, Volume 17, Issue 3, pp 357–366 | Cite as

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

Original Paper


An extremely thermophilic bacterial isolate that produces a high titer of thermostable endoxylanase and β-xylosidase extracellularly in an inducible manner was identified as Geobacillus thermodenitrificans TSAA1. The distinctive features of this strain are alkalitolerance and halotolerance. The endoxylanase is active over a broad range of pH (5.0–10.0) and temperatures (30–100 °C) with optima at pH 7.5 and 70 °C, while β-xylosidase is optimally active at pH 7.0 and 60 °C. The T 1/2 values of the endoxylanase and β-xylosidase are 30 min at 80 °C, and 180 min at 70 °C, respectively. The endoxylanase activity is stimulated by dithiothreitol, but inhibited strongly by EDAC and Woodward’s reagent K. N-BS and DEPC strongly inhibited β-xylosidase. MALDI-ToF (MS/MS) analysis of tryptic digest of β-xylosidase revealed similarity with that of G. thermodenitrificans NG 80-2, and suggested that this belongs to the GH 52 glycosyl hydrolase super family. The action of endoxylanase on birch wood xylan and agro-residues such as wheat bran and wheat straw liberated xylooligosaccharides similar to endoxylanases of the family 10 glycoside hydrolases, while the enzyme preparation having both endoxylanase and β-xylosidase liberated xylose as main hydrolysis product.


Extreme thermophile Geobacillus thermodenitrificans Endoxylanase Thermostability β-Xylosidase Agro-residues Xylooligosaccharides Xylose 



Authors are grateful to the Ministry of Environment and Forests, Indian Council of Medical Research and Department of Biotechnology, Government of India, New Delhi, for providing financial assistance while carrying out the work presented in the manuscript.

Supplementary material

792_2013_524_MOESM1_ESM.docx (78 kb)
Supplementary material 1 (DOCX 79 kb)


  1. Aachary AA, Prapulla SG (2011) Xylooligosaccharides (XOS) as an emerging prebiotic: microbial synthesis, utilization, structural characterization, bioactive properties and applications. Compr Rev Food Sci Saf 10:2–16CrossRefGoogle Scholar
  2. Adsul MG, Bastawde KB, Gokhale DV (2009) Biochemical characterization of two xylanases from yeast Pseudozyma hubeiensis producing only xylooligosaccharides. Biores Technol 100:6488–6495CrossRefGoogle Scholar
  3. Alvira P, Tomas-Pejo E, Ballesteros M, Negro MJ (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Biores Technol 101:4851–4861CrossRefGoogle Scholar
  4. Archana A, Satyanarayana T (2003) Purification and characterization of a cellulase free xylanase of a moderate thermophile Bacillus licheniformis A99. World J Microbiol Biotechnol 19:53–57CrossRefGoogle Scholar
  5. Bravman T, Zolotnitsky G, Shulami S, Belakhov V, Solomon D, Baasov T, Shoham G, Shoham Y (2001a) Stereochemistry of family 52 glycosyl hydrolases: a β-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme. FEBS Lett 495:39–43PubMedCrossRefGoogle Scholar
  6. Bravman T, Mechaly A, Shulami S, Belakhov V, Baasov T, Shoham G, Shoham Y (2001b) Glutamic acid 160 is the acid–base catalyst of β-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase. FEBS Lett 495:115–119PubMedCrossRefGoogle Scholar
  7. Brüx C, David AB, Shezifi DS, Leon M, Niefind K, Shoham G, Shoham Y, Schomburg D (2006) The structure of an inverting GH43 beta-xylosidase from Geobacillus stearothermophilus with its substrate reveals the role of the three catalytic residues. J Mol Biol 359:97–109PubMedCrossRefGoogle Scholar
  8. 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):1262–1770PubMedGoogle Scholar
  9. 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(5):1981–1988PubMedCrossRefGoogle Scholar
  10. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. J Nucleic Acids Res 37(database issue):D233–D238CrossRefGoogle Scholar
  11. Collin T, Gerday C, Feller G (2005) Xylanase, xylanase families and extremophilic xylanase. FEMS Microbiol Rev 29:3–23CrossRefGoogle Scholar
  12. Cournoyer B, Faure D (2003) Radiation and functional specialization of the family-3 glycoside hydrolases. J Mol Microbiol Biotechnol 5:190–198PubMedCrossRefGoogle Scholar
  13. 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 104:5602–5607PubMedCrossRefGoogle Scholar
  14. Garg N, Tang W, Goto Y, van der Donk WA (2012) Geobacillins: lantibiotics from Geobacillus thermodenitrificans. Proc Natl Acad Sci USA 109:5241–5246PubMedCrossRefGoogle Scholar
  15. Gerasimova J, Kuisiene N (2012) Characterization of the novel xylanase from the thermophilic Geobacillus thermodenitrificans JK11. Microbiology 81:418–424CrossRefGoogle Scholar
  16. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268Google Scholar
  17. Gibson GR, Roberfroid MB (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 87:S287–S291Google Scholar
  18. Gupta S, Kuhad RC, Bhushan B, Hoondal GS (2001) Improved xylanase production from a haloalkalophilic Staphylococcus sp. SG-13 using inexpensive agricultural residues. World J Microbiol Biotechnol 17:5–8CrossRefGoogle Scholar
  19. Hansen SA (1975) TLC method for identification of mono, di and trisaccharides. J Chromatogr 107:224–226CrossRefGoogle Scholar
  20. Khasin A, Alchanati I, Shoham Y (1993) Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl Environ Microbiol 59:1725–1730PubMedGoogle Scholar
  21. Kumar V, Satyanarayana T (2011) Applicability of thermo-alkali-stable and cellulase free xylanase from novel thermo-halo-alkaliphilic Bacillus halodurans in producing xylooligosaccharides. Biotechnol Lett 33:2279–2285PubMedCrossRefGoogle Scholar
  22. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680Google Scholar
  23. Lee YE, Zeikus JG (1993) Genetic organization, sequence and biochemical characterization of recombinant β-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. J Gen Microbiol 139:1235–1243PubMedCrossRefGoogle Scholar
  24. Lorenz W, Wiegel J (1997) Isolation, analysis, and expression of two genes from Thermoanaerobacterium saccharolyticum strain JW/SL-YS485: a β-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity. J Bacteriol 179:5436–5441PubMedGoogle Scholar
  25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275Google Scholar
  26. Mai V, Wiegel J, Lorenz W (2000) Cloning, sequencing, and characterization of the bifunctional xylose-arabinosidase from the anaerobic thermophile Thermoanaerobacter ethanolicus. Gene 247:137–143PubMedCrossRefGoogle Scholar
  27. Mamo G, Hatti-Kaul R, Mattiasson B (2006) A thermostable alkaline active endo β-1-4-xylanase from Bacillus halodurans S7: purification and characterization. Enzyme Microb Technol 39:1492–1498CrossRefGoogle Scholar
  28. Manelius Å, Dahlberg L, Holst O (1994) Some properties of a thermostable β-xylosidase from Rhodothermus marinus. Appl Biochem Biotechnol 44:39–48CrossRefGoogle Scholar
  29. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428CrossRefGoogle Scholar
  30. Mohana S, Shah A, Madamwar D (2008) Xylanase production by Burkholderia sp. DMAX strain under solid state fermentation using distillery spent wash. Biores Technol 99:7553–7564CrossRefGoogle Scholar
  31. Nanmori T, Watanabe T, Shinke R, Kohno A, Kawamura Y (1990) Purification and properties of thermostable xylanase and β-xylosidase produced by a newly isolated Bacillus stearothermophilus strain. J Bacteriol 172:6669–6672PubMedGoogle Scholar
  32. Okazaki W, Akiba T, Horikoshi K, Akahoshi R (1985) Purification and characterization of xylanases from alkaliphilic thermophilic Bacillus sp. Agric Biol Chem 49:2033–2039CrossRefGoogle Scholar
  33. Park YS, Kang SW, Lee JS, Hong SI, Kim SW (2002) Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs. Appl Microbiol Biotechnol 58:761–766PubMedCrossRefGoogle Scholar
  34. Quintero D, Velasco Z, Hurtado-Gomez E, Neira JL, Contreras LM (2007) Isolation and characterization of a thermostable β-xylosidase in the thermophilic bacterium Geobacillus pallidus. Biochim Biophys Acta 1774:510–518PubMedCrossRefGoogle Scholar
  35. Rajoka MI, Khan S (2005) Hyper-production of a thermotolerant β-xylosidase by a deoxy-D glucose and cycloheximide resistant mutant derivative of Kluyveromyces marxianus PPY 125. Electron J Biotechnol 8:177–184CrossRefGoogle Scholar
  36. Roberge M, Shareck F, Morosoli R, Kluepfel D, Dupont C (1997) Characterization of two important histidine residues in the active site of xylanase A from Streptomyces lividans, a family 10 glycanase. Biochemistry 36:7769–7775PubMedCrossRefGoogle Scholar
  37. Ruttersmith LD, Daniel RM (1993) Thermostable α-glucosidase and β-xylosidase from Thermotoga sp. strain FjSS3-B.1. Biochim Biophys Acta 1156:167–172PubMedCrossRefGoogle Scholar
  38. Samanta AK, Jayapal N, Kolte AP, Senani S, Sridhar M, Suresh KP, Sampath KT (2012) Enzymatic production of xylooligosaccharides from alkali solubilized xylan of natural grass (Sehima nervosum). Biores Technol 112:199–205CrossRefGoogle Scholar
  39. Sanchez OJ, Cardona CA (2008) Trends in biotechnological production of fuel ethanol from different feedstocks. Biores Technol 99:5270–5295CrossRefGoogle Scholar
  40. Sá-Pereira P, Paveia H, Costa-Ferreira M, Aires-Barros MR (2003) A new look at xylanases: an overview of purification strategies. Appl Biochem Biotechnol Part B Mol Biotechnol 24:257–281Google Scholar
  41. 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, pp 343–379Google Scholar
  42. Shallom D, Leon M, Bravman T, Ben-David A, Zaide G, Belakhov V, Shoham G, Schomburg D, Baasov T, Shoham Y (2005) Biochemical characterization and identification of the catalytic residues of a family 43 β-d-xylosidase from Geobacillus stearothermophilus T-6. Biochemistry 44:387–397PubMedCrossRefGoogle Scholar
  43. Shao W, Wiegel J (1992) Purification and characterization of a thermostable β-xylosidase from Thermoanaerobacter ethanolicus. J Bacteriol 174:5848–5853PubMedGoogle Scholar
  44. Sharma A, Adhikari S, Satyanarayana T (2007) Alkali-thermostable and cellulase free xylanase production by an extreme thermophile Geobacillus thermoleovorans. World J Microbiol Biotechnol 23:483–490CrossRefGoogle Scholar
  45. Sunna A, Antranikian G (1997) Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol 17:39–67PubMedCrossRefGoogle Scholar
  46. Sunna A, Puls J, Antranikian G (1997) Characterization of the xylanolytic enzyme system of the extremely thermophilic anaerobic bacteria Thermotoga maritima, T. neapolitana, and T. thermarum. Comp Biochem Physiol A Physiol 118:453–461CrossRefGoogle Scholar
  47. Suzuki T, Kitagawa E, Sakakibara F, Ibata K, Usui K, Kawai K (2001) Cloning, expression, and characterization of a family 52 β-xylosidase gene (xysB) of a multiple-xylanase-producing bacterium, Aeromonas caviae ME-1. Biosci Biotechnol Biochem 65:487–494PubMedCrossRefGoogle Scholar
  48. Vafiadi C, Christakopoulos P, Topakas E (2010) Purification, characterization and mass spectrometric identification of two thermophilic xylanases from Sporotrichum thermophile. Process Biochem 45:419–424CrossRefGoogle Scholar
  49. Vazquez MJ, Alonso JL, Dominguez H, Parajo JC (2000) Xylooligosaccharides: manufacture and applications. Trends Food Sci Technol 11:387–393CrossRefGoogle Scholar
  50. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes; sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–42PubMedCrossRefGoogle Scholar
  51. Xu ZH, Bail YL, Xu X, Shi JS, Tao WI (2005) Production of alkali-tolerant cellulase-free xylanase by Pseudomonas sp. UN024 with wheat bran as the main substrate. World J Microbiol Biotechnol 21:575–581CrossRefGoogle Scholar
  52. Xue YM, Shao WL (2004) Expression and characterization of a thermostable β-xylosidase from hyperthermophile Thermotoga maritima. Biotechnol Lett 26:1511–1515PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2013

Authors and Affiliations

  1. 1.Department of MicrobiologyUniversity of Delhi South CampusNew DelhiIndia

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