Molecular and Biochemical Characterization of a Novel Xylanase from Massilia Sp. RBM26 Isolated from the Feces of Rhinopithecus

J. Microbiol. Biotechnol. (2016), 26(1), 9–19 http://dx.doi.org/10.4014/jmb.1504.04021 Research Article Review jmb

Molecular and Biochemical Characterization of a Novel Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti S Bo Xu1,2,3†, Liming Dai1†, Junjun Li1,2,3, Meng Deng1, Huabiao Miao1, Junpei Zhou1,2,3, Yuelin Mu1,2,3, Qian Wu1,2,3, Xianghua Tang1,2,3, Yunjuan Yang1,2,3, Junmei Ding1,2,3, Nanyu Han1,2,3, and Zunxi Huang1,2,3*

1School of Life Science, Yunnan Normal University, Kunming 650500, P.R. China 2Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Kunming 650500, P.R. China 3Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming 650500, P.R. China

Received: April 8, 2015 Revised: July 17, 2015 Xylanases sourced from different bacteria have significantly different enzymatic properties. Accepted: September 17, 2015 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

First published online is the first to report a xylanase (XynRBM26) belonging to the glycosyl hydrolase family (GH10) September 18, 2015 from the genus Massilia. The gene encodes a 383-residue polypeptide (XynRBM26) with the

*Corresponding author highest identity of 62% with the endoxylanase from uncultured bacterium BLR13. The Phone: +86-871-5920830; XynRBM26 expressed in Escherichia coli BL21 is a monomer with a molecular mass of 45.0 kDa. Fax: +86-871-5920952; According to enzymatic characteristic analysis, pH 5.5 is the most appropriate for XynRBM26, E-mail: [email protected] which could maintain more than 90% activity between pH 5.0 and 8.0. Moreover, XynRBM26 † These authors contributed is stable at 37°C and could maintain at least 96% activity after being placed at 37°C for 1 h. equally to this work. 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,

Km, Vmax, and kcat of XynRBM26 to beechwood xylan are 9.49 mg/ml, 65.79 µmol/min/mg, and S upplementary data for this 47.34 /sec, respectively. Considering that XynRBM26 comes from an animal GIT, this xylanase paper are available on-line only at has potential application in feedstuff. Moreover, XynRBM26 is applicable to high-salt food and http://jmb.or.kr. seafood processing, as well as other high-salt environmental biotechnological fields, because pISSN 1017-7825, eISSN 1738-8872 of its high catalytic activity in high-concentration NaCl.

Introduction industrial fields, such as food, feedstuff, wine, papermaking, linen degumming, and fuel production. Xylan is a main component of hemicelluloses in plant cell The gastrointestinal tract (GIT) of herbivorous animals walls. The complete hydrolysis of xylan needs the has rich enzymes related with the degradation of synergistic effect of multiple enzymes such as xylanase and lignocelluloses. However, different animals have different xylosidase. Xylanases mainly include endo-1,4-β-D-xylanase enzymes [5, 14, 29]. Several researchers have recently (E.C. 3.2.1.8), β-D-xylosidase (E.C. 3.2.1.37), and α-L- acquired various xylanases from the GIT of longicorn arabinofuranosidase (E.C. 3.2.1.55) [4]. Endo-1,4-β-D-xylanase beetle, cow rumen, and goat rumen through isolated could hydrolyze the β-1,4 glucosidic bonds of the xylan culture of microbes and metagenomics [38, 33, 32]. As a main chain. This xylanase is the most important enzyme in typical herbivorous primate, Rhinopithecus bieti is fed on the degradation of xylan. Considering its potential to lichen, wild fruit, tender leaves of needle-leaved tree, degrade plant xylan, xylanase is widely used in various overwintering cataphyll, and sunglo [19, 22]. Given the

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different feeding types and species, the GIT of R. bieti may The results showed that XynRBM26 has catalytic activity in contain new microorganism xylanase gene resources high-concentration NaCl and is applicable to high-salt food different from other animals. No report on enzymes in the and seafood processing and other high-salt environmental GIT of R. bieti has been reported yet. biotechnological fields. Moreover, this xylanase has application Xylanase has extensive sources, mainly including bacteria, potential in feedstuff materials because XynRBM26 comes fungus, terrestrial plant tissues, and animal digestive juice. from an animal GIT. Among microorganisms, xylanase mainly comes from Bacillus, Aspergillus, Trichoderma, Ruminococci, and Fibrobacteres Materials and Methods [4]. Potential xylan degrading genes in Massilia were recently discovered through genomic sequencing [9]. However, no Main Reagents and Vectors research on the activity of xylanase has been reported yet. The DNA polymerase and dNTP were bought from TaKaRa (Otsu, A number of previous research demonstrated that xylanase Japan). Beechwood xylan and p-nitrophenyl-β-D-xylopyranoside from different bacterial sources have significantly different were bought from Sigma (St. Louis, MO, USA). Escherichia coli enzymatic properties [10, 18]. Therefore, studying xylanase BL21 and the expression vector pEasy-E2 were bought from TransGen (Beijing, China). Isopropyl-β-D-1-thiogalactopyranoside from different bacterial sources is significant to their (IPTG) was purchased from Amresco (Solon, OH, USA). The applications in different fields. Genomic DNA Clean and Concentration Kit was bought from Salt, playing a key role in food safety and preservation Zymo Research (Orange, CA, USA). The Tureseq DNA Sample by retarding the growth of spoilage microorganisms, is the Preparation Kit was bought from Illumima (San Diego, CA, USA). world’s oldest food additive. Xylanases that are magically Nickel-NTA resin was bought from Qiagen (Valencia, CA, USA). active and stable at high salt concentrations could be used All other chemicals were of analytic grade. in harsh industrial processes, such as food processing and washing [24]. Furthermore, fermentation and materials Microorganism Isolation and Identification processing under high salt condition could reduce cost Massilia sp. RBM26 was extracted from fecal microorganism of because sterilization is unnecessary [24]. In recent years, R. bieti. Feces samples were collected from the National Nature salt-tolerant xylanases have become an interesting research Reserve in Baima Snow Mountain in Weixi County, Yunnan topic. Previous research studied reported that xylanases Province, People’s Republic of China. Two grams of the feces was suspended in 0.7% (w/v) NaCl and spread onto screening agar with salt resistance have been acquired from extreme plates containing 0.2% (w/v) carboxymethyl cellulose sodium environments (seawater and high-salt soil). Occasionally, salt, 0.1% (w/v) peptone, 0.1% (w/v) yeast extract, and 0.02% (w/v) the properties of the enzymes are different from the source Congo red. The pure culture of strain RBM26 was obtained environments, such as a halotolerant cellulase isolated through repeated streaking on the screening agar plates at 30°C. from non extreme soil [31]. In this study, a GH10 salt- Strains were identified on the basis of their 16S rDNA. Two tolerant endoxylanase was revealed from Massilia harbored universal primers of bacteria, namely, 27F (AGAGTTTGATCC in an animal GIT. TGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT), were used Genomic sequencing could comprehensively reveal for 16S rDNA amplification. Amplification conditions included genetic information on microbial genomics. With the rapid 94°C for 5 min, 30 cycles at 94°C for 30 sec, 52°C for 30 sec, and development of sequencing techniques, genomes of 72°C for 2 min; and then, 72°C for 10 min. numerous microorganisms have been sequenced. Based on genomic sequencing, xylanase genes from different Genomic Sequencing We accomplished the genomic sequencing of Massilia sp. RBM26 microorganisms have been cloned, heterologously expressed, in the authors’ laboratory. Library construction, sequencing, and and characterized. Zhou et al. [37] cloned and heterologous data analysis were similar to those reported by Zhou et al. [37]. expressed a xylanase gene with multiple structural domains Library preparation. Genomic DNA of RBM26 was extracted from Arthrobacter sp. GN16, an isolate in feces of Grus using the Tiangen Genomic DNA Isolation Kit (Beijing, China), nigricollis. Bhalla et al. [2] gained a heat-resistant GH10 assessed using NanoDrop-2000 (Thermo Scientific, Waltham, MA, xylanase from Geobacillus sp. WSUCF1 and made heterologous USA), quantified using the Qubit DNA Quantification Kit expression. In the present paper, a GH10 xylanase gene (Invitrogen, Carlsbad, CA, USA), randomly fragmented using the was cloned from Massilia for the first time through genomic Bioruptor sonicator (Diagenode, Liège, Belgium), and purified sequencing. Sequence analysis, phylogenetic analysis, and using the Zymo Genomic DNA Clean & Concentration Kit heterologous expression of this xylanase were conducted. (Orange, CA, USA). Then, the DNA library was prepared using Enzymatic characteristics of its recombinase were analyzed. the Illumina TruSeq DNA Sample Preparation Kit according to the

J. Microbiol. Biotechnol. Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti 11

manufacturer’s instruction (San Diego, CA, USA). After adapter albumin as the standard. The protein within the gel was identified ligation, a library fragment size of 400–600 bp was chosen and using matrix-assisted laser desorption/ionization time-of-flight- PCR enriched. The library quantity and quality were confirmed mass spectrometry (MALDI-TOF/MS) performed by Tianjin Biochip using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). (Tianjin, China). Sequencing. The MiSeq Reagent Kit V2 provided reagents for the cluster amplification and sequencing on a Miseq sequencer Enzyme Assay and Substrate Specificity (Illumina). The activity of XynRBM26 was determined by measuring the Data analysis. Real-time image analysis and base calling were release of reducing sugar or p-nitrophenol from the substrate. The performed using the compatible sequencing software RTA reaction system contained 100 µl of enzyme solution (concentration (Illumina). of XynRBM26: 0.02mg/ml) and 900 µl of McIlvaine buffer (pH 5.5) containing 0.5% (w/v) beechwood xylan, oat spelts xylan, Sequence Analysis and Phylogenetic Analysis carboxymethyl cellulose sodium salt, laminarin and β-gulcan, The open reading frame (ORF) was predicted using microcrystalline cellulose, or 2 mmol/l p-nitrophenyl-β-D- GeneMark.hmm (Ver. 2.4; http://exon.gatech.edu/GeneMark/ xylopyranoside. The substrate was pre-heated for 5 min at 45°C gmhmm2_prok.cgi). DNA comparison and protein comparison and added with enzyme solution for another 10 min reaction. were conducted using BLASTn and BLASTp programs (http:// Then 1.5 ml of DNS was added to end the reaction and boiled for blast.ncbi.nlm.nih.gov/Blast.cgi). The signal peptide was predicted 5 min, or the reaction was stopped with 1.5 ml of 1 mol/l Na2CO3. by SignalP (http://www.cbs.dtu.dk/services/SignalP/). Glucoside The absorption at 540 nm or 405 nm was measured when the hydrolases were classified by InterPro (http://www.ebi.ac.uk/ above mixture was cooled down to room temperature. One unit of interpro/). enzyme activity (U) was defined as the amount of enzyme that Sequences were compared by ClustalX. A phylogenetic tree was produced 1 µmol of xylose or p-nitrophenol equivalent per minute. built by the neighbor-joining algorithm and Poission correction matrix in the MEGA 6.0 software package. The bootstrap value Analysis of Enzymatic Characteristics was 1,000. Characterization of the purified XynRBM26 activity was determined using beechwood xylan as the substrate. The buffer Heterologous Expression of XynRBM26 solutions used were 0.1 mol/l McIlvaine buffer (pH 3.0–8.0), PCR amplification was implemented using XynRBM26F (ATG 0.1 mol/l Tris-HCl (pH 8.0–9.0), and 0.1 mol/l glycine-NaOH (pH ACATCTCGACGCGATAC) and XynRBM26R (GGCTTTACG 9.0–12.0). CATCGGCATCG) as primers and genome DNA of Massilia sp. The optimal pH for the purified XynRBM26 was determined at RBM26 as template. Touchdown-PCR was performed as follows 37°C in buffers with pH ranging from 3.0 to 12.0. The enzyme to amplify the xylanase gene: 94°C for 5 min, and then 20 touchdown stability at different pH values was estimated by measuring the cycles of 94°C for 30 sec, 72°C for 30 sec (decreasing by 1°C each residual enzyme activity after incubating the enzyme solution at cycle), and 72°C for 1.5 min; followed by 10 cycles of 94°C for 37°C for 60 min, with the untreated enzyme defining 100% activity. 30sec, 52°C for 30 sec, and 72°C for 1.5 min, and one final The optimal temperature for purified XynRBM26 activity was extension at 72°C for 7 min. The PCR product was gel purified, determined over the range of 0–70°C in McIlvaine buffer (pH 5.5). ligated to pEasy-E2 vector, transformed into E. coli BL21, and The thermostability of purified XynRBM26 was determined after sequenced by Beijing Genomics Institute (Guangzhou, China). pre-incubation of the enzyme in McIlvaine buffer (pH 7.0) at different temperatures (37°C, 45°C, 50°C, and 55°C) without Purification of Recombinant XynRBM26 substrate for various periods, with the untreated enzyme defining The transformed strains were grown in LB medium (100 µg/ml 100% activity. Amp) until the OD600 reached 0.6–1.0. Protein expression was The NaCl resistance test of XynRBM26 was determined by induced by adding IPTG to 0.05 mM, and the culture was shaken enzymatic reaction in the presence of 0.5–5 M NaCl at pH 5.5 and for 20 h at 20°C. Cells were harvested by centrifugation at 45°C; without adding NaCl was set as the control group. For the 12,000 ×g for 5 min. After being suspended by an appropriate NaCl stability test of XynRBM26, purified enzyme solution was amount of pH 7.0 McIlvaine buffer solution, the collected cells placed in 0.5–5 M NaCl for 1 h at 37°C, and the residual enzyme were disrupted by ultrasonic wave in a low-temperature water activity was determined. The enzyme solution without added bath. The processed intracellular concentrated initial enzyme NaCl was kept at 37°C for 1 h, set as the control group. solution was centrifuged for 12 min at 12,000 ×g. The supernatant To examine its resistance to proteinases, the purified XynRBM26 was extracted and purified into target protein by Nickel-NTA was incubated at 37°C for 60 min with proteinase K (30 U/mg) or resin (XynRBM26; His6-tagged at C terminal). The purified trypsin (250 U/mg) at a ratio of 1 : 0.1 (proteinase : xylanase (w/w)), protein was separated by sodium dodecyl sulfate-polyacrylamide and the residual enzyme activity was measured in McIlvaine gel electrophoresis (12% SDS-PAGE) and the protein concentration buffer (pH 5.5) at 45°C. A similar experiment without proteinase was determined by the Bradford method using bovine serum K and trypsin was done as a control experiment.

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To investigate the effects of different metal ions and chemical XynRBM26 is highly consistent (96%) with the putative reagents on the purified XynRBM26 activity measured in McIlvaine protein (WP005666746) from M. timonae in GenBank. The buffer (pH 5.5) at 45°C, 1 or 10 mM (final concentration) K+, Ca2+, second most consistent protein with XynRBM26 is the + 2+ 2+ 2+ 2+ 3+ 2+ 2+ 2+ + 2+ Co , Ni , Cu , Mg , Fe , Fe , Mn , Zn , Pb , Ag , Hg , EDTA, endo-1,4-beta-xylanase A precursor (ACN58881) of Tween-80, Triton-100, β-mercaptoethanol, or SDS was individually uncultured bacterium BLR13 from soil microorganism, added to the reaction solution. The reaction without metal ions or showing a consistency of 62%. However, the enzymatic chemical reagents was used as a control. properties of this gene are unidentified. For the kinetic parameter test of XynRBM26, using 0.05%–2% According to multi-alignment of the XynRBM26 protein (w/v) beechwood xylan as the substrate, Km, Vmax, and kcat were determined by the Lineweaver-Burk method at pH 5.5 and 45°C. sequence with others in GenBank (Fig. 1), XynRBM26 has reported conserved regions of GH10 xylanase (DVVNE Hydrolysis Products and TEXD) [3, 26]. E169 and E288 are predicted to be the For the hydrolysis product analysis, 0.5% (w/v) beechwood catalytic sites. xylan was incubated at 45°C, pH 5.5, for 10–60 min with 1 U of the purified XynRBM26 in a reaction volume of 1 ml. Enzyme Phylogenetic Analysis inactivated at 90°C was used as the control group. Products were In recent years, several researchers have gained various analyzed through thin-layer chromatography (TLC) [37]. An xylanases from GIT microorganisms of longicorn beetle, aliquot (4 µl) of each sample was spotted on the TLC plates and cow, goat, and human beings through isolated culture of developed at room temperature with the solvent system of n- microbes and metagenomics. Considering the different butanol/water/acetic acid (2:1:1 (v/v/v)). Oligosaccharides and feeding types and species, xylanase from different animal monosaccharides were located using aniline-diphenylamine- GITs may differ from one another. A phylogenetic tree was phosphoric acid-acetone reagent [37]. Xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose were used as standards. built with XynRBM26 from Massilia sp. RBM26 of R. bieti and GH10 xylanase from GIT of other animals in GenBank Nucleotide Sequence Accession Numbers (Fig. 2). The results showed that XynRBM26 is closer to The nucleotide sequences of the 16S rDNA and endoxylanase xylanase from cow GIT gained through metagenomic gene (XynRBM26) were deposited in GenBank under the accession technology [33] and could cluster with xylanase from numbers KP677390 and KP677391, respectively. Ampullaria gigas [6], but has far genetic relationship with xylanase from other GITs. Results Heterologous Expression and Purification of XynRBM26 Identification of Strains The xylanase gene gained from PCR amplification was Based on the BLASTn analysis, 16S rDNA (1, 398 bp) of ligated with the expression vector pEasy-E2. Thus, the RBM26 was consistent with those of Massilia aurea strain recombinant expression vector containing XynRBM26 was AP13 (NR_042502), Massilia oculi strain CCUG 43427A acquired. Positive clone was proven by sequencing, and (NR_117180), and Massilia timonae strain UR/MT95 (NR_026014) the activity of xylanase in ultrasonic disruption enchylema at 99%, 97%, and 97%, respectively. The distance tree created was detected. After being purified by nickel-NTA resin, the by the neighbor-joining method also revealed the closest purified XynRBM26 migrated as a single band on SDS- phylogenetic position of RBM26 with Massilia strains PAGE with a molecular mass of 45.0 kDa (Fig. 3), which is (Fig. S1). Thus, strain RBM26 was classified into the genus close to the calculated value (43.17 kDa). The deduced Massilia. molecular mass of three internal peptides from XynRBM26 (SFRDPAYR, SKPVAHAEAILR, and MGAVEQIEFAFR) Gene Clone and Sequence Analysis matched three peaks of MALDI-TOF/MS (Fig. S2), confirming XynRBM26 was obtained on the basis of ORF prediction that the purified enzyme was indeed XynRBM26. and BLAST comparison of the genomic sequencing data. The full-length XynRBM26 (1,152 bp) gene starts with the Substrate Specificity putative codon ATG, ends with TGA, and encodes a 383- Determined at pH 5.5 and 45°C, specific activities of the residue polypeptide with a calculated mass of 43.17 kDa. purified XynRBM26 towards substrates of 0.5% (w/v) oat No signal peptide was discovered. A BLASTp analysis spelt xylan and beechwood xylan were 20.14 ± 0.4 U/mg showed that XynRBM26 was most similar to a number of and 20.42 ± 0.9 U/mg, respectively. However, no activity of hypothetical proteins and putative endo-1,4-beta-xylanase. XynRBM26 was detected towards substrates of 0.5% (w/v)

J. Microbiol. Biotechnol. Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti 13

Fig. 1. 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 Bacillus sp. NG-27; ABN52146, endo-1, 4-beta-xylanase from Ruminiclostridium thermocellum ATCC 27405; AEE64767, Xyn10A from Ruminococcus albus 8; this study, XynRBM26 protein sequence (KP677391). barley β-glucan, carboxymethyl cellulose sodium salt, the maximum activity when assayed at 30°C–50°C, 27% at laminarin, microcrystalline cellulose, or 2 mmol/l p- 20°C, and 10.7% at 10°C. (Fig. 4C). After being processed at nitrophenyl-β-D-xylopyranoside. 37°C, 45°C, and 50°C for 60 min, XynRBM26 maintained 95.6%, 81.2%, and 59.7% activity, respectively (Fig. 4D). Enzyme Characterization Purified XynRBM26 exhibited good salt tolerance, retaining When assayed at 37°C, the purified XynRBM26 showed greater than 96% xylanase activity in the presence of 0.5– apparent optimal xylanase activity at pH 5.5, and more 3 M NaCl (Fig. 4E), and more than 100% xylanase activity than 90% of the maximum activity between pH 5.0 and 8.0 after 60 min incubation with 0.5–3.5 M NaCl at 45°C and (Fig. 4A). The enzyme retained more than 80% of the initial pH 5.5 (Fig. 4F). activity after incubation in buffers ranging from pH 5.0 to XynRBM26 was resistant to trypsin, as it did not lose 10.0 at 37°C for 60 min (Fig. 4B). The purified XynRBM26 xylanase activity after incubation with trypsin at 37°C for activity was optimal at 45°C, retaining more than 62% of 60 min. However, XynRBM26 was moderately resistant to

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Fig. 2. Phylogenetic tree of XynRBM26 with GH10 family proteins using the neighbor-joining method. The bootstrap values (n = 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.

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Table 1. Effects of metal ions and chemical reagents on the xylanase activity of purified XynRBM26. Relative activity (%) Chemicals 1 mMa 10 mMa Control 100 ± 0.8100 ± 1 Ca2+ 100.1 ± 0.2 99.7 ± 0.8 Ni2+ 101.7 ± 0.4 97.9 ± 1 Mg2+ 101.6 ± 0.9 96.8 ± 0.3 EDTA 96.9 ± 0.4 93.0 ± 0.5 Cu2+ 106.0 ± 0.3 89.5 ± 0.6 Fe3+ 88.4 ± 1 75.3 ± 0.3 Co2+ 99.1 ± 0.7 71.9 ± 0.3 Fe2+ 88.2 ± 0.5 61.8 ± 0.4 Mn2+ 115.1 ± 0.4 50.3 ± 0.8 β-mercaptoethanol 105.5 ± 0.1 111.9 ± 0.2 Fig. 3. SDS-PAGE analysis of XynRBM26. Tween-80 (0.5% (v/v)) 96.8 ± 0.9 107.3 ± 1 Lanes: M, marker from 35–116 kDa; 1, cell lysate of E. coli harboring + empty pEasy-E2 following IPTG induction; 2, cell lysate from induced Na 92.1 ± 0.1 101.4 ± 0.3 transformant harboring pEasy-XynRBM26; 3, purified protein. Zn2+ 100.3 ± 0.3 100.7 ± 0.2 K+ 101.4 ± 0.5 100.3 ± 0.6 Triton-100 (0.5 % (v/v)) 94.9 ± 0.9 100.1 ± 0.5 proteinase K, losing 82.4% of xylanase activity at the same Pb2+ 93.9 ± 0.4 0 treatment condition. Ag+ 0 0 In the presence of different metal ions or chemical SDS 0 0 reagents (Table 1), the activity of XynRBM26 was 2+ completely inhibited by 1 or 10 mM Ag+, SDS, and Hg2+. At Hg 0 0 a concentration of 10 mM, Pb2+ inhibited XynRBM26 activity XynRBM26 activity was measured in McIlvaine buffer (pH 5.5) at 45°C. aFinal concentration. completely; activity was strongly inhibited (retaining less than 60% activity) by 10 mM Mn2+ and Fe2+, and partially inhibited (retaining 71.9–75.3% activity) by 10 mM Co2+, microbes, whose composition ultimately reflects their and Fe3+. β-Mercaptoethanol enhanced the activity. The rest coevolution. Non-human primates and human beings have of the metal ions or chemical agents exerted no or small close relationship in system evolution. Hence, studying the influences on XynRBM26 activity. microorganism composition in a non-human primate GIT XynRBM26 was quantified by the Bradford method, is significantly important to understand the microbial showing a protein concentration of 0.9786 mg/ml. Based on evolution in the human GIT. Enzymes related to a Lineweaver-Burk plot, the Km, Vmax, and kcat values were lignocelluloses degrading in rumen microorganism of 9.49 mg/ml, 65.79 µmol/min/mg, and 47.34 /sec, respectively. herbivorous animals have recently been widely studied. However, only a few research studies on non-human Hydrolysis Products primates are reported. In this paper, a cellulose-degrading Hydrolysis products of 0.5% (w/v) beechwood xylan by bacterium (Massilia sp. RBM26) was isolated from feces of XynRBM26 were analyzed by TLC (Fig. 5). Xylobiose, R. bieti by using pure culture technique. Based on genomic xylopentaose, and xylose were released after 10 min and sequencing of Massilia sp. RBM26, the GH10 xylanase gene their amounts increased with increasing time of incubation. XynRBM26 was cloned. Sequence analysis, phylogenetic The result revealed that the purified XynRBM26 was of analysis, and heterologous expression of XynRBM26 were endo-acting nature. conducted, and a recombinase was identified. Massilia has extensive sources, including water [8], soil Discussion [17], and air [27]. However, no research on acquisition of Massilia from animal GIT has been reported yet. To the best The animal GIT contains a complex community of of our knowledge, none of the glycosyl hydrolases from

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Fig. 4. Characterization of purified XynRBM26. (A) Effect of pH on xylanase activity. The enzyme activity was determined at 37°C from pH 3.0–12.0. (B) pH stability assay. After pre-incubation of the enzyme at pH 3.0–12.0 at 37°C for 1 h, the enzyme activity was determined in McIlvaine buffer (pH 5.5) at 37°C. (C) Effect of temperature on XynRBM26 activity measured in McIlvaine buffer (pH 5.5) at 0–70°C. (D) Thermostability assay. Purified XynRBM26 was pre-incubated in McIlvaine buffer (pH 5.5) at 37°C, 45°C, 50°C, or 55°C, and aliquots were removed at specific time points for the measurement of residual activity at 45°C. (E) Effect of NaCl on XynRBM26 activity (pH 5.5, 45°C). The activity of the enzyme was measured in the presence of 0–5 M NaCl. (F) Stability in NaCl. The enzyme was incubated at 37°C for 1 h with 0–5 M NaCl, and the residual enzyme activity was determined in McIlvaine buffer (pH 5.5) at 45°C. The error bars represent the mean ± SD (n = 3).

Massilia has been characterized for function and none of GenBank. The phylogenetic analysis revealed that XynRBM26 the GH10 xylanases from Massilia has been found in has a close genetic relationship with GH10 xylanase in cow available literatures and databases. Only a few putative rumen, whereas far from xylanase from other GITs. glycosyl hydrolases have been revealed in Massilia genome Many known xylanases from the GIT showed optimal sequences [9]. This study first reported the identification activity at pH 5.5–6.5 and 35–45°C (Table 2), which is and characterization of a GH10 xylanase from Massilia sp. approximately equivalent to the environmental condition According to comparison, the XynRBM26 sequence is novel in the animal GIT. Several xylanases are low-temperature and has a low consistency (62%) with the endo-1,4-beta- active. The xylanase XynAGN16 from the feces of Grus xylanase A precursor from uncultured bacterium BLR13 in nigricollis showed activity at 0–70°C and retained 27.6%

J. Microbiol. Biotechnol. Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti 17

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. In previous reports, most of the xylanases were found to be inhibited by Cu2+ ions [13]. Therefore, Cu2+ is considered as one of the main adverse factors for xylanase industrial application [35]. However, the activity of XynRBM26 was not markedly affected by Cu2+, 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 Fig. 5. Hydrolysis product analysis of beechwood xylan after had better thermostability. The protease resistance and XynRBM26 treatment for different times. thermostability of XynRBM26 might make it a candidate The reaction system contained 900 µl of 0.5% (w/v) beechwood xylan for potential applications in the animal feed industry. and 100 µl of appropriately diluted enzyme solution (1 U). Enzyme Currently, considerable research studies have reported inactivated at 90°C was used as the control group. Lanes M: xylohexaose, xylopentaose, xylotetraose, xylotriose, xylobiose, and that salt-resistant xylanase has been acquired from seawater xylose; 10–60: beechwood xylan hydrolyzed by XynRBM26 for and high-salt environments [2, 12, 13, 15, 16, 23]. No research different times (min); CK: beechwood xylan with the inactivated on a salt-tolerant xylanase from animal GIT containing a (90°C for 5 min) XynRBM26. low concentration of salt environment condition had been reported. XynRBM26 showed the highest activity in 0.5 M NaCl, activity at 10°C. After treatment at 37°C for 1 h, the which is approximately equivalent to seawater salinity. It remaining enzyme activity was 52.4% [37]. The xylanase still retained 98% activity in 2.5 M NaCl, showing its good xynGR40 from the environmental DNA of goat rumen salt-tolerant ability. The xylanase (XynA) from marine contents showed optimal temperature at 30°C, much lower bacterium Glaciecola mesophila KMM 241 could remain 120% than other xylanases from rumen. After treatment at 55°C of its maximum activity in the presence of 0.5 M NaCl [12]. for 1 h, the enzyme activity decreased rapidly, losing A xylanase (XynAHJ3) from a saline environment retained

Table 2. Comparative study of XynRBM26 with other xylanases from the gastrointestinal tract. Sample Name Mol. wt. Optimum pH Optimum T (°C) Specific characterization References Feces of Rhinopithecus bieti XynRBM26 43.17 5.5 45 Tolerance to proteases and salt Present study Gut of Batocera horsfieldi larvae XynB119 35.9 5 50 Tolerance to proteases and SDS [38] Goat rumen xynGR67 43 6 40 ND [32] Holstein cattle rumen XyIn-SH1 39.5 6.5 40 ND [3] Bovine rumen xyn10N1854.5 6.5 35 ND [11] Human gut Xyn10A 40 6 37 ND [25] ND : No data.

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Table 3. Comparison of amino acid sequences of salt-tolerant endoxylanases. Parameters XynRBM26 XynAHJ3 XynA Xyn10A XynA Acidic amino acids (%) 12.02 10.35 12.06 15.6812.88 Hydrophobic amino acids (%)a 39.95 38.69 37.59 33.14 33.1 Highest activity with NaCl (%) 100 <100 120 134 190 Concentration of NaCl (M)b 0.5 None 0.5 0.5 0.4 Reference Present work [36] [12] [1] [15] 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).

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