Skip to main content
SpringerLink
Log in

Engineering the thermostability of Trichoderma reesei endo-1,4-β-xylanase II by combination of disulphide bridges

  • Original Paper
  • Published:
Extremophiles Aims and scope Submit manuscript

Abstract

Disulphide bridges were introduced in different combinations into the N-terminal region and the single α-helix of mesophilic Trichoderma reesei xylanase II (TRX II). We used earlier disulphide-bridge data and designed new disulphide bridges for the combination mutants. The most stable mutant contained two disulphide bridges (between positions 2 and 28 and between positions 110 and 154, respectively) and the mutations N11D, N38E, and Q162H. With a half-life of ~56 h at 65°C, the thermostability of this sevenfold mutant was ~5,000 times higher than that of TRX II, and the half-life was 25 min even at 75°C. The thermostability of this mutant was ~30 times higher than that of the corresponding mutant missing the bridge between positions 2 and 28. The extensive stabilization at two protein regions did not alter the kinetic properties of the sevenfold mutant from that of the wild-type TRX II. The combination of disulphide bridges enhanced significantly the pH-dependent stability in a wide pH range.

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.

Fig. 1
Fig. 2
Fig. 3
Fig. 4a, b
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Arase A, Yomo T, Urabe I, Hata Y, Katsube Y, Okada H (1993) Stabilization of xylanase by random mutagenesis. FEBS Lett 316:123–127

    Article  CAS  PubMed  Google Scholar 

  • Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23:257–270

    Article  CAS  Google Scholar 

  • Betz SF (1993) Disulfide bonds and the stability of globular proteins. Protein Sci 2:1551–1558

    CAS  PubMed  Google Scholar 

  • Biely P, Mislovicova D, Toman R (1985) Soluble chromogenic substrates for the assay of endo-1,4-β-xylanases and endo-1,4-β-glucanases. Anal Biochem 144:142–146

    CAS  PubMed  Google Scholar 

  • Chen Y-L, Tang T-Y, Cheng K-J (2001) Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase. Can J Microbiol 47:1088–1094

    Article  CAS  PubMed  Google Scholar 

  • Daniel RM, Dines M, Petach HH (1996) The denaturation and degradation of stable enzymes at high temperatures. Biochem J 317:1–11

    CAS  PubMed  Google Scholar 

  • Davoodi J, Wakarchuk WW, Surewicz WK, Carey PR (1998) Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant. Protein Sci 7:1538–1544

    CAS  PubMed  Google Scholar 

  • Doran PM (2000) Bioprocess engineering principles. Academic Press, New York

  • Fenel F, Leisola M, Jänis J, Turunen O (2004) A de novo designed N-terminal disulfide bridge stabilizes the Trichoderma reesei endo-1,4-β-xylanase II. J Biotechnol 108:137–143

    Article  CAS  PubMed  Google Scholar 

  • Fushinobu S, Ito K, Konno M, Wagaki T, Matsuzawa H (1998) Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for the catalysis at low pH. Protein Eng 11:1121–1128

    Article  CAS  PubMed  Google Scholar 

  • Georis J, de Lemos Esteves F, Lamotte-Brasseur J, Bougnet V, Devreese B, Giannotta F, Granier B, Frére J-M (2000) An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study. Protein Sci 9:466–475

    CAS  PubMed  Google Scholar 

  • Grüber K, Klintschar G, Hayn M, Schlacher A, Steiner W, Kratky C (1998) Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies. Biochemistry 37:13475–13485

    Article  PubMed  Google Scholar 

  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modelling. Electrophoresis 18:2714–2723

    CAS  PubMed  Google Scholar 

  • Hakulinen N, Turunen O, Jänis J, Leisola M, Rouvinen J (2003) Three-dimensional structures of thermophilic β-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

    CAS  PubMed  Google Scholar 

  • Jänis J, Rouvinen J, Leisola M, Turunen O, Vainiotalo P (2001) Thermostability of endo-1,4-β-xylanase II from Trichoderma reesei studied by electrospray ionization Fourier-transform ion cyclotron resonance MS, hydrogen/deuterium-exchange reactions and dynamic light scattering. Biochem J 356:453–460

    Article  PubMed  Google Scholar 

  • Jänis J, Turunen O, Leisola M, Derrick P, Rouvinen J, 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 (in press)

    Google Scholar 

  • Krengel U, Dijkstra BW (1996) Three-dimensional structure of endo-1,4-β-xylanase I from Aspergillus niger: molecular basis for its low pH optimum. J Mol Biol 263:70–78

    Article  CAS  PubMed  Google Scholar 

  • Kumar PR, Eswaramoorthy S, Vithayathil PJ, Viswamitra MA (2000) The tertiary structure at 1.59 Å resolution and the proposed amino acid sequence of a family-11 xylanase from the thermophilic fungus Paecilomyces varioti Bainier. J Mol Biol 295:581–593

    Article  CAS  PubMed  Google Scholar 

  • Li Y, Coutinho PM, Ford C (1998) Effect on thermostability and catalytic activity of introducing disulfide bonds into Aspergillus awamori glucoamylase. Protein Eng 11:661–667

    Article  CAS  PubMed  Google Scholar 

  • Liu H-L, Wang W-C (2003) Protein engineering to improve the thermostability of glucoamylase from Aspergillus awamori based on molecular dynamics simulations. Protein Eng 16:19–25

    Article  PubMed  Google Scholar 

  • McCarthy AA, Morris DD, Bergquist PL, Baker EN (2000) Structure of XynB, a highly thermostable β-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8 Å resolution. Acta Crystallogr D 56:1367–1375

    Article  PubMed  Google Scholar 

  • Morris DD, Gibbs MD, Chin CW, Koh MH, Wong KKY, Allison RW, Nelson PJ, Bergquist PL (1998) Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on Kraft pulp. Appl Environ Microbiol 64:1759–1765

    CAS  PubMed  Google Scholar 

  • Pace CN (1990) Measuring and increasing protein stability. Trends Biotechnol 8:93–98

    Article  CAS  PubMed  Google Scholar 

  • Pikkemaat MG, Linssen ABM, Berendsen HJC, Janssen DB (2002) Molecular dynamics simulations as a tool for improving protein stability. Protein Eng 15:185–192

    Article  CAS  PubMed  Google Scholar 

  • Prade RA (1996) Xylanases: from biology to biotechnology. Biotechnol Genet Eng Rev 13:101–131

    CAS  PubMed  Google Scholar 

  • Sandgren M, Shaw A, Ropp TH, Wu S, Bott R, Cameron AD, Ståhlberg J, Mitchinson C, Jones AT (2001) The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, cel12A, at 1.9 Å resolution. J Mol Biol 308:295–310

    Article  CAS  PubMed  Google Scholar 

  • Shibuya H, Kaneko S, Hayashi K (2000) Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling. Biochem J 349:651–656

    Article  CAS  PubMed  Google Scholar 

  • Sung WL (2003) Xylanases with enhanced thermophilicity and alkalophilicity. Patent WO 03/046169

  • Tatu U, Murthy SK, Vithayathil PJ (1990) Role of a disulfide cross-link in the conformational stability of a thermostable xylanase. J Protein Chem 9:641–646

    CAS  PubMed  Google Scholar 

  • Tenkanen M, Puls J, Poutanen K (1992) Two major xylanases of Trichoderma reesei. Enzyme Microbiol Technol 14:566–574

    Article  CAS  Google Scholar 

  • Tidor B, Karplus M (1993) The contribution of cross-links to protein stability: a normal mode analysis of the configurational entropy of the native state. Proteins 15:71–79

    CAS  PubMed  Google Scholar 

  • Törrönen A, Rouvinen J (1997) Structural and functional properties of low molecular weight endo-1,4-β-xylanases. J Biotechnol 57:137–149

    Article  PubMed  Google Scholar 

  • Turunen O, Etuaho K, Fenel F, Vehmaanperä J, Wu X, Rouvinen J, Leisola M (2001) A combination of weakly stabilizing mutations with a disulfide bridge in the α-helix region of Trichoderma reesei endo-1,4-β-xylanase II increases the thermal stability through synergism. J Biotechnol 88:37–46

    Article  CAS  PubMed  Google Scholar 

  • Turunen O, Vuorio M, Fenel F, Leisola M (2002) Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-β-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH. Protein Eng 15:141–145

    Article  CAS  PubMed  Google Scholar 

  • Van den Burg B, Vriend G, Veltman OR, Venema G, Eijsink VGH (1998) Engineering an enzyme to resist boiling. Proc Natl Acad Sci USA 95:2056–2060

    Article  PubMed  Google Scholar 

  • Viikari L, Kantelinen A, Sundquist J, Linko M (1994) Xylanases in bleaching: from an idea to the industry. FEMS Microbiol Rev 13:335–350

    Article  CAS  Google Scholar 

  • Vogl T, Brengelmann R, Hinz H-J, Scharf M, Lötzbeyer M, Engels JW (1995) Mechanism of protein stabilization by disulfide bridges: calorimetric unfolding studies on disulfide-deficient mutants of the α-amylase inhibitor Tendamistat. J Mol Biol 254:481–496

    Article  CAS  PubMed  Google Scholar 

  • Wakarchuk WW, Sung WL, Campbell RL, Cunningham A, Watson DC, Yaguchi M (1994) Thermostabilization of the Bacillus circulans xylanase by the introduction of disulfide bonds. Protein Eng 7:1379–1386

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Johanna Aura for technical assistance and Dr. Xiaoyan Wu for assistance in protein purification. Financial support from the Academy of Finland, the National Technology Agency of Finland, TEKES, and the Research Foundation of Helsinki University of Technology is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Laboratory of Bioprocess Engineering, Helsinki University of Technology, P.O. Box 6100, 02015-HUT, Helsinki, Finland

    Hairong Xiong, Matti Leisola & Ossi Turunen

  2. Carbozyme, Ltd., Keilaranta 16, 02150, Espoo, Finland

    Fred Fenel

Authors
  1. Hairong Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fred Fenel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matti Leisola
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ossi Turunen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ossi Turunen.

Additional information

Communicated by G. Antranikian

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xiong, H., Fenel, F., Leisola, M. et al. Engineering the thermostability of Trichoderma reesei endo-1,4-β-xylanase II by combination of disulphide bridges. Extremophiles 8, 393–400 (2004). https://doi.org/10.1007/s00792-004-0400-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00792-004-0400-9

Keywords

Search

Navigation