Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Alkaliphilic Endoxylanase from Lignocellulolytic Microbial Consortium Metagenome for Biobleaching of Eucalyptus Pulp

  • Weerachavangkul, Chawannapak (Institute of Molecular Biosciences, Mahidol University) ;
  • Laothanachareon, Thanaporn (Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology) ;
  • Boonyapakron, Katewadee (Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology) ;
  • Wongwilaiwalin, Sarunyou (Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology) ;
  • Nimchua, Thidarat (Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology) ;
  • Eurwilaichitr, Lily (Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology) ;
  • Pootanakit, Kusol (Institute of Molecular Biosciences, Mahidol University) ;
  • Igarashi, Yasuo (Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo) ;
  • Champreda, Verawat (Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology)
  • Received : 2012.06.19
  • Accepted : 2012.08.10
  • Published : 2012.12.28
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Abstract

Enzymatic pre-bleaching by modification of pulp fibers with xylanases is an attractive approach to reduce the consumption of toxic bleaching chemicals in the paper industry. In this study, an alkaliphilic endoxylanase gene was isolated from metagenomic DNA of a structurally stable thermophilic lignocellulose-degrading microbial consortium using amplification with conserved glycosyl hydrolase family 10 primers and subsequent genome walking. The full-length xylanase showed 78% sequence identity to an endo-${\beta}$-1,4-xylanase of Clostridium phytofermentans and was expressed in a mature form with an N-terminal His6 tag fusion in Escherichia coli. The recombinant xylanase Xyn3F was thermotolerant and alkaliphilic, working optimally at $65-70^{\circ}C$ with an optimal pH at 9-10 and retaining >80% activity at pH 9, $60^{\circ}C$ for 1 h. Xyn3F showed a $V_{max}$ of 2,327 IU/mg and $K_m$ of 3.5 mg/ml on birchwood xylan. Pre-bleaching of industrial eucalyptus pulp with no prior pH adjustment (pH 9) using Xyn3F at 50 IU/g dried pulp led to 4.5-5.1% increase in final pulp brightness and 90.4-102.4% increase in whiteness after a single-step hypochlorite bleaching over the untreated pulp, which allowed at least 20% decrease in hypochlorite consumption to achieve the same final bleaching indices. The alkaliphilic xylanase is promising for application in an environmentally friendly bleaching step of kraft and soda pulps with no requirement for pH adjustment, leading to improved economic feasibility of the process.

Keywords

References

  1. Bajpai, P. 1999. Application of enzymes in the pulp and paper industry. Biotechnol. Prog. 15: 147-157. https://doi.org/10.1021/bp990013k
  2. Betini, J. H., M. Michelin, S. C. Peixoto-Nogueira, J. A. Jorge, H. F. Terenzi, and M. L. Polizeli 2009. Xylanases from Aspergillus niger, Aspergillus niveus and Aspergillus ochraceus produced under solid-state fermentation and their application in cellulose pulp bleaching. Bioprocess Biosyst. Eng. 32: 819-824. https://doi.org/10.1007/s00449-009-0308-y
  3. Buchert, J., M. Tenkanen, A. Kantelinen, and L. Viikari 1994. Application of xylanases in the pulp and paper industry. Bioresour. Technol. 50: 65-72. https://doi.org/10.1016/0960-8524(94)90222-4
  4. Christov, L. P., G. Szakacs, and H. Balakrishnan 1999. Production, partial characterization and use of fungal cellulase-free xylanases in pulp bleaching. Process Biochem. 34: 511-517. https://doi.org/10.1016/S0032-9592(98)00117-4
  5. Collins, T., C. Gerday, and G. Feller 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29: 3-23. https://doi.org/10.1016/j.femsre.2004.06.005
  6. Daniel, R. 2004. The soil metagenome--a rich resource for the discovery of novel natural products. Curr. Opin. Biotechnol. 15: 199-204. https://doi.org/10.1016/j.copbio.2004.04.005
  7. Duarte, M. C. T., A. C. A. Pellegrino, E. P. Portugal, A. N. Ponezi, and T. T. Franco 2000. Characterization of alkaline xylanases from Bacillus pumilus. Braz. J. Microbiol. 31: 90-94.
  8. Duarte, M. C., E. C. da Silva, I. M. de Bulhoes Gomes, A. N. Ponezi, E. P. Portugal, J. R. Vicente, et al. 2003. Xylanhydrolyzing enzyme system from Bacillus pumilus CBMAI 0008 and its effects on Eucalyptus grandis kraft pulp for pulp bleaching improvement. Bioresour. Technol. 88: 9-15. https://doi.org/10.1016/S0960-8524(02)00270-5
  9. El-Shahed, K. Y. I. and M. El-Sakhawy 2003. Improvement of kraft bagasse pulp bleachability by treatment with fungi and their enzymes. IPPTA: Quarterly J. Indian Pulp and Paper Technical Association 15: 45-51.
  10. Fushinobu, S., K. Ito, M. Konno, T. Wakagi, and H. Matsuzawa 1998. Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH. Protein Eng. 11: 1121-1128. https://doi.org/10.1093/protein/11.12.1121
  11. Gessesse, A. 1998. Purification and properties of two thermostable alkaline xylanases from an alkaliphilic Bacillus sp. Appl. Environ. Microbiol. 64: 3533-3535.
  12. Giridhar, P. V. and T. S. Chandra 2010. Production of novel halo-alkali-thermo-stable xylanase by a newly isolated moderately halophilic and alkali-tolerant Gracilibacillus sp. TSCPVG. Proc. Biochem. 45:1730-1737. https://doi.org/10.1016/j.procbio.2010.07.012
  13. Gupta, S., B. Bhushan, and G. S. Hoondal 2000. Isolation, purification and characterization of xylanasefrom Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. J. Appl. Microbiol. 88: 325-334. https://doi.org/10.1046/j.1365-2672.2000.00974.x
  14. Hakulinen, N., O. Turunen, J. Janis, M. Leisola, and J. Rouvinen 2003. Three-dimensional structures of thermophilic beta-1,4- xylanases from Chaetomium thermophilum and Nonomuraea flexuosa. Comparison of twelve xylanases in relation to their thermal stability. Eur. J. Biochem. 270: 1399-1412. https://doi.org/10.1046/j.1432-1033.2003.03496.x
  15. Iefuji, H., M. Chino, M. Kato, and Y. Iimura 1996. Acid xylanase from yeast Cryptococcus sp. S-2: purification, characterization, cloning, and sequencing. Biosci. Biotechnol. Biochem. 60: 1331-1338. https://doi.org/10.1271/bbb.60.1331
  16. Inagaki, K., K. Nakahira, K. Mukai, T. Tamura, and H. Tanaka 1998. Gene cloning and characterization of an acidic xylanase from Acidobacterium capsulatum. Biosci. Biotechnol. Biochem. 62: 1061-1067. https://doi.org/10.1271/bbb.62.1061
  17. Jeya, M., S. Thiagarajan, J. K. Lee, and P. Gunasekaran 2009. Cloning and expression of GH11 xylanase gene from Aspergillus fumigatus MKU1 in Pichia pastoris. J. Biosci. Bioeng. 108: 24-29. https://doi.org/10.1016/j.jbiosc.2009.02.003
  18. Jiang, Z., X. Li, S. Yang, L. Li, Y. Li, and W. Feng 2006. Biobleach boosting effect of recombinant xylanase B from the hyperthermophilic Thermotoga maritima on wheat straw pulp. Appl. Microbiol. Biotechnol. 70: 65-71. https://doi.org/10.1007/s00253-005-0036-4
  19. Kimura, T., J. Ito, A. Kawano, T. Makino, H. Kondo, S. Karita, et al. 2000. Purification, characterization, and molecular cloning of acidophilic xylanase from Penicillium sp.40. Biosci. Biotechnol. Biochem. 64: 1230-1237. https://doi.org/10.1271/bbb.64.1230
  20. Khonzue, P., T. Laothanachareon, N. Rattanaphan, P. Tinnasulanon, S. Apawasin, A. Paemanee, et al. 2011. Optimization of xylanase production from Aspergillus niger for biobleaching of eucalyptus pulp. Biosci. Biotechnol. Biochem. 75: 1129-1134. https://doi.org/10.1271/bbb.110032
  21. Krengel, U. and B. W. Dijkstra 1996. Three-dimensional structure of Endo-1,4-beta-xylanase I from Aspergillus niger: molecular basis for its low pH optimum. J Mol. Biol. 263: 70-78. https://doi.org/10.1006/jmbi.1996.0556
  22. Li, C., Yuzhi, H., Zongze, S., Ling, L., Xiaoluo, H., Pengfu, L., Gaobing, W., Xin, M., and L. Ziduo 2010. Novel alkali-stable, cellulase-free xylanase from deep-sea Kocuria sp. Mn22. J. Microbiol. Biotechnol. 19: 873-880.
  23. Mamo, G., Thunnissen, M., Hatti-Kaul, R., and B. Mattiasson 2009. An alkaline active xylanase: Insights into mechanisms of high pH adaptation. Biochemie 91: 1187-1196. https://doi.org/10.1016/j.biochi.2009.06.017
  24. Medeiros, R. G., F. G. Silva Jr., S. N. Báo, R. Hanada, and E. X. Ferreira Filho 2007. Application of xylanases from Amazon Forest fungal species in bleaching of eucalyptus kraft pulps. Braz. Arch. Biol. Technol. 50: 231-238.
  25. Miller, G. L. 1959. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal. Chem. 31: 426-428. https://doi.org/10.1021/ac60147a030
  26. Ninawe, S. and R. C. Kuhad 2006. Bleaching of wheat strawrich soda pulp with xylanase from a thermoalkaliphilic Streptomyces cyaneus SN32. Bioresour. Technol. 97: 2291-2295. https://doi.org/10.1016/j.biortech.2005.10.035
  27. Paice, M. G., N. Gurnagul, D. H. Page, and L. Jurasek 1992. Mechanism of hemicellulose-directed prebleaching of kraft pulps. Enzyme Microb. Technol. 14: 272-276. https://doi.org/10.1016/0141-0229(92)90150-M
  28. Ragauskas, A. J., K. M. Poll, and A. J. Cesternino 1994. Effects of xylanase pretreatment procedures on nonchlorine bleaching. Enzyme Microb. Technol. 16: 492-495. https://doi.org/10.1016/0141-0229(94)90019-1
  29. Raghukumar, C., U. Muraleedharan, V. R. Gaud, and R. Mishra 2004. Xylanases of marine fungi of potential use for biobleaching of paper pulp. J. Ind. Microbiol. Biotechnol. 31: 433-441. https://doi.org/10.1007/s10295-004-0165-2
  30. Rattanachomsri, U., S. Tanapongpipat, L. Eurwilaichitr, and V. Champreda 2009. Simultaneous non-thermal saccharification of cassava pulp by multi-enzyme activity and ethanol fermentation by Candida tropicalis. J. Biosci. Bioeng. 107: 488-493. https://doi.org/10.1016/j.jbiosc.2008.12.024
  31. Sandrim, V. C., A. C. S. Rizzatti, H. F. Terenzi, J. A. Jorge, A. M. F. Milagres, and M. L. T. M. Polizeli 2005. Purification and biochemical characterization of two xylanases produced by Aspergillus caespitosus and their potential for kraft pulp bleaching. Process Biochem. 40: 1823-1828. https://doi.org/10.1016/j.procbio.2004.06.061
  32. Senthilkumar, S. R., M. Dempsey, C. Krishnan, and P. Gunasekaran 2008. Optimization of biobleaching of paper pulp in an expanded bed bioreactor with immobilized alkali stable xylanase by using response surface methodology. Bioresour. Technol. 99: 7781-7787. https://doi.org/10.1016/j.biortech.2008.01.058
  33. Shedova, E. N., O. V. Berezina, N. A. Lunina, V. V. Zverlov, W. H. Schwartz, and G. A. Velikodvorskaia 2009. Cloning and characterization of a large metagenomic DNA fragment containing glycosyl-hydrolase genes. Mol. Gen. Mikrobiol. Virusol. 11-15.
  34. Shrinivas, D., Savitha, G., Raviranjan, K., and G. R. Naik 2010. A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic bacillus sp. JB 99 suitable for paper and pulp industry: Purification and characterization. Appl. Biochem. Biotechnol. 162: 2049-2057. https://doi.org/10.1007/s12010-010-8980-6
  35. Torronen, A. and J. Rouvinen 1995. Structural comparison of two major endo-1,4-xylanases from Trichoderma reesei. Biochemistry. 34: 847-856. https://doi.org/10.1021/bi00003a019
  36. Vielle, C., and G. J. Zeikus 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65: 1-43. https://doi.org/10.1128/MMBR.65.1.1-43.2001
  37. Voget, S., H. L. Steele, and W. R. Streit 2006. Characterization of a metagenome-derived halotolerant cellulase. J. Biotechnol. 126: 26-36. https://doi.org/10.1016/j.jbiotec.2006.02.011
  38. Warnecke, F., P. Luginbuhl, N. Ivanova, M. Ghassemian, T. H. Richardson, J. T. Stege, et al. 2007. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature. 450: 560-565. https://doi.org/10.1038/nature06269
  39. Warnick, T. A., B. A. Methe, and S. B. Leschine 2002. Clostridium phytofermentans sp. nov., a cellulolytic mesophile from forest soil. Int. J. Syst. Evol. Microbiol. 52: 1155-1160. https://doi.org/10.1099/ijs.0.02125-0
  40. Wongwilaiwalin, S., U. Rattanachomsri, T. Laothanachareon, L. Eurwilaichitr, Y. Igarashi, and V. Champreda 2010. Analysis of a thermophilic lignocellulose degrading microbial consortium and multi-species lignocellulolytic enzyme system. Enzyme Microb. Technol. 47: 283-290. https://doi.org/10.1016/j.enzmictec.2010.07.013
  41. Zhao, J., X. Li, and Y. Qu 2006. Application of enzymes in producing bleached pulp from wheat straw. Bioresour. Technol. 97: 1470-1476. https://doi.org/10.1016/j.biortech.2005.07.012
  42. Zhu, H., F. Qu, and L. H. Zhu 1993. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucleic Acids Res. 21: 5279-5280. https://doi.org/10.1093/nar/21.22.5279

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