified XynRBM26 was of endo-acting nature.
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Hydrolysis product analysis of beechwood xylan after XynRBM26 treatment for different times.

The reaction system contained 900 μl of 0.5% (w/v) beechwood xylan and 100 μl of appropriately diluted enzyme solution (1 U). Enzyme inactivated at 90℃ was used as the control group. Lanes M: xylohexaose, xylopentaose, xylotetraose, xylotriose, xylobiose, and xylose; 10–60: beechwood xylan hydrolyzed by XynRBM26 for different times (min); CK: beechwood xylan with the inactivated (90℃ for 5 min) XynRBM26.

Discussion
The animal GIT contains a complex community of microbes, whose composition ultimately reflects their coevolution. Non-human primates and human beings have close relationship in system evolution. Hence, studying the microorganism composition in a non-human primate GIT is significantly important to understand the microbial evolution in the human GIT. Enzymes related to lignocelluloses degrading in rumen microorganism of herbivorous animals have recently been widely studied. However, only a few research studies on non-human primates are reported. In this paper, a cellulose-degrading bacterium ( Massilia sp. RBM26) was isolated from feces of R. bieti by using pure culture technique. Based on genomic sequencing of Massilia sp. RBM26, the GH10 xylanase gene XynRBM26 was cloned. Sequence analysis, phylogenetic analysis, and heterologous expression of XynRBM26 were conducted, and a recombinase was identified.
Massilia has extensive sources, including water [8] , soil [17] , and air [27] . However, no research on acquisition of Massilia from animal GIT has been reported yet. To the best of our knowledge, none of the glycosyl hydrolases from Massilia has been characterized for function and none of the GH10 xylanases from Massilia has been found in available literatures and databases. Only a few putative glycosyl hydrolases have been revealed in Massilia genome sequences [9] . This study first reported the identification and characterization of a GH10 xylanase from Massilia sp. According to comparison, the XynRBM26 sequence is novel and has a low consistency (62%) with the endo-1,4-beta-xylanase A precursor from uncultured bacterium BLR13 in GenBank. The phylogenetic analysis revealed that XynRBM26 has a close genetic relationship with GH10 xylanase in cow rumen, whereas far from xylanase from other GITs.
Many known xylanases from the GIT showed optimal activity at pH 5.5–6.5 and 35–45°C ( Table 2 ), which is approximately equivalent to the environmental condition in the animal GIT. Several xylanases are low-temperature active. The xylanase XynAGN16 from the feces of Grus nigricollis showed activity at 0–70°C and retained 27.6% activity at 10°C. After treatment at 37°C for 1 h, the remaining enzyme activity was 52.4% [37] . The xylanase xynGR40 from the environmental DNA of goat rumen contents showed optimal temperature at 30°C, much lower than other xylanases from rumen. After treatment at 55°C for 1 h, the enzyme activity decreased rapidly, losing almost all of the activity after 5 min [32] . Compared with these two xylanase, the xylanase from Massilia sp. RBM26 was more stable at 37°C than XynAGN16 and had a longer half-life at 55°C than xynGR40.
Comparative study of XynRBM26 with other xylanases from the gastrointestinal tract.
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ND : No data.
In previous reports, most of the xylanases were found to be inhibited by Cu 2+ ions [13] . Therefore, Cu 2+ is considered as one of the main adverse factors for xylanase industrial application [35] . However, the activity of XynRBM26 was not markedly affected by Cu 2+ , although the mechanism needs to be further studied.
In the feed additive industry, an endoxylanase showing good proteinase resistance is very attractive from economical and technical view points. Endogenous proteinases are secreted in the digestive tract and exogenous proteinases are commonly added in feeds [36] . Only the XynRBM26 from Massilia sp. RBM26 and xylanase (XynB119) from the gut of Batocera horsfieldi larvae had protease resistance, compared with other xylanases from the GIT ( Table 2 ). XynRBM26 was less resistant to proteinase K than XynB119, which retained more than 91.7% activity after treatment with acid and neutral proteases (half-life of the enzyme at 50°C was ca. 20 min) [38] , but the XynRBM26 had better thermostability. The protease resistance and thermostability of XynRBM26 might make it a candidate for potential applications in the animal feed industry.
Currently, considerable research studies have reported that salt-resistant xylanase has been acquired from seawater and high-salt environments [2 , 12 , 13 , 15 , 16 , 23] . No research on a salt-tolerant xylanase from animal GIT containing a low concentration of salt environment condition had been reported.
XynRBM26 showed the highest activity in 0.5 M NaCl, which is approximately equivalent to seawater salinity. It still retained 98% activity in 2.5 M NaCl, showing its good salt-tolerant ability. The xylanase (XynA) from marine bacterium Glaciecola mesophila KMM 241 could remain 120% of its maximum activity in the presence of 0.5 M NaCl [12] . A xylanase (XynAHJ3) from a saline environment retained 85% of its original activity when 0.5 M NaCl was added to the reaction system [36] . The XynRBM26 showed inferior salt tolerance over XynA, but superior salt tolerance over XynAHJ3. Salt-tolerant xylanases have potential application in the processing of seafood and saline food, which have 0.5 to 2.5 M NaCl, such as marine algae, pickles, and sauce [12 , 15 , 24] , suggesting that XynRBM26 might be applied in high-salt food and seafood processing.
It has been reported that salt-tolerant proteins usually consist of an excess of acidic amino acids, which are sually on the surface of the protein [21] . The acidic amino acids have a high water-binding capacity and could form a salvation shell on the proteins’ surface to keep them being hydrated, thus facilitating their adaptation to the environmental pressure that represents the high salt concentration [7 , 30] . Compared with the amino acid sequence of salt-tolerant endoxylanases ( Table 3 ), the acidic amino acids of XynRBM26 were equal with XynA [12] , and the maximum percentage of hydrophobic amino acids was found in XynRBM26. An excess of acidic amino acids and a deficiency of hydrophobic amino acids could result in structural instability [21] , implying that the structural stability of XynRBM26 is better than endoxylanases from Table 3 .
Comparison of amino acid sequences of salt-tolerant endoxylanases.
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aHydrophobic amino acids: A I L F W V. bThe concentration of NaCl at which the enzymes display the highest activity (NaCl was added to the reaction system).
In conclusion, a GH10 xylanase gene was cloned from Massilia sp. RBM26 for the first time. XynRBM26 is the first reported salt-tolerant xylanase from isolated bacteria in an animal GIT, which could be used in high-salt food and seafood processing and other high-salt environmental biotechnological fields. Considering the animal GIT source, the optimum temperature, and the protease resistance, this xylanase shows great potential for utility in the feedstuff fields.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (31360268 and 31160229).
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