Skip to main content
SpringerLink
Log in

Characterization of a xylanase from a thermophilic strain of Anoxybacillus pushchinoensis A8

  • Published:
Biologia Aims and scope Submit manuscript

Abstract

A facultatively anaerobic, thermophilic, xylanolytic bacterium was isolated from a sample collected from the Diyadin Hot Springs, Turkey. According to morphological, biochemical and molecular identification, this new strain was suggested to be representative of the Anoxybacillus pushchinoensis and it was designated as Anoxybacillus pushchinoensis strain A8. It exhibited 97% similarity to 16S rRNA gene sequence of A. pushchinoensis and 77% DNA homology by DNA-DNA hybridization studies. Q-sepharose and CM-sepharose chromatography was used to purify an extracellular xylanase to >90% purity from this species. The enzyme had a molecular mass of approximately 83 kDa. The enzyme showed optimum activity at pH 6.5 and it was 96% stable over a broad pH range of 6.5–11 for 24 hours. The enzyme had optimum activity at 55°C and it was 100% stable at temperature between 50–60°C up to 24 hours. Kinetic characterization of the enzyme was performed at temperature optima (55°C) and Vmax and K m were found to be 59.88 U/mg protein and 0.909 mg/mL, respectively. Oat spelt xylan but not xylooligosaccharides was degraded by the enzyme and xylose was the only product detected from oat xylan degradation. This suggested that the enzyme was an exo-acting xylanase.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Ahmad S., Scopes R.K., Rees G. & Patel B.K.C. 2000. Saccharococcus caldoxylolyticus sp. nov., an obligately thermophilic, xylose-utilizing, endospore-forming bacterium. Int. J. Syst. Evol. Microbiol. 50: 517–523.

    PubMed  CAS  Google Scholar 

  • Becker P., Abu-Reesh I. & Markossian S. 1997. Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil. Appl. Microbiol. Biotechnol. 48: 184–190.

    Article  PubMed  CAS  Google Scholar 

  • Beffa T., Blanc M., Lyon P.F., Vogt G., Marchiani M., Fischer J.L. & Aragno M. 1996. Isolation of Thermus strains from hot composts (60 to 80°C). Appl. Environ. Microbiol. 62: 1723–1727.

    PubMed  CAS  Google Scholar 

  • Beg O.K., Bhushan B., Kapoor M. & Hoondal G.S. 2000. Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3. J. Ind. Microbiol. Biotechnol. 24: 396–402.

    Article  CAS  Google Scholar 

  • Belduz A.O., Dulger S. & Demirbag Z. 2003. Anoxybacillus gonensis sp. nov., a moderately thermophilic, xylose-utilizing, endospore-forming bacterium. Int. J. Syst. Evol. Microbiol. 53: 1315–1320.

    Article  PubMed  CAS  Google Scholar 

  • Benson D.A., Karsch-Mizrachi I., Lipman D.J., Ostell J. & Wheeler D.L. 2007. GenBank. Nucleic Acids Res. 35 (Database Issue): D21–D25.

    Article  CAS  Google Scholar 

  • Bergquist P.L. & Morgan H.W. 1992. The molecular genetics and biotechnological application of enzyme from extremely thermophilic eubacteria, pp. 44–75. In: Herbert R.A. & Sharp R.J. (eds), Molecular Biology and Biotechnology of Extremophiles, Chapman & Hall, New York.

    Google Scholar 

  • Blanco A., Diaz P., Zueco J., Parascandola P. & Pastor F.I.J.A. 1999. A multidomain xylanase from a Bacillus sp. with a region homologous to thermostabilizing domains of thermophilic enzymes. Microbiology 145: 2163–2170.

    Article  PubMed  CAS  Google Scholar 

  • Brosius J., Palmer M.L., Kennedy P.J. & Noller H.F. 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75: 4801–4805.

    Article  PubMed  CAS  Google Scholar 

  • Charnock S.J., Bolam D.N., Turkenburg J.P., Gilbert H.J., Ferreira L.M.A., Davies G.J. & Fontes C.M.G.A. 2000. The X6 “thermostabilizing” domains of xylanases are carbohydratebinding modules: structure and biochemistry of the Clostridium thermocellum X6b domain. Biochemistry 39: 5013–5021.

    Article  PubMed  CAS  Google Scholar 

  • Coutinho P.M. & Henrissat B. 1999b. The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach, pp. 15–23. In: Genetics, Biochemistry and Ecology of Cellulose Degradation (Ohmiya K., Hayashi K., Sakka K., Kobayashi Y., Karita S. & Kimura T., eds), Uni Publishers Company, Tokyo.

    Google Scholar 

  • De Ley J., Cattoir H. & Reynaerts A. 1970. The quantitative measurement of DNA hybridization from renaturation rates. Eur. J. Biochem. 12: 133–142.

    Article  PubMed  Google Scholar 

  • Dulger S., Demirbag Z. & Belduz A.O. 2004. Anoxybacillus ayderensi ssp. nov. and Anoxybacillus kestanbolensis sp. nov. Int. J. Syst. Evol. Microbiol. 54: 1499–1503.

    Article  PubMed  CAS  Google Scholar 

  • Dupont C., Roberge M., Shareck F., Morosoli R. & Kluepfel D. 1998. Substratebinding domains of glycanases from Streptomyces lividans: characterization of a new family of xylanbinding domains. Biochem. J. 330: 41–45.

    PubMed  CAS  Google Scholar 

  • Escara J.F. & Hutton J.R. 1980. Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: acceleration of the renaturation rate. Biopolymers 19: 1315–1327.

    Article  PubMed  CAS  Google Scholar 

  • Fernandes A.C., Fontes C.M.G.A., Gilbert H.J., Hazlewood G.P. & Fernandes T.H. 1999. Homologous xylanases from Clostridium thermocellum: evidence for bifunctional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes. Biochem. J. 342: 105–110.

    Article  PubMed  CAS  Google Scholar 

  • Gasparic A., Martin J., Daniel A.S. & Flint H.J. 1995. A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and β-(1,4)-xylosidase activities. Appl. Environ. Microbiol. 61: 2958–2964.

    PubMed  CAS  Google Scholar 

  • Gessesse A. 1998. Purification and properties of two thermostable alkaline xylanases from an alkaliphilic Bacillus sp. Appl. Environ. Microbiol. 64: 3533–3535.

    PubMed  CAS  Google Scholar 

  • Gessesse A. & Gashe B.A. 1997. Production of alkaline xylanases by an alkaliphilic Bacillus sp. isolated from na alkaline soda lake. J. Appl. Microbiol. 83: 402–406.

    Article  CAS  Google Scholar 

  • Huss V.A.R., Festl H. & Schleifer K.H. 1983. Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst. Appl. Microbiol. 4: 184–192.

    CAS  Google Scholar 

  • Johnson J.L. 1985. Determination of DNA base composition. Methods Microbiol. 18: 1–29.

    CAS  Google Scholar 

  • Kalogeris E., Christakopoulos P., Kekos D. & Macris B.J. 1998. Studies on the solid-state production of thermostable endoxylanases from Thermoascus aurantiacus: characterization of two isozymes. J. Biotechnol. 6: 155–163.

    Article  Google Scholar 

  • Kambourova M., Mandeva R., Fiume I., Maurelli L., Rossi M. & Morana A. 2006. Hydrolysis of xylan at high temperature by co-action of the xylanase from Anoxybacillus flavithermus BC and the β-xylosidase/α-arabinosidase from Sulfolobus solfataricus Oα. J. Appl. Microbiol. 102: 1586–1593.

    Article  CAS  Google Scholar 

  • Khasin A., Alchanati I. & Shoham Y. 1993. Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl. Environ. Microbiol. 59: 1725–1730.

    PubMed  CAS  Google Scholar 

  • Kubata B.K., Suzuki T., Horitsu H., Kawai K. & Takamizawa K. 1994. Purification and characterization of Aeromonas caviae ME-1 xylanase V, which produces exclusively xylobiose from xylan. Appl. Environ. Microbiol. 60: 531–535.

    PubMed  CAS  Google Scholar 

  • Kubata B.K., Takamizawa K., Kawai K., Suzuki T. & Horitsu H. 1995. Xylanase IV, an exoxylanase of Aeromonas caviae ME-1 which produces xylotetraose as the only low-molecular-weight oligosaccharide from xylan. Appl. Environ. Microbiol. 61: 1666–1668.

    PubMed  CAS  Google Scholar 

  • Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  PubMed  CAS  Google Scholar 

  • Lee D., Koh Y.S., Kim K.J., Kim B.C., Choi H.J., Kim D.S., Suhartono M.T. & Pyun Y.R. 1999. Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1. FEMS Microbiol. Lett. 179: 393–400.

    Article  PubMed  CAS  Google Scholar 

  • Lineweawer H. & Burk D. 1934. The determination of enzyme dissociation constant. J. Amer. Chem. Soc. 56: 658–661.

    Article  Google Scholar 

  • Mandel M. & Marmur J. 1968. Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. Methods Enzymol. 12: 195–206.

    Article  CAS  Google Scholar 

  • Miller G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal. Chem. 31: 426–428.

    Article  CAS  Google Scholar 

  • Pikuta E., Cleland D. & Tang J. 2003. Aerobic growth of Anoxybacillus pushchinensis K1T: emended descriptions of A. pushchinensis and the genus Anoxybacillus. Int. J. Syst. Evol. Microbiol. 53: 1561–1562.

    Article  PubMed  CAS  Google Scholar 

  • Pikuta E., Lysenko A., Chuvilskaya N., Mendrock U., Hippe H., Suzina N., Nikitin D., Osipov G. & Laurinavichus K. 2000. Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavithermus comb. nov. Int. J. Syst. Evol. Microbiol. 50: 2109–2117.

    PubMed  CAS  Google Scholar 

  • Ratanakhanokchai K., Kyu K.L. & Tanticharoen M. 1999. Purification and properties of a xylan-binding endoxylanase from alkaliphilic Bacillus sp. strain K-1. App. Environ. Microbiol. 65: 694–697.

    CAS  Google Scholar 

  • Sneath P.H.A. 1986. Endospore-forming gram-positive rods and cocci, pp. 1104–1207. In Sneath P.H.A., Mair N.S., Sharpe M.S. & Holt J.G. (eds), Bergey’s Manual of Systematic Bacteriology, Vol. 2, Williams & Wilkins, Baltimore.

    Google Scholar 

  • Somogyi M. 1952. Notes on sugar determination. J. Biol. Chem. 195: 19–23.

    CAS  Google Scholar 

  • Sonnleitner B. & Fiechter A. 1983. Advantages of using thermophiles in biotechnological processes: expectations and reality. Trends Biotechnol. 1: 74–80.

    Article  Google Scholar 

  • Stackebrandt E. & Goebel B.M. 1994. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Sys. Bacteriol. 44: 846–849.

    Article  CAS  Google Scholar 

  • Sunna A., Gibbs M.D. & Bergquist P.L. 2000. The thermostabilizing domain, XynA, of Caldibacillus cellulovorans xylanase is a xylan binding domain. Biochem. J. 346: 583–586.

    Article  PubMed  CAS  Google Scholar 

  • Sunna A., Gibbs M.D. & Bergquist P.L. 2001. Identification of novel β-mannan-and β-glucan-binding modules: evidence for a superfamily of carbohydrate-binding modules. Biochem. J. 356: 791–798.

    Article  PubMed  CAS  Google Scholar 

  • Teather R.M. & Wood P.J. 1982. Use of Congo red polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43: 777–780.

    PubMed  CAS  Google Scholar 

  • Touzel J.P., O’Donohue M., Debeire P., Samain E. & Breton C. 2000. Thermobacillus xylanilyticus gen. nov., sp. nov., a new aerobic thermophilic xylan-degrading bacterium isolated from farm soil. Int. J. Syst. Evol. Microbiol. 50: 315–320.

    PubMed  CAS  Google Scholar 

  • Vandamme P., Pot B., Gillis M., De Vos P., Kersters K. & Swings J. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev. 60: 407–438.

    PubMed  CAS  Google Scholar 

  • Wayne L.G., Brenner D.J., Colwell R.R., Grimont P.A.D., Kandler P., Krichevsky M.I., Moore L.H., Moore W.E.C., Murray R.G.E., Stackebrandt E., Starr M.P. & Truper H.G. 1987. International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Sys. Bacteriol. 37: 463–464.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, Faculty of Arts & Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey

    Murat Kacagan, Sabriye Canakci, Kadriye Inan, Dilsat N. Colak & Ali O. Belduz

  2. Microbiology & Molecular Biology Research Laboratory, Department of Biology, Faculty of Arts & Sciences, Rize University, 53100, Rize, Turkey

    Cemal Sandalli

Authors
  1. Murat Kacagan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sabriye Canakci
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cemal Sandalli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kadriye Inan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dilsat N. Colak
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ali O. Belduz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sabriye Canakci.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kacagan, M., Canakci, S., Sandalli, C. et al. Characterization of a xylanase from a thermophilic strain of Anoxybacillus pushchinoensis A8. Biologia 63, 599–606 (2008). https://doi.org/10.2478/s11756-008-0134-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11756-008-0134-8

Key words

Search

Navigation