Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B

  • Sanni P. Voutilainen
  • Harry Boer
  • Marika Alapuranen
  • Janne Jänis
  • Jari Vehmaanperä
  • Anu Koivula
Biotechnologically Relevant Enzymes and Proteins


Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔTm = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔTm = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔTm = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.


Site-directed mutagenesis Cellulase Saccharomyces cerevisiae expression Protein engineering Cellulose 


  1. Aho S, Olkkonen V, Jalava T, Paloheimo M, Buhler R, Niku-Paavola ML, Bamford DH, Korhola M (1991) Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I. Eur J Biochem 200:643–649CrossRefGoogle Scholar
  2. Baker JO, McCarley JR, Lovett R, Yu CH, Adney WS, Rignall TR, Vinzant TB, Decker SR, Sakon J, Himmel ME (2005) Catalytically enhanced endocellulase Cel5A from Acidothermus cellulolyticus. Appl Biochem Biotechnol 121:129–148CrossRefGoogle Scholar
  3. Betz SF (1993) Disulfide bonds and the stability of globular proteins. Protein Sci 2:1551–1558CrossRefGoogle Scholar
  4. Boer H, Teeri TT, Koivula A (2000) Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters. Biotech Bioeng 69:486–494CrossRefGoogle Scholar
  5. Boer H, Munck N, Natunen J, Wohlfahrt G, Soderlund H, Renkonen O, Koivula A (2004) Differential recognition of animal type beta4-galactosylated and alpha3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum. Glycobiology 14:1303–1313CrossRefGoogle Scholar
  6. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ (2004) Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J 382:769–781CrossRefGoogle Scholar
  7. Divne C, Ståhlberg J, Reinikainen T, Ruohonen L, Pettersson G, Knowles JK, Teeri TT, Jones TA (1994) The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. Science 265:524–528CrossRefGoogle Scholar
  8. Divne C, Ståhlberg J, Teeri TT, Jones TA (1998) High-resolution crystal structures reveal how a cellulose chain is bound in the 50 Å long tunnel of cellobiohydrolase I from Trichoderma reesei. J Mol Biol 275:309–325CrossRefGoogle Scholar
  9. Gilkes NR, Henrissat B, Kilburn DC, Miller RC Jr., Warren RAJ (1991) Domains in microbial beta-1,4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev 55:303–315Google Scholar
  10. Grassick A, Murray PG, Thompson R, Collins CM, Byrnes L, Birrane G, Higgins TM, Tuohy MG (2004) Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii. Eur J Biochem 271:4495–4506CrossRefGoogle Scholar
  11. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723CrossRefGoogle Scholar
  12. Haakana H, Miettinen-Oinonen A, Joutsjoki V, Mäntylä A, Suominen P, Vehmaanperä J (2004) Cloning of cellulase genes from Melanocarpus albomyces and their efficient expression in Trichoderma reesei. Enzyme Microb Techol 34:159–167CrossRefGoogle Scholar
  13. Henrissat B, Bairoch A (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316(Pt 2):695–696Google Scholar
  14. Jänis J, Rouvinen J, Leisola M, Turunen O, Vainiotalo P (2001) Thermostability of endo-1,4-beta-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 356:453–460CrossRefGoogle Scholar
  15. Karhunen T, Mäntylä A, Nevalainen KM, Suominen PL (1993) High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Mol Gen Genet 241:515–522CrossRefGoogle Scholar
  16. Kraulis J, Clore GM, Nilges M, Jones TA, Pettersson G, Knowles J, Gronenborn AM (1989) Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing. Biochemistry 28:7241–7257CrossRefGoogle Scholar
  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685CrossRefGoogle Scholar
  18. Lehmann M, Wyss M (2001) Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution. Curr Opin Biotechnol 12:371–375CrossRefGoogle Scholar
  19. Lever M (1972) A new reaction for colorimetric determination of carbohydrates. Anal Biochem 47:276–279CrossRefGoogle Scholar
  20. Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol Rev 64:461–488CrossRefGoogle Scholar
  21. Matsumura M, Signor G, Matthews BW (1989) Substantial increase of protein stability by multiple disulphide bonds. Nature 342:291–293CrossRefGoogle Scholar
  22. Merino ST, Cherry J (2007) Progress and challenges in enzyme development for biomass utilization. Adv Biochem Engin Biotechnol 108:95–120Google Scholar
  23. Miettinen-Oinonen A, Londesborough J, Joutsjoki V, Lantto R, Vehmaanperä J (2004) Three cellulases from Melanocarpus albomyces with applications in textile industry. Enzyme Microb Technol 34:332–341CrossRefGoogle Scholar
  24. Munoz IG, Ubhayasekera W, Henriksson H, Szabo I, Pettersson G, Johansson G, Mowbray SL, Ståhlberg J (2001) Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 A resolution and homology models of the isozymes. J Mol Biol 314:1097–1111CrossRefGoogle Scholar
  25. Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation-a model system for the study of recombination. Proc Natl Acad Sci 78:6354–6358CrossRefGoogle Scholar
  26. Pace CN (1990) Measuring and increasing protein stability. Trends Biotechnol 8:93–98CrossRefGoogle Scholar
  27. Pace CN, Vajdos F, Fe L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423CrossRefGoogle Scholar
  28. Paloheimo M, Mäntylä A, Kallio J, Suominen P (2003) High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure. Appl Environ Microbiol 69:7073–7082CrossRefGoogle Scholar
  29. Parkkinen T, Koivula A, Vehmaanperä J, Rouvinen J (2008) Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding. Protein Sci 17:1383–1394CrossRefGoogle Scholar
  30. Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164CrossRefGoogle Scholar
  31. Sambrook J, Russel DW (2001) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  32. Schülein M (2000) Protein engineering of cellulases. Biochim Biophys Acta 1543:239–252Google Scholar
  33. Sherman F (1991) Getting started with yeast. Methods Enzymol 194:3–21CrossRefGoogle Scholar
  34. Shoseyov O, Shani Z, Levy I (2006) Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev 70:283–295CrossRefGoogle Scholar
  35. Singh R, Blattler WA, Collinson AR (1993) An amplified assay for thiols based on reactivation of papain. Anal Biochem 213:49–56CrossRefGoogle Scholar
  36. Stals I, Sandra K, Geysens S, Contreras R, Van Beeumen J, Claeyssens M (2004) Factors influencing glycosylation of Trichoderma reesei cellulases. I: postsecretorial changes of the O- and N-glycosylation pattern of Cel7A. Glycobiology 14:713–24CrossRefGoogle Scholar
  37. Suurnäkki A, Tenkanen M, Siika-Aho M, Niku-Paavola M-L, Viikari L, Buchert J (2000) Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp. Cellulose 7:189–209CrossRefGoogle Scholar
  38. Tomme P, Van Tilbeurgh H, Pettersson G, Van Damme J, Vandekerckhove J, Knowles J, Teeri TT, Claeyssens M (1988) Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J Biochem 170:575–581CrossRefGoogle Scholar
  39. Tuohy MG, Walsh DJ, Murray PG, Claeyssens M, Cuffe MM, Savage AV, Coughlan MP (2002) Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii. Biochim Biophys Acta 1596:366–380Google Scholar
  40. Turner P, Mamo G, Karlsson EN (2007) Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb Cell Fact 6:9CrossRefGoogle Scholar
  41. van Tilbeurgh H, Tomme P, Claeyssens M, Bhikhabbai R, Pettersson G (1986) Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei. FEBS Lett 204:223–227CrossRefGoogle Scholar
  42. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43CrossRefGoogle Scholar
  43. von Ossowski I, Ståhlberg J, Koivula A, Piens K, Becker D, Boer H, Harle R, Harris M, Divne C, Mahdi S et al (2003) Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D. J Mol Biol 333:817–829CrossRefGoogle Scholar
  44. Voutilainen SP, Boer H, Linder MB, Puranen T, Rouvinen J, Vehmaanperä J, Koivula A (2007) Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random mutagenesis to improve its thermostability. Enzyme Microb Technol 41:234–243CrossRefGoogle Scholar
  45. Voutilainen SP, Puranen T, Siika-Aho M, Lappalainen A, Alapuranen M, Kallio J, Hooman S, Viikari L, Vehmaanperä J, Koivula A (2008) Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases. Biotechnol Bioeng 101:515–528CrossRefGoogle Scholar
  46. Wohlfahrt G, Pellikka T, Boer H, Teeri TT, Koivula A (2003) Probing pH-dependent functional elements in proteins: modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A. Biochemistry 42:10095–10103CrossRefGoogle Scholar
  47. Xiong H, Fenel F, Leisola M, Turunen O (2004) Engineering the thermostability of Trichoderma reesei endo-1,4-beta-xylanase II by combination of disulphide bridges. Extremophiles 8:393–400CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Sanni P. Voutilainen
    • 1
  • Harry Boer
    • 1
  • Marika Alapuranen
    • 2
  • Janne Jänis
    • 3
  • Jari Vehmaanperä
    • 2
  • Anu Koivula
    • 1
  1. 1.VTT Technical Research Centre of FinlandEspooFinland
  2. 2.ROAL OyRajamäkiFinland
  3. 3.Department of ChemistryUniversity of JoensuuJoensuuFinland

Personalised recommendations