Properties of an alkali-thermo stable xylanase from Geobacillus thermodenitrificans A333 and applicability in xylooligosaccharides generation

  • Loredana Marcolongo
  • Francesco La Cara
  • Alessandra Morana
  • Anna Di Salle
  • Giovanni del Monaco
  • Susana M. Paixão
  • Luis Alves
  • Elena Ionata
Original Paper


An extracellular thermo-alkali-stable and cellulase-free xylanase from Geobacillus thermodenitrificans A333 was purified to homogeneity by ion exchange and size exclusion chromatography. Its molecular mass was 44 kDa as estimated in native and denaturing conditions by gel filtration and SDS-PAGE analysis, respectively. The xylanase (GtXyn) exhibited maximum activity at 70 °C and pH 7.5. It was stable over broad ranges of temperature and pH retaining 88 % of activity at 60 °C and up to 97 % in the pH range 7.5–10.0 after 24 h. Moreover, the enzyme was active up to 3.0 M sodium chloride concentration, exhibiting at that value 70 % residual activity after 1 h. The presence of other metal ions did not affect the activity with the sole exceptions of K+ that showed a stimulating effect, and Fe2+, Co2+ and Hg2+, which inhibited the enzyme. The xylanase was activated by non-ionic surfactants and was stable in organic solvents remaining fully active over 24 h of incubation in 40 % ethanol at 25 °C. Furthermore, the enzyme was resistant to most of the neutral and alkaline proteases tested. The enzyme was active only on xylan, showing no marked preference towards xylans from different origins. The hydrolysis of beechwood xylan and agriculture-based biomass materials yielded xylooligosaccharides with a polymerization degree ranging from 2 to 6 units and xylobiose and xylotriose as main products. These properties indicate G. thermodenitrificans A333 xylanase as a promising candidate for several biotechnological applications, such as xylooligosaccharides preparation.


Thermo-alkali stable xylanase Geobacillus thermodenitrificans A333 Agro-derived lignocellulosics Xylooligosaccharides 


  1. Anand A, Kumar V, Satyanarayana T (2013) Characteristics of thermostable endoxylanase and β-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues. Extremophiles 17:357–366. doi:10.1007/s00792-013-0524-x CrossRefGoogle Scholar
  2. Archana A, Satyanarayana T (1997) Xylanase production by thermophilic Bacillus licheniformis A99 in solid state fermentation. Enzyme Microb Technol 21:12–17. doi:10.1016/S0141-0229(96)00207-4 CrossRefGoogle Scholar
  3. Bastawde KB (1992) Xylan structure, microbial xylanases, and their mode of action. World J Microbiol Biotechnol 8:353–368. doi:10.1007/BF01198746 CrossRefGoogle Scholar
  4. Biely P, Mislovicová D, Toman R (1985) Soluble chromogenic substrates for the assay of endo-1,4-beta-xylanases and endo-1,4-beta-glucanases. Anal Biochem 144(1):142–146. doi:10.1016/0003-2697(85)90095-8 CrossRefGoogle Scholar
  5. Biely P, Hirsch J, la Grange DC, Van Zyl WH, Prior BA (2000) A chromogenic substrate for β-xylosidase coupled assay of α-glucuronidase. Anal Biochem 286:289–294. doi:10.1006/abio.2000.4810 CrossRefGoogle Scholar
  6. 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 72:248–254CrossRefGoogle Scholar
  7. Canakci S, Inan K, Kacagan M, Belduz AO (2007) Evaluation of arabinofuranosidase and xylanase activities of Geobacillus spp. isolated from some hot springs in Turkey. J Microbiol Biotechnol 17(8):1262–1270Google Scholar
  8. Canakci S, Cevher Z, Inan K, Tokgoz M, Bahar F, Kacagan M, Sal FA, Belduz AO (2012) Cloning, purification and characterization of an alkali-stable endoxylanase from thermophilic Geobacillus sp. 71. World J Microbiol Biotechnol 28:1981–1988. doi:10.1007/s11274-011-1000-3 CrossRefGoogle Scholar
  9. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomic. Nucleic Acids Res 37:D233–D238. doi:10.1093/nar/gkn663 CrossRefGoogle Scholar
  10. Coleri A, Cokmus C, Ozcan B, Akkoc N, Akcelik M (2009) Isolation of alpha-glucosidase-producing thermophilic bacilli from hot springs of Turkey. Mikrobiologiia 78:68–78Google Scholar
  11. Collins T, Gerday C, Feller G (2005) Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 29:3–23. doi:10.1016/j.femsre.2004.06.005 CrossRefGoogle Scholar
  12. Feng L, Wang W, Cheng J, Ren Y, Zhao G, Gao C, Tang Y, Liu X, Han W, Peng X, Liu R, Wang L (2007) Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proc Natl Acad Sci USA 104(13):5602–5607. doi:10.1073/pnas.0609650104 CrossRefGoogle Scholar
  13. Fontes CM, Hall J, Hirst BH, Hazlewood GP, Gilbert HJ (1995) The resistance of cellulases and xylanases to proteolytic inactivation. Appl Microbiol Biotechnol 43:52–57. doi:10.1007/s002530050369 CrossRefGoogle Scholar
  14. Gerasimova J, Kuisiene N (2012) Characterization of the novel xylanase from the thermophilic Geobacillus thermodenitrificans JK11. Mikrobiologiia 81(4):418–424. doi:10.1134/S0026261712040066 Google Scholar
  15. Gessesse A (1998) Purification and properties of two thermostable alkaline xylanases from an alkaliphilic Bacillus sp. Appl Environ Microbiol 64:3533–3535Google Scholar
  16. Guo B, Chen XL, Sun CY, Zhou BC, Zhang YZ (2009) Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-β-1,4-xylanase from marine Glaciecola mesophila KMM 241. Appl Microbiol Biotechnol 84:1107–1115. doi:10.1007/s00253-009-2056-y CrossRefGoogle Scholar
  17. Juturu V, Wu JC (2012) Microbial xylanases: engineering, production and industrial applications. Biotechnol Adv 30(6):1219–1227. doi:10.1016/j.biotechadv.2011.11.006 CrossRefGoogle Scholar
  18. Kaya F, Heitmann JA, Joyce TW (1995) Influence of surfactants on the enzymatic hydrolysis of xylan and cellulose. Tappi J 78:150–157Google Scholar
  19. Khandeparker R, Verma P, Deobagkar D (2011) A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing. New Biotechnol 28(6):814–821. doi:10.1016/j.nbt.2011.08.001 CrossRefGoogle Scholar
  20. Khasin A, Alchanati I, Shoham Y (1993) Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl Environ Microbiol 59(6):1725–1730Google Scholar
  21. Krasuska E, Cardonica C, Tenorio JL, Testa G, Scordia D (2010) Potential land availability for energy crops production in Europe. Biofuels, Bioprod Biorefin 4:658–673. doi:10.1002/bbb.259 CrossRefGoogle Scholar
  22. Kulkarni N, Shendye A, Rao M (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23(4):411–456. doi:10.1111/j.1574-6976.1999.tb00407.x CrossRefGoogle Scholar
  23. Kumar V, Satyanarayana T (2011) Applicability of thermo-alkali-stable and cellulase-free xylanase from a novel thermo-halo-alkaliphilic Bacillus halodurans in producing xylooligosaccharides. Biotechnol Lett 33:2279–2285. doi:10.1007/s10529-011-0698-1 CrossRefGoogle Scholar
  24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature 227:680–685. doi:10.1038/227680a0 CrossRefGoogle Scholar
  25. Li N, Yang P, Wang Y, Luo H, Meng K, Wu N, Fan Y, Yao B (2008) Cloning, expression, and characterization of protease-resistant xylanase from Streptomyces fradiae var. k11. J Microbiol Biotechnol 18:410–416Google Scholar
  26. Liu B, Zhang N, Zhao C, Lin B, Xie L, Huang Y (2012) Characterization of a recombinant thermostable xylanase from hot spring thermophilic Geobacillus sp. TC-W7. J Microbiol Biotechnol 22(10):1388–1394. doi:10.4014/jmb.1203.03045 CrossRefGoogle Scholar
  27. Mamo G, Hatti-Kaul R, Mattiasson B (2007) Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7. Extremophiles 11:169–177. doi:10.1007/s00792-006-0023-4 CrossRefGoogle Scholar
  28. Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262(21):10035–10038Google Scholar
  29. Maurelli L, Ionata E, La Cara F, Morana A (2013) Chestnut shell as unexploited source of fermentable sugars: effect of different pretreatment methods on enzymatic saccharification. Appl Biochem Biotechnol 170:1104–1118. doi:10.1007/s12010-013-0264-5 CrossRefGoogle Scholar
  30. Menon G, Mody K, Keshri J, Jha B (2010) Isolation, purification, and characterization of haloalkaline xylanase from a marine Bacillus pumilus strain, GESF-1. Biotechnol Bioprocess Eng 15:998–1005. doi:10.1007/s12257-010-0116-x CrossRefGoogle Scholar
  31. Morrison D, van Dyk JS, Pletschke BI (2011) The effect of alcohols, lignin and phenolic compounds on the enzyme activity of Clostridium cellulovorans XynA. BioResources 6(3):3132–3141Google Scholar
  32. Moure A, Gullon P, Dominguez H, Parajo JC (2006) Advances in the manufacture, purification and applications of xylooligosaccharides as food additives and nutraceuticals. Process Biochem 41:1913–1923. doi:10.1016/j.procbio.2006.05.011 CrossRefGoogle Scholar
  33. Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153:375–380Google Scholar
  34. Nielsen H, Brunak S, von Heijne G (1999) Machine learning approaches for the prediction of signal peptides and other protein sorting signals. Protein Eng 12:3–9. doi:10.1093/protein/12.1.3 CrossRefGoogle Scholar
  35. Sato Y, Fukuda H, Zhou Y, Mikami S (2010) Contribution of ethanol-tolerant xylanase G2 from Aspergillus oryzae on Japanese sake brewing. J Biosci Bioeng 110:679–683. doi:10.1016/j.jbiosc.2010.07.015 CrossRefGoogle Scholar
  36. Satyanarayana T, Sharma A, Mehta D, Puri AK, Kumar V, Nisha M, Joshi S (2012) Biotechnological applications of biocatalysts from the firmicutes Bacillus and Geobacillus species. In: Satyanarayana T, Johri BN, Prakash A (eds) Microorganisms in sustainable agriculture and biotechnology, part 2. Springer, Dordrecht, pp 343–379CrossRefGoogle Scholar
  37. Scordia D, Cosentino SL, Lee JW, Jeffries TW (2011) Dilute oxalic acid pretreatment for biorefining giant reed (Arundo donax L.). Biomass Bioenergy 35:3018–3024. doi:10.1016/j.biombioe.2011.03.046 CrossRefGoogle Scholar
  38. Sellek GA, Chaudhuri JB (1999) Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb Technol 25:471–482. doi:10.1016/S0141-0229(99)00075-7 CrossRefGoogle Scholar
  39. Sharma A, Adhikari S, Satyanarayana T (2007) Alkali-thermostable and cellulasefree xylanase production by an extreme thermophile Geobacillus thermoleovorans. World J Microbiol Biotechnol 23:483–490. doi:10.1007/s11274-006-9250-1 CrossRefGoogle Scholar
  40. Subramaniyan S, Prema P (2000) Cellulase-free xylanases from Bacillus and other microorganisms. FEMS Microbiol Lett 183:1–7. doi:10.1111/j.1574-6968.2000.tb08925.x CrossRefGoogle Scholar
  41. Tan SS, Li DY, Jiang ZQ, Zhu YP, Shi B, Li LT (2008) Production of xylobiose form the autohydrolysis explosion liquor of corncob using Thermotoga maritima xylanase B (XynB) immobilized on nickel-chelated Eupergit C. Bioresour Technol 99:200–204. doi:10.1016/j.biortech.2006.12.005 CrossRefGoogle Scholar
  42. Timell TE (1967) Recent progress in the chemistry of wood hemicelluloses. Wood Sci Technol 1:45–70. doi:10.1007/BF00592255 CrossRefGoogle Scholar
  43. Tseng MJ, Yap MN, Ratanakhanokchai K, Kyu KL, Chen ST (2002) Purification and characterization of two cellulase free xylanases from an alkaliphilic Bacillus firmus. Enzyme Microb Technol 30:590–595. doi:10.1016/S0141-0229(02)00018-2 CrossRefGoogle Scholar
  44. Vazquez MJ, Alonso JL, Domınguez H, Parajo JC (2000) Xylooligosaccharides: manufacture and applications. Trend Food Sci Technol 11:387–393. doi:10.1016/s0924-2244(01)00031-0 CrossRefGoogle Scholar
  45. Verma D, Satyanarayana T (2012) Cloning, expression and applicability of thermo-alkali-stable xylanase of Geobacillus thermoleovorans in generating xylooligosaccharides from agro-residues. Bioresour Technol 107:333–338. doi:10.1016/j.biortech.2011.12.055 CrossRefGoogle Scholar
  46. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65(1):1–43. doi:10.1128/MMBR.65.1.1-43.2001 CrossRefGoogle Scholar
  47. Viikari L, Kantelinen A, Sundquist J, Linko M (1994) Xylanases in bleaching: from an idea to the industry. FEMS Microbiol Rev 13:335–350. doi:10.1111/j.1574-6976.1994.tb00053.x CrossRefGoogle Scholar
  48. Woldesenbet F, Gupta N, Sharma P (2012) Statistical optimization of the production of a cellulase-free, thermo-alkali-stable, salt- and solvent-tolerant xylanase from Bacillus halodurans by solid state fermentation. Arch Appl Sci Res 4(1):524–535Google Scholar
  49. Wu S, Liu B, Zhang X (2006) Characterization of a recombinant thermostable xylanase from deep-sea thermophilic Geobacillus sp. MT-1 East Pacific. Appl Microbiol Biotechnol 72:1210–1216. doi:10.1007/s00253-006-0416-4 CrossRefGoogle Scholar
  50. Yang R, Xu S, Wang Z, Yang W (2005) Aqueous extraction of corncob xylan and production of xylooligosaccharides. Food Sci Technol LEB 38:677–682. doi:10.1016/j.lwt.2004.07.023 CrossRefGoogle Scholar
  51. Zhou J, Gao Y, Dong Y, Tang X, Li J, Xu B, Mu Y, Wu Q, Huang Z (2012) A novel xylanase with tolerance to ethanol, salt, protease, SDS, heat, and alkali from actinomycete Lechevalieria sp. HJ3. J Ind Microbiol Biotechnol 39:965–975. doi:10.1007/s10295-012-1113-1 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Loredana Marcolongo
    • 1
  • Francesco La Cara
    • 1
  • Alessandra Morana
    • 1
  • Anna Di Salle
    • 1
  • Giovanni del Monaco
    • 1
  • Susana M. Paixão
    • 2
  • Luis Alves
    • 2
  • Elena Ionata
    • 1
  1. 1.Institute of Biosciences and BioresourcesNational Research CouncilNaplesItaly
  2. 2.Laboratório Nacional de Energia e Geologia, Instituto Nacional de Energia e Geologia IPUnidade de BioenergiaLisbonPortugal

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