Current Genetics

, Volume 29, Issue 3, pp 227–233 | Cite as

Secretion ofTrichoderma reeseiβ-glucosidase bySaccharomyces cerevisiae

  • C. Cummings
  • T. Fowler
Original Paper


An intronless form of thebgl1 gene encoding an extracellularβ-glucosidase fromTrichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL 1 promoter. Transformation of a yeast strain with this vector resulted in transformants that produce and secrete activeβ-glucosidase into the growth medium. Additionally, active recombinantβ-glucosidase protein was shown to be localized predominantly in the periplasmic space by using ap-nitrophenylβ-D-glycoside hydrolysis assay against fractionated yeast cells. The apparent size of the recombinant enzyme was 10–15 kDa larger than that of the native form. Treatment of the recombinantβ-glucosidase with endoglycosidase-H indicated the apparent increase in size was due to N-linked glycosylation.

Key words

Trichoderma reesei β-Glucosidase Cellulase Saccharomyces cerevisiae 


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  1. Barnett CC, Berka RM, Fowler T (1991) Cloning and amplification of the gene encoding an extracellularβ-glucosidase fromTrichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates. Bio/Technol 9:562–567Google Scholar
  2. Chen H, Hayn M, Esterbauer H (1992) Purification and characterization of two extracellularβ-glucosidases fromTrichoderma reesei. Biochim Biophys Acta 1121:54–60PubMedGoogle Scholar
  3. Chirico WJ, Brown RD Jr (1987) Purification and characterization of aβ-glucosidase fromTrichoderma reesei. Eur J Biochem 165:333–341PubMedGoogle Scholar
  4. Enari TM, Paavola MLN, Harju L, Lappalainen A, Nummi M (1981) Purification ofTrichoderma reesei andAspergillus niger β-glucosidase. J Appl Biochem 3:157–163Google Scholar
  5. Fowler T (1993) Recombinantβ-glucosidase ofTrichoderma reesei. In: Himmel ME, Georgiou G (eds) Biocatalyst design for stability and specificity. Washington DC, Symposium Series, American Chemical Society 516:233–243Google Scholar
  6. Fowler T, Brown RD Jr (1992) Thebgl1 gene encoding extracellularβ-glucosidase fromTrichoderma reesei is required for rapid induction of the cellulase complex. Mol Microbiol 6:3225–3235PubMedGoogle Scholar
  7. Fowler T, Grizali M, Brown RD Jr (1993) Regulation of the cellulose genes ofTrichoderma reesei. In: Suominen P, Reinikained T (eds)Trichoderma reesei cellulases and other hydrolases. Proc 2nd TRICEL Symp, Espoo, Finland. Foundation for Biotechnical and Industrial Fermentation Research 8:199–210Google Scholar
  8. Gong CS, Ladisch MR, Tsao GT (1977) Cellobiases fromTrichoderma viride: purificaion, properties, kinetics and mechanism. Biotechnol Bioeng 19:959–981PubMedGoogle Scholar
  9. Harlow E, Lane D (1988) Antibodies, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  10. Hofer F, Weissinger E, Mischak H, Messner R, Meixner-Monori G, Blaas D, Visser J, Kubicek CP (1989) A monoclonal antibody against the alkaline extracellularβ-glucosidase fromTrichoderma reesei: reactivity with otherTrichoderma β-glucosidases. Biochim Biophys Acta 992:298–306Google Scholar
  11. Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation ofEscherichia coli. Gene 57:267–272PubMedGoogle Scholar
  12. Inglin M, Feinberg BA, Loewenberg JR (1980) Partial purification and characterization of a new intracellularβ-glucosidase ofTrichoderma reesei. Biochem J 185:515–519PubMedGoogle Scholar
  13. Jackson MA, Talburt DE (1988) Mechanism forβ-glucosidase release into cellulose-grownTrichoderma reesei culture supernatants. Exp Mycol 12:203–216Google Scholar
  14. Korman DR, Bayliss FT, Barnett CC, Carmona CL, Kodama KH, Royer TJ, Thompson SA, Ward M, Wilson LJ, Berka RM (1990) Cloning, characterization, and expression of two alpha-amylase genes fromAspergillus niger varawamori. Curr Genet 17:203–212PubMedGoogle Scholar
  15. Kowamori M, Ado Y, Takasawa S (1986) Preparation and application ofTrichoderma reesei mutants with enhancedβ-glucosidase. Agric Biol Chem 50:2477–2482Google Scholar
  16. Kubicek CP, Messner R, Gruber F, Mach RL, Kubicek-Pranz EM (1993) TheTrichoderma cellulase regulatory puzzle: from the interior life of a secretory fungus. Enzyme Microb Technol 15:90–99PubMedGoogle Scholar
  17. Labudova I, Farkas V (1983) Multiple enzyme forms in the cellulase system ofTrichoderma reesei during its growth on cellulose. Bioch Biophys Acta 744:135–140Google Scholar
  18. Meselson M, Yuan R (1968) DNA restriction enzyme fromE. coli. Nature 217:1110–1114PubMedGoogle Scholar
  19. Messner R, Kubicek CP (1990) Evidence for a single, specificβ-glucosidase in cell walls fromTrichoderma reesei QM9414. Enzyme Microb Technol 12:685–690Google Scholar
  20. Messner R, Hagspiel K, Kubicek CP (1990) Isolation of aβ-glucosidase-binding and activating polysaccharide from cell walls ofTrichoderma reesei. Arch Microbiol 154:150–155Google Scholar
  21. Nasmyth K (1983) Molecular analysis of a cell lineage. Nature 302:670–676PubMedGoogle Scholar
  22. Penttila ME, André L, Saloheimo M, Lehtovaara P, Knowles JKC (1987) Expression of twoTrichoderma reesei endoglucanases in the yeastSaccharomyces cerevisiae. Yeast 3:175–185PubMedGoogle Scholar
  23. Penttila ME, André L, Lehtovaara P, Bailey M, Teeri TT, Knowles JKC (1988) Efficient secretion of two fungal cellobiohydrolases bySaccharomyces cerevisiae. Gene 63:103–112PubMedGoogle Scholar
  24. Penttila L, Lehtovaara P, Knowles JKC (1989) Cellulolytic yeasts and their application. In: Barr PJ, Brake AJ, Valenzuela P (eds) Yeast genetic engineering. Butterworths, London, pp 247–267Google Scholar
  25. Pines O, London A (1991) Expression and secretion of staphylococcal nuclease in yeast: effects of amino-terminal sequences. J Gen Microbiol 137:771–778PubMedGoogle Scholar
  26. Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8:423–488PubMedGoogle Scholar
  27. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  28. Schagger H, Von Jagow G (1987) Tricine-sodium dodecyl sulphate-polyacrylamide-gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379PubMedGoogle Scholar
  29. Schmid G, Wandrey C (1987) Purification and partial characterization of cellodextrin glucohydrolase (β-glucosidase) fromTrichoderma reesei strain QM9414. Biotechnol Bioeng 30:571–585Google Scholar
  30. Sprey B (1986) Localization ofβ-glucosidase inTrichoderma reesei cells walls with immunoelectron microscopy. FEMS Microbiol Lett 36:287–292Google Scholar
  31. Sternberg D (1976)β-Glucosidase ofTrichoderma: its biosynthesis and role in saccharification of cellulose. Appl Environ Microbiol 31:648–654PubMedGoogle Scholar
  32. Towbin H, Steahlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc Natl Acad Sci USA 76:4350–4354PubMedGoogle Scholar
  33. Umile C, Kubicek CP (1986) A constitutive, plasma membraneboundβ-glucosidase inTrichoderma reesei. FEMS Microbiol Letts 34:291–295Google Scholar
  34. Usami S, Kirimura K, Imura M, Morikawa S (1990) Cellular localization of the constitutiveβ-glucosidase inTrichoderma viride. J Ferm Bioeng 70:185–187Google Scholar
  35. Van Arsdell JN, Kwok S, Schweickart VL, Ladner MB, Gelfand DH, Innis MA (1987) Cloning, characterization, and expression inSaccharomyces cerevisae of endoglucanase I fromTrichoderma reesei. Bio/Technol 5:60–64Google Scholar
  36. Yanisch-Perron C, Vicira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • C. Cummings
    • 1
  • T. Fowler
    • 1
  1. 1.Genencor InternationalSouth San FranciscoUSA

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