Applied Microbiology and Biotechnology

, Volume 86, Issue 1, pp 143–154

Characterization and kinetic analysis of a thermostable GH3 β-glucosidase from Penicillium brasilianum

  • Kristian B. R. M. Krogh
  • Paul V. Harris
  • Carsten L. Olsen
  • Katja S. Johansen
  • Jesper Hojer-Pedersen
  • Johan Borjesson
  • Lisbeth Olsson
Biotechnologically Relevant Enzymes and Proteins


A GH3 β-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60°C at pH 4–6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-β-d-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-13C6 as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.


Cellulose hydrolysis Glucose inhibition Purification Fungal expression Kinetics 


  1. Barnett CC, Berka RM, Fowler T (1991) Cloning and amplification of the gene encoding an extracellular β-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates. Biotechnology (N Y) 9:562–567CrossRefGoogle Scholar
  2. Bhatia Y, Mishra S, Bisaria VS (2002) Microbial β-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol 22:375–407CrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  4. Carlsen M (1994) α-amylase production by Aspergillus oryzae. PhD thesis. Department of Biotechnology, The Technical University of DenmarkGoogle Scholar
  5. Chirico WJ, Brown RD Jr (1987) Purification and characterization of a β-glucosidase from Trichoderma reesei. Eur J Biochem 165:333–341CrossRefGoogle Scholar
  6. Christakopoulos P, Goodenough PW, Kekos D, Macris BJ, Claeyssens M, Bhat MK (1994) Purification and characterisation of an extracellular β-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum. Eur J Biochem 224:379–385CrossRefGoogle Scholar
  7. Christensen T, Woeldike H, Boel S, Mortensen SB, Hjortshoej K, Thim L, Hansen MT (1988) High level expression of recombinant genes in Aspergillus oryzae. Nat Biotechnol 6:1419–1422CrossRefGoogle Scholar
  8. Copa-Patino JL, Rodriguez J, Perez-Leblic MI (1990) Purification and properties of a β-glucosidase from Penicillium oxalicum autolysates. FEMS Microbiol Lett 55:191–196CrossRefGoogle Scholar
  9. Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. In: Gilbert HJ, Davies G, Henrissat B, Svensson B (eds) Recent advances in carbohydrate bioengineering. The Royal Society of Chemistry, Cambridge, pp 3–12Google Scholar
  10. Dan S, Marton I, Dekel M, Bravdo BA, He S, Withers SG, Shoseyov O (2000) Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger β-glucosidase. J Biol Chem 275:4973–4980CrossRefGoogle Scholar
  11. Decker CH, Visser J, Schreier P (2000) β-glucosidases from five black Aspergillus species: study of their physico-chemical and biocatalytic properties. J Agric Food Chem 48:4929–4936CrossRefGoogle Scholar
  12. Decker CH, Visser J, Schreier P (2001) β-glucosidase multiplicity from Aspergillus tubingensis CBS 643.92: purification and characterization of four β-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance. Appl Microbiol Biotechnol 55:157–163CrossRefGoogle Scholar
  13. Hidalgo M, Steiner J, Eyzaguirre J (1992) β-glucosidase from Penicillium purpurogenum: purification and properties. Biotechnol Appl Biochem 15:185–191Google Scholar
  14. Hrmova M, Streltsov VA, Smith BJ, Vasella A, Varghese JN, Fincher GB (2005) Structural rationale for low-nanomolar binding of transition state mimics to a family GH3 β-d-glucan glucohydrolase from barley. Biochem 44:16529–16539CrossRefGoogle Scholar
  15. Agency IE (2005) Land use and feedstock availability issues. In: Difiglio C (ed) Biofuels for transport. International Energy Agency, Paris, pp 123–145Google Scholar
  16. Kantham L, Vartak HG, Jagannathan V (1984) β-glucosidase of Penicillium funiculosum.II. Properties and mycelial binding. Biotechnol Bioeng 27:786–791CrossRefGoogle Scholar
  17. Kawai R, Igarashi K, Kitaoka M, Ishii T, Samejima M (2004) Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1–>3)-β-glucosidase from the white-rot fungus Phanerochaete chrysosporium. Carbohydr Res 339:2851–2857CrossRefGoogle Scholar
  18. Krogh KB, Morkeberg A, Jorgensen H, Frisvad JC, Olsson L (2004) Screening genus Penicillium for producers of cellulolytic and xylanolytic enzymes. Appl Biochem Biotechnol 113–116:389–401CrossRefGoogle Scholar
  19. Kubicek CP (1992) The cellulase proteins of Trichoderma reesei: structure, multiplicity, mode of action and regulation of formation. Adv Biochem Eng Biotechnol 45:1–27Google Scholar
  20. Lee YH, Fan LT (1983) Kinetic studies of enzymatic hydrolysis insoluble cellulose. Biotechnol Bioeng 25:939–966CrossRefGoogle Scholar
  21. Lin J, Pillay B, Singh S (1999) Purification and biochemical characteristics of β-d-glucosidase from a thermophilic fungus, Thermomyces lanuginosus-SSBP. Biotechnol Appl Biochem 30:81–87Google Scholar
  22. Mandels M, Weber J (1969) The production of cellulases. Adv Chem Ser 95:394–414Google Scholar
  23. McCarter JD, Withers SG (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol 4:885–892CrossRefGoogle Scholar
  24. Montero MA, Romeu A (1992) Kinetic study on the β-glucosidase-catalysed reaction of Trichoderma viride cellulase. Appl Microbiol Biotechnol 38:350–353CrossRefGoogle Scholar
  25. Murray P, Aro N, Collins C, Grassick A, Penttila M, Saloheimo M, Tuohy M (2004) Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii. Protein Expr Purif 38:248–257CrossRefGoogle Scholar
  26. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6CrossRefGoogle Scholar
  27. Parr SR (1983) Some kinetic properties of the β-d-glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose. Enzyme Microb Technol 5:457–462CrossRefGoogle Scholar
  28. Parry NJ, Beever DE, Owen E, Vandenberghe I, Van BJ, Bhat MK (2001) Biochemical characterization and mechanism of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus. Biochem J 353:117–127CrossRefGoogle Scholar
  29. Reese ET, Levinson HS, Downing MH (1950) Quartermaster culture collection. Farlowia 4:45–86Google Scholar
  30. Riou C, Salmon J-M, Vallier M-J, Günata Z, Barre P (1998) Purification, characterization, and substrate specificity of a novel highly glucose-tolerant β-glucosidase from Aspergillus oryzae. Appl Environ Microbiol 64:3607–3614Google Scholar
  31. Rose TM, Henikoff JG, Henikoff S (2003) CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res 31:3763–3766CrossRefGoogle Scholar
  32. Saha BC, Bothast RJ (1996) Production, purification, and characterization of a highly glucose-tolerant novel β-glucosidase from Candida peltata. Appl Environ Microbiol 62:3165–3170Google Scholar
  33. Saloheimo M, Kuja-Panula J, Ylosmaki E, Ward M, Penttilä M (2002) Enzymatic properties and intracellular localization of the novel Trichoderma reesei β-glucosidase BGLII (Cel1A). Appl Environ Microbiol 68:4546–4553CrossRefGoogle Scholar
  34. Sambrook J, Fritsch EF, Maniatis T (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Plainview, NYGoogle Scholar
  35. Seidle HF, Huber RE (2005) Transglucosidic reactions of the Aspergillus niger family 3 β-glucosidase: qualitative and quantitative analyses and evidence that the transglucosidic rate is independent of pH. Arch Biochem Biophys 436:254–264CrossRefGoogle Scholar
  36. Seidle HF, Marten I, Shoseyov O, Huber RE (2004) Physical and kinetic properties of the family 3 β-glucosidase from Aspergillus niger which is important for cellulose breakdown. Protein J 23:11–23CrossRefGoogle Scholar
  37. Shewale JG (1982) β-Glucosidase: its role in cellulase synthesis and hydrolysis of cellulose. Int J Biochem 14:435–443CrossRefGoogle Scholar
  38. Sinnott ML (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem Rev 90:1171–1202CrossRefGoogle Scholar
  39. Skoog B, Wichman A (1986) Calculation of the isoelectric points of polypeptides from the amino acid composition. Trends Anal Chem 3:82–83CrossRefGoogle Scholar
  40. Sternberg D, Vijayakumar P, Reese ET (1977) β-Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose. Can J Microbiol 23:139–147CrossRefGoogle Scholar
  41. Stokvis E, Rosing H, Beijnen JH (2005) Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? Rapid Commun Mass Spectrom 19:401–407CrossRefGoogle Scholar
  42. Tengborg C, Galbe M, Zacchi G (2001) Influence of enzyme loading and physical parameters on the enzymatic hydrolysis of steam-pretreated softwood. Biotechnol Prog 17:110–117CrossRefGoogle Scholar
  43. Tu M, Zhang X, Kurabi A, Gilkes N, Mabee W, Saddler J (2006) Immobilization of β-glucosidase on Eupergit C for lignocellulose hydrolysis. Biotechnol Lett 28:151–156CrossRefGoogle Scholar
  44. Varghese JN, Hrmova M, Fincher GB (1999) Three-dimensional structure of a barley β-d-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure 7:179–190CrossRefGoogle Scholar
  45. Wood TM (1985) Properties of cellulolytic enzyme systems. Biochem Soc Trans 13:407–410Google Scholar
  46. Xie Y, Gao Y, Chen Z (2004) Purification and characterization of an extracellular β-glucosidase with high transglucosylation activity and stability from Aspergillus niger No. 5.1. Appl Biochem Biotechnol 119:229–240CrossRefGoogle Scholar
  47. Zorov IN, Gusakov AV, Baraznenok VA, Bekkarevich AO, Okunev ON, Sinitsyn AP, Kondrat'eva EG (2001) Isolation and properties of cellobiase from Penicillium verruculosum. Appl Biochem Micro 37:587–592CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Kristian B. R. M. Krogh
    • 1
  • Paul V. Harris
    • 2
  • Carsten L. Olsen
    • 3
  • Katja S. Johansen
    • 4
  • Jesper Hojer-Pedersen
    • 1
  • Johan Borjesson
    • 5
  • Lisbeth Olsson
    • 1
  1. 1.Center for Microbial BiotechnologyTechnical University of DenmarkLyngbyDenmark
  2. 2.Novozymes IncDavisUSA
  3. 3.Fungal Gene TechnologyBagsvaerdDenmark
  4. 4.Detergent Applications IIBagsvaerdDenmark
  5. 5.Department of BiochemistryLund UniversityLundSweden
  6. 6.Protein BiochemistryNovozymes A/SBagsvaerdDenmark
  7. 7.Fermentation OptimizationNovozymes A/SCopenhagen NDenmark
  8. 8.Biofuels-DK, Novozymes A/SBagsvaerdDenmark
  9. 9.Industrial Biotechnology, Department of Chemical and Biological EngineeringChalmers University of TechnologyGöteborgSweden

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