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. Copyright© 2016 by The Korean Society for Microbiology Keywords: Gastrointestinal tract, Massilia, Rhinopithecus bieti, salt tolerant, xylanase and Biotechnology 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 January 2016 ⎪ Vol. 26⎪ No. 1 10 Xu et al. 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