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Purification and characterization of two thermostable cellulase-free xylanases from workers of the termite Macrotermes subhyalinus (Isoptera: Termitidae)

  • Betty Meuwiah Faulet
  • Sébastien NiamkéEmail author
  • Jean Tia Gonnety
  • Lucien Patrice Kouamé
Article

Abstract

Termite workers, Macrotermes subhyalinus (Rambur), produced two cellulase-free xylanases, namely Xyl A and Xyl B. DEAE-Sepharose CL-6B, Sephacryl S-200 HR, CM-Sepharose CL-6B and Phenyl-Sepharose CL-4B chromatographies purified these enzymes. They exhibited molecular masses of 63–66.1 (Xyl A) and 60.7–62.4 (Xyl B) kDa. Both enzymes appeared to be endo-xylanases, which produced oligomers of xylose from xylan and did not hydrolyse them to xylose. They had different optimum pH (pH 4.6–5.0 for Xyl A and pH 5.0 for Xyl B) and different optimum temperatures (60 °C for Xyl A and 55 °C for Xyl B). However, they had the same pH stability (4.0–5.6). Both enzymes were stable at 50 °C for more than 4 h. At a pH ranging from 4.6–5.0 and 60 °C, Xyl A and Xyl B possessed the half-life of 115 and 60 min, respectively. The xylanase activities were stimulated by Na+, Mn2+ and dithiol-reducing agents and were sensitive to Cu2+ and detergent agents. Their enzymatic activity was slightly reduced by the presence of urea at 1% (w/v) concentration. The two enzymes could be used in the presence of organic solvents such as acetone (up to 10% v/v) without loss of activity.

Key words

xylanases cellulase-free thermostable Macrotermes subhyalinus termite worker Macrotermitinae Termitidae 

Mots clés

xylanases sans cellulase thermostable Macrotermes subhyalinus ouvrier de termite Macrotermitinae Termitidae 

Résumé

Les ouvriers du termite Macrotermes subhyalinus (Rambur) produisent deux cellulases sans xylanases, à savoir Xyl A et Xyl B. Ces enzymes ont été purifiées en chromatographies DEAE-Sepharose CL-6B, Sephacryl S-200 HR, CM-Sepharose CL-6B et Phenyl-Sepharose CL-4B. Leurs masses moléculaires sont de 63–66,1 (Xyl A) et de 60,7–62,4 (Xyl B) kDa. Les deux enzymes semblent être des endo-xylanases, qui produisent des oligomères du xylose à partir du xylane mais ne les hydrolisent pas en xylose. Elles ont des pH optima différents (pH 4,6–5,0 pour Xyl A et pH de 5,0 pour Xyl B) et des températures optimales différentes (60°C pour Xyl A et 55°C pour Xyl B). Elles ont cependant le même pH de stabilité (4,0–5,6). Les deux enzymes sont stables à 50°C pendant 4 h. A un pH compris entre 4,6–5,0 et une température de 60°C, Xyl A et Xyl B ont une durée de demivie de 115 et 60 min respectivement. Les activités xylanases sont stimulées par Na+, Mn2+ et des agents réducteurs de type dithiol mais réduites par le Cu2+ et des agents détergents. Leur activité enzymatique est légèrement altérée par la présence d’urée à la concentration de 1% (w/v). Les deux enzymes pourraient être utilisées en présence de solvants organiques tels que l’acétone (jusqu’à 10% v/v) sans perte d’activité.

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References

  1. Alconada T. M. and Martinez M. J. (1994) Purification and characterization of an extracellular endo-1, 4-(ß-xyla-nase from Fusarium oxysporum f. sp. melonis. FEMS Microbiology Letters 118, 305–310.PubMedGoogle Scholar
  2. Baba T., Scinke R. and Nanmori T. (1994) Identification and characterization of clustered genes for thermostable xylan-degrading enzymes, ß-xylosidase and xylanase, of Bacillus stearothermophilus 21. Applied and Environmental Microbiology 151, 2252–2258.Google Scholar
  3. Bataillon M., Nunes Cardinali A.-P., Castillon N. and Duchiron F. (2000) Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-O. Enzyme and Microbial Technology 26, 187–192.CrossRefGoogle Scholar
  4. Bedford M. R. and Classen H. L. (1992) The influence of dietary xylanase on intestinal viscosity and molecular weight distribution of cardohydrates in rye-fed broiler chicks, pp. 361–370. In Xylans and Xylanases (Edited by J. Visser, G. Beldman, M. A. K. van Someren and A. G. J. Voragen). Elsevier, Amsterdam.Google Scholar
  5. Beg Q. R., Kapoor M., Mahajan L. and Hoondal G. S. (2001) Microbial xylanases and their industrial applications: A review. Applied Microbiology and Biotechnology 56, 326–338.CrossRefGoogle Scholar
  6. Bennett C. (1967) Denaturation of polypeptide substrates. Methods in Enzymology 11, 211–213.CrossRefGoogle Scholar
  7. Bernfeld P. (1955) Amylase α and ß, pp. 149–154. In Mehods in Enzymology 11 (Edited by S. P. Colswick and N. O. Kaplan).CrossRefGoogle Scholar
  8. Biely P. (1985) Microbial xylanolytic systems. Trends in Biotechnology 3, 286–290.CrossRefGoogle Scholar
  9. Blum H., Beier H. and Gross B. (1987) Improved silver staining of plant proteins, RNA and DNA in Polyacrylamide gels. Electrophoresis 8, 93–99.CrossRefGoogle Scholar
  10. Brennan Y., Callen W. N, Christoffersen L., Dupree P., Goubet R, Healey S., Hernandez M., Keller M., Li K., Palackal N., Sittenfeld A., Tamayo G., Wells S., Hazlewood G. P., Mathur E. J., Short J. M., Robertson D. E. and Steer B. A. (2004) Unusual microbial xylanases from insect guts. Applied Microbiology and Biotechnology 70, 3609–3618.Google Scholar
  11. Brückner H. (1955) Estimation of monosaccharides by the orcinol-sulphuric acid reaction. Biochemistry Journal 60, 200–205.CrossRefGoogle Scholar
  12. Butler J. H. A. and Buckerfield J. C. (1973) Digestion of lignin by termites. Soil Biology and Biochemistry 11, 507–513.CrossRefGoogle Scholar
  13. Cannio R., Di Prizito N, Rossi M. and Morana A. (2004) A xylan-degrading strain of Sulfolobus solfataricus: Isolation and characterization of the xylanase activity. Extrem 1121, 67–71.Google Scholar
  14. Cesar T. and Mrsa V. (1996) Purification and properties of the xylanase produced by Thermomyces lanuginosus. Enzyme and Microbial Technology 19, 289–296.CrossRefGoogle Scholar
  15. Chandra K. R. and Chandra T. S. (1996) Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn 1. FEMS Microbiology Letters 145, 457–461.CrossRefGoogle Scholar
  16. Cookson L. J. (1992) Studies of lignin degradation in mound material of the termite Nasutitermes exitiosus. Australian Journal of Soil Research 30, 189–193.CrossRefGoogle Scholar
  17. Dekker R. E H. (1983) Bioconversion of hemicelluloses: Aspects of hemicellulase production by Trichoderma resei Qm 9414 and enzymatic saccharification of hemicellulose. Biotechnology and Bioengineering 25, 1127–1146.CrossRefGoogle Scholar
  18. Eriksson K. E. and Wood T. M. (1985) Biodegradation of cellulose, pp. 469–503. In Biosynthesis and Biodegradation of Wood Components (Edited by T. Higuchi). Academic Press, Orlando, Florida.CrossRefGoogle Scholar
  19. Fialho M. B. and Carmona E. C. (2004) Purificaton and characterization of xylanases from Aspergillus giganteusa. Folia Microbiologica 49, 13–18.CrossRefGoogle Scholar
  20. Franco P. E, Ferreira H. M. and Filho E. X. (2004) Production and characterization of hemicellulase activities from Trichoderma harzianum strain T4. Biotechnology and Applied Biochemistry 40, 255–259.CrossRefGoogle Scholar
  21. Gawande P. V. and Kamat M. Y. (1998) Preparation, characterization and application of Aspergillus sp. xylanase immobilized on Eudragit S-100. Journal of Biotechnology 66, 165–175.CrossRefGoogle Scholar
  22. Grassé P. P. (1982) Anatomie, Physiologie, Reproduction des Termites. Masson, Paris. 676 pp.Google Scholar
  23. Gupta S., Bhushan B. and Hoondal G. S. (2000) Isolation, purification and characterization of xylanase from Staphylococcus sp. SG-13 and its application in biobleaching of Kraft pulp. Journal of Applied Microbiology 88, 325–334.CrossRefGoogle Scholar
  24. Higashi M. and Abe T. (1996). Global diversification of termites driven by the evolution of symbiosis and sociality, pp. 83–112. In Biodiversity: An Ecological Perspective. (Edited by Abe T., Levin S. A. and Higashi M.). Springer-Verlag, New York.CrossRefGoogle Scholar
  25. Jänis J., Turunen O., Leisola M., Derrick P. J., Rouvinen J. and Vainiotalo P. (2004) Characterization of mutant xylanases using fourier transform ion cyclotron resonance mass spectrometry: Stabilizing contributions of disulfide bridges and N-terminal extensions. Biochemistry 43, 9556–9566.CrossRefGoogle Scholar
  26. Khasin A., Alchnati I. and Shoam Y. (1993) Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Applied and Environmental Microbiology 59, 1725–1730.PubMedPubMedCentralGoogle Scholar
  27. König H., Fröhlich J., Bechtold M. and Wenzel M. (2002) Diversity and microhabitats of the hindgut flora of termites. Recent Research Developments in Microbiology 146, 125–156.Google Scholar
  28. Kouamé L. P. (2006) Identification de protéases du termite Macrotermes subhyalinus (Rambur) et de son champignon symbiotique Termitomyces sp. Caractérisation des exo-glycosidases thermophiles ou possédant une bonne activité de transglycosylation. Thèse de Doctorat d’Etat, Université Abobo-Adjamé, Abidjan, Côte d’Ivoire. 210 pp.Google Scholar
  29. Kouamé L. P., Kouamé A. F., Niamké S. L., Faulet M. B. and Kamenan A. (2005) Biochemical and catalytic properties of two ß-glycosidases purified from workers of the termite Macrotermes subhyalinus (Isoptera: Termitidae). International Journal of Tropical Insect Science 25, 103–113.CrossRefGoogle Scholar
  30. Kouamé L. P., Niamké S., Diopoh J. and Colas B. (2001) Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus. Biotechnology Letters 23, 1575–1581.CrossRefGoogle Scholar
  31. Kuhad R. C. and Singh A. (1993) Lignocellulosic biotechnology: Current and future prospects. Critical Reviews in Biotechnology 13, 152–172.CrossRefGoogle Scholar
  32. Laemmli U. K. (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefGoogle Scholar
  33. Lama L., Calandrelli V., Gambacorta A. and Nicolaus B. (2004) Purification and characterization of thermostable xylanase and ß-xylosidase by the thermophilic bacterium Bacillus thermantarcticus. Research in Microbiology 155, 283–289.CrossRefGoogle Scholar
  34. Lee S. F., Forsberg C. W. and Gibbins L. N. (1985) Xylanolytic activity of Clostridium acetobutylicum. Applied and Environmental Microbiology 50, 1068–1076.PubMedPubMedCentralGoogle Scholar
  35. Li L., Fröhlich J., Pfeiffer P. and König H. (2003) Termite gut symbiotic Archaezoa are becoming living metabolic fossils. Eukaryotic Cell 2, 1091–1098.CrossRefGoogle Scholar
  36. Li X-L., Zhang Z.-Q., Dean J. F D., Eriksson K.-E. L. and Ljungdahl L. G. (1993) Purification and characterization of a new xylanase (APX-II) from the fungus Aureobasidium pullulans Y-2311-1. Applied and Environmental Microbiology 59, 3212–3218.PubMedPubMedCentralGoogle Scholar
  37. Maat J., Roza M., Verbakel J., Stam H., da Silra M. J. S., Egmond M. R., Hagemans M. D. L., Van Garcom R. F M., Hessing J. G. M., Van Derhondel C. A. M. J. J. and Van Roterdam C. (1992) Xylanases and their application in baking, pp. 349–360. In Xylans and Xylanases (Edited by J. Visser, G. Beldman, M. A. K. Van Someran and A. G. J. Voragen). Elsevier, Amsterdam.Google Scholar
  38. Marrone L. A., McAllister K. and Clarke J. A. (2000) Characterization of the function and activity of domains A, B and C of xylanase C from Fibrobacter succinogenes S85. Protein Engineering 13, 593–601.CrossRefGoogle Scholar
  39. Martin M. M. (1991) The evolution of cellulose digestion in insects. Philosophical Transactions of the Royal Society of London 333, 281–288.CrossRefGoogle Scholar
  40. Matoub M. (1993) La symbiose termite champignon chez Macrotermes bellicosus (Termitidae, Macrotermitinae) Thèse de Doctorat. Université Paris XII Val de Marne. 187 pp.Google Scholar
  41. Matoub M. and Rouland C. (1995) Purification and properties of the xylanases from the termite Macrotermes bellicosus and its symbiotic fungus Termitomyces sp. Comparative Biochemistry and Physiology 112B, 629–635.CrossRefGoogle Scholar
  42. Nakashima K., Watanabe H., Saitoh H., Tokuda G. and Azuma J.-J. (2002) Dual cellulose-digesting system of the wood-feeding termite Coptotermes formosanus. Insect Biochemistry and Molecular Biology 32, 777–784.CrossRefGoogle Scholar
  43. Niamké S., Guionie O., Guével-David L., Moallic C, Dabonne S., Sine J.-P and Colas B. (2003) Physico-chemical and immunological properties and partial amino acid sequencing of a new metalloprotease: Endoprotease Thr-N. Biochimica et Biophysica Acta 1623, 21–28.CrossRefGoogle Scholar
  44. Niamké S., Sine J.-P, Guionie O. and Colas B. (1999) A novel endopeptidase with a strict specificity for threonine residues at the PI, position. Biochemical and Biophysical Research Communications 256, 307–312.CrossRefGoogle Scholar
  45. Noirot C. (1992) From wood to humus feeding: An important trend in termite evolution, pp 107–119. In Biology and Evolution of Social Insects (Edited by J. Billen). Leuven University Press, Leuven, Belgium.Google Scholar
  46. Pountanen K. and Puis J. (1988) Characteristics of Trichoderma reesei ß-xylosidase and its use in the hydrolysis of solubilized xylans. Applied Microbiology and Biotechnology 28, 425–432.CrossRefGoogle Scholar
  47. Rogalski J., Oleszek M. and Tokarzewska-Zadora J. (2001) Purification and characterization of two endo-1, 4-beta-xylanases and a 3-xylosidase from Phlebia radiata. Acta Microbiologica Polonika 50, 117–128.Google Scholar
  48. Rouland C., Braumann A., Keleke S., Labat M., Mora P. and Renoux J. (1990) Endosymbiosis and exosymbio-sis in the fungus-growing termites, pp. 78–82. In Microbiology of Poecilotherms (Edited by R. Lesel). Elsevier Science Publishers, Amsterdam.Google Scholar
  49. Rouland C., Civas A., Renoux J. and Petek F. (1988a) Purification and properties of cellulases from termite Macrotermes mulleri (Termitidae, Macrotermitinae) and its symbiotic fungus Termitomyces sp. Comparative Biochemistry and Physiology 91B, 449–458.Google Scholar
  50. Rouland C., Renoux J. and Petek F. (1988b) Purification and properties of two xylanases from Macrotermes mulleri (Termitidae, Macrotermitinae) and its symbiotic fungus Termitomyces sp. Insect Biochemistry 18, 709–715.CrossRefGoogle Scholar
  51. Shao W. and Wiegel J. (1992) Purification and characterization of a thermostable ß-xylosidase from Thermo-anaerobacter ethanolicus. Journal of Bacteriology 174, 5848–5853.CrossRefGoogle Scholar
  52. Shareck E C. R., Yaguchi M., Morosoli R. and Kluepfel D. (1991) Sequences of three genes specifying xylanases in Streptomyces lividans. Gene 107, 75–82.CrossRefGoogle Scholar
  53. Smith P. K., Krohm R. L, Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke M. N., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150, 76–85.CrossRefGoogle Scholar
  54. Subramaniyan S. and Prema P. (2000) Cellulase-free xylanases from Bacillus and other microorganisms. FEMS Microbiology Letters 183, 1–7.CrossRefGoogle Scholar
  55. Taneja K., Gupta S. and Kuhad R. C. (2002) Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK-99. Bioresource Technology 85, 39–42.CrossRefGoogle Scholar
  56. Viikari L., Kantelineo A., Bundquist J. and Linko M. (1994) Xylanase in bleaching: From an idea to the industry. FEMS Microbiology Reviews 13, 335–350.CrossRefGoogle Scholar
  57. Wong K. K. Y, Tan L. U. L. and Saddler J. N. (1988) Multiplicity of ß-1,4-xylanase in microorganisms: Function and applications. Microbiology Reviews 52, 305–317.Google Scholar
  58. Woodward J. (1984) Xylanase functions, properties and applications. Topics in Enzyme and Fermentation Biotechnology. 178, 9–30.Google Scholar
  59. Wong K. K. Y and Saddler J. N. (1992) Trichoderma xylanases, their properties and purification. Critical Reviews in Biotechnology 12, 413–435.CrossRefGoogle Scholar

Copyright information

© ICIPE 2006

Authors and Affiliations

  • Betty Meuwiah Faulet
    • 1
  • Sébastien Niamké
    • 2
    Email author
  • Jean Tia Gonnety
    • 1
  • Lucien Patrice Kouamé
    • 1
  1. 1.Laboratoire de Biochimie et Technologie des Aliments de l’Unité de Formation et de Recherche en Sciences et Technologie des Aliments de l’Université d’Abobo-AdjaméCôte d’Ivoire
  2. 2.Laboratoire de Biotechnologies, Filière Biochimie-Microbiologie de l’Unité de Formation et de Recherche en Biosciences de l’Université de CocodyCôte d’Ivoire

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