Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Characterization of a Multidomain GH10 Xylanase from Paenibacillus sp. DG-22

  • Lee, Sun Hwa (Department of Biotechnology, Dongguk University) ;
  • Lee, Yong-Eok (Department of Biotechnology, Dongguk University)
  • Received : 2014.07.28
  • Accepted : 2014.08.10
  • Published : 2014.11.28
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Abstract

The xynC gene, which encodes high molecular weight xylanase from Paenibacillus sp. DG-22, was cloned and expressed in Escherichia coli, and its nucleotide sequence was determined. The xynC gene comprised a 4,419bp open reading frame encoding 1,472 amino acid residues, including a 27 amino acid signal sequence. Sequence analysis indicated that XynC is a multidomain enzyme composed of two family 4_9 carbohydrate-binding modules (CBMs), a catalytic domain of family 10 glycosyl hydrolases, a family 9 CBM, and three S-layer homologous domains. Recombinant XynC was purified to homogeneity by heat treatment, followed by Avicel affinity chromatography. SDS-PAGE and zymogram analysis of the purified enzyme identified three active truncated xylanase species. Protein sequencing of these truncated proteins showed that all had identical N-terminal sequences. In the protein characterization, recombinant XynC exhibited optimal activity at pH 6.5 and $65^{\circ}C$ and remained stable at neutral to alkaline pH (pH 6.0-10.0). The xylanase activity of recombinant XynC was strongly inhibited by 1 mM $Cu^{2+}$ and $Hg^{2+}$, whereas it was noticeably enhanced by 10 mM dithiothreitol. The enzyme exhibited strong activity towards xylans, including beechwood xylan and arabinoxylan, whereas it showed no cellulase activity. The hydrolyzed product patterns of birchwood xylan and xylooligosaccharides by thin-layer chromatography confirmed XynC as an endoxylanase.

Keywords

References

  1. Ali MK, Hayashi H, Karita S, Goto M, Kimura T, Sakka K, Ohmiya K. 2001. Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis. Biosci. Biotechnol. Biochem. 65: 41-47. https://doi.org/10.1271/bbb.65.41
  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402. https://doi.org/10.1093/nar/25.17.3389
  3. Bataillon M, Nunes Cardinali AP, Castillon N, Duchiron F. 2000. Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-0. Enzyme Microb. Technol. 26: 187-192. https://doi.org/10.1016/S0141-0229(99)00143-X
  4. Beg QK, Kapoor M, Mahajan L, Hoondal GS. 2001. Microbial xylanases and their industrial applications: a review. Appl. Microbiol. Biotechnol. 56: 326-338. https://doi.org/10.1007/s002530100704
  5. Biely P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3: 286-290. https://doi.org/10.1016/0167-7799(85)90004-6
  6. Boraston AB, Creagh AL, Alam MM, Kormos JM, Tomme P, Haynes CA, et al. 2001. Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A. Biochemistry 40: 6240-6247. https://doi.org/10.1021/bi0101695
  7. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  8. Breccia JD, Sineriz F, Baigori MD, Castro GR, Hatti-Kaul R. 1998. Purification and characterization of a thermostable xylanase from Bacillus amyloliquefaciens. Enzyme Microb. Technol. 22: 42-49. https://doi.org/10.1016/S0141-0229(97)00102-6
  9. Cheng YM, Hong TY, Liu CC, Meng M. 2009. Cloning and functional characterization of a complex endo-$\beta$-1,3-glucanase from Paenibacillus sp. Appl. Microbiol. Biotechnol. 81: 1051- 1061. https://doi.org/10.1007/s00253-008-1617-9
  10. Collins T, Gerday C, Feller G. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29: 3-23. https://doi.org/10.1016/j.femsre.2004.06.005
  11. Feng JX, Karita S, Fujino E, Fujino T, Kimura T, Sakka K, Ohmiya K. 2000. Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product. Biosci. Biotechnol. Biochem. 64: 2614-2624. https://doi.org/10.1271/bbb.64.2614
  12. Gouet P, Robert X, Courcelle E. 2003. ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res. 31: 3320-3323. https://doi.org/10.1093/nar/gkg556
  13. Guillen D, Sanchez S, Rodriguez-Sanoja R. 2010. Carbohydratebinding domains: multiplicity of biological roles. Appl. Microbiol. Biotechnol. 85: 1241-1249. https://doi.org/10.1007/s00253-009-2331-y
  14. Han Y, Agarwal V, Dodd D, Kim J, Bae B, Mackie RI, et al. 2012. Biochemical and structural insights into xylan utilization by the thermophilic bacterium Caldanaeobius polysaccharolyticus. J. Biol. Chem. 287: 34946-34960. https://doi.org/10.1074/jbc.M112.391532
  15. Henrissat B, Bairoch A. 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316: 695-696. https://doi.org/10.1042/bj3160695
  16. Hung KS, Liu SM, Fang TY, Tzou WS, Lin FP, Sun KH, Tang SJ. 2011. Characterization of a salt-tolerant xylanase from Thermoanaerobacterium saccharolyticum NTOU1. Biotechnol. Lett. 33: 1441-1447. https://doi.org/10.1007/s10529-011-0579-7
  17. Ihsanawati, Kumasaka T, Kaneko T, Morokuma C, Yatsunami R, Sato T, et al. 2005. Structural basis of the substrate subsite and the highly thermal stability of xylanase 10B from Thermotoga maritima MSB8. Proteins 61: 999-1009. https://doi.org/10.1002/prot.20700
  18. Ito Y, Tomita T, Roy N, Nakano A, Sugawara-Tomita N, Watanabe S, et al. 2003. Cloning, expression, and cell surface localization of Paenibacillus sp. strain W-61 xylanase 5, a multidomain xylanase. Appl. Environ. Microbiol. 69: 6969-6978. https://doi.org/10.1128/AEM.69.12.6969-6978.2003
  19. Kosugi A, Murashima K, Tamaru Y, Doi RH. 2002. Cellsurface- anchoring role of N-terminal surface layer homology domains of Clostridium cellulovorans EngE. J. Bacteriol. 184: 884-888. https://doi.org/10.1128/jb.184.4.884-888.2002
  20. Kulkarni N, S hendye A , Rao M. 1999. Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Rev. 23: 411-456. https://doi.org/10.1111/j.1574-6976.1999.tb00407.x
  21. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  22. Lee TH, Lim PO, Lee YE. 2007. Cloning, characterization, and expression of xylanase A gene from Paenibacillus sp. DG-22 in Escherichia coli. J. Microbiol. Biotechnol. 17: 29-36.
  23. Lee YE, Lim PO. 2004. Purification and characterization of two thermostable xylanases from Paenibacillus sp. DG-22. J. Microbiol. Biotechnol. 14: 1014-1021.
  24. Mesnage S, Fontaine T, Mignot T, Delepierre M, Mock M, Fouet A. 2000. Bacterial SLH domain proteins are noncovalently anchored to the cell surface via a c onserved mechanism involving wall polysaccharide pyruvylation. EMBO J. 19: 4473-4484. https://doi.org/10.1093/emboj/19.17.4473
  25. Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786. https://doi.org/10.1038/nmeth.1701
  26. Selvaraj T, Kim SK, Kim YH, Jeong YS, Kim YJ, Phuong ND, et al. 2010. The role of carbohydrate-binding module (CBM) repeat of a multimodular xylanase (XynX) from Clostridium thermocellum in cellulose and xylan binding. J. Microbiol. 48: 856-861. https://doi.org/10.1007/s12275-010-0285-5
  27. Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, Li W, et al. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7: 539.
  28. Solomon V, Teplitsky A, Shulami S, Zolotnitsky G, Shoham Y, Shoham G. 2007. Structure-specificity relationships of an intracellular xylanase from Geobacillus stearothermophilus. Acta Crystallogr. D63: 845-859.
  29. St. John FJ, Rice JD, Preston JF. 2006. Paenibacillus sp. strain JDR-2 and XynA1: a novel system for methylglucuronoxylan utilization. Appl. Environ. Microbiol. 72: 1496-1506. https://doi.org/10.1128/AEM.72.2.1496-1506.2006
  30. Subramaniyan S, Prema P. 2002. Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit. Rev. Biotechnol. 22: 33-64. https://doi.org/10.1080/07388550290789450
  31. Sunna A, Antranikian G. 1997. Xylanolytic enzymes from fungi and bacteria. Crit. Rev. Biotechnol. 17: 39-67. https://doi.org/10.3109/07388559709146606
  32. van Roosmalen ML, Geukens N, Jongbloed JDH, Tjalsma H, Dubois JYF, Bron S, et al. 2004. Type I signal peptidases of gram-positive bacteria. Biochim. Biophys. Acta 1694: 279-297. https://doi.org/10.1016/j.bbamcr.2004.05.006
  33. Waeonukul R, Pason P, Kyu KL, Sakka K, Kosugi A, Mori Y, Ratanakhanokchai K. 2009. Cloning, sequencing, and expression of the gene encoding a multidomain endo-$\beta$-1,4- xylanase from Paenibacillus curdlanolyticus B-6, and characterization of the recombinant enzyme. J. Microbiol. Biotechnol. 19: 277-285.
  34. Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol. Microbiol. 15: 431-444. https://doi.org/10.1111/j.1365-2958.1995.tb02257.x
  35. Wong KY, Tan L, Saddler JN. 1988. Multiplicity of $\beta$-1,4 xylanase in microorganism: functions and applications. Microbiol. Rev. 52: 305-317.
  36. Wood PJ, Erfle JD, Teather RM. 1988. Use of complex formation between Congo red and polysaccharide in detection and assay of polysaccharide hydrolases. Methods Enzymol. 160: 59-74. https://doi.org/10.1016/0076-6879(88)60107-8
  37. Zhao Y, Meng K, Luo H, Huang H, Yuan T, Yang P, Yao B. 2013. Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge. World J. Microbiol. Biotechnol. 29: 327-334. https://doi.org/10.1007/s11274-012-1186-z

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