Current Genetics

, Volume 29, Issue 4, pp 404–409 | Cite as

Secretion of an enzymatically activeTrichoderma harzianum endochitinase bySaccharomyces cerevisiae

  • Henriette Draborg
  • Stephan Christgau
  • Torben Halkier
  • Grethe Rasmussen
  • Henrik Dalbøge
  • Sakari Kauminen
Original Paper

Abstract

A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from aTrichoderma harzianum cDNA library by expression in yeast. The 1473-bpchil cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 fromBacillus circulans. TheT. harzianum endochitinase I was secreted into the culture medium by the yeastSaccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.

Key words

Biocontrol Secretion Chitinase Expression cloning Saccharomyces cerevisiae Trichoderma harzianum 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Becker DM, Guarente L (1991) High-efficiency transformation of yeast by electroporation. Methods Enzymol 194:182–187Google Scholar
  2. Blaiseau P, Lafay J (1992) Primary structure of a chitinase-encoding gene (chil) from the filamentous fungusAphanocladium album: similarity to bacterial chitinases. Gene 120:243–248Google Scholar
  3. Christgau S, Kauppinen S, Vind J, Kofod LV, Dalbøge H (1994) Expression cloning, purification and characterization of a β-1,4-mannanase fromAspergillus aculeatus. Biochem Mol Biol Int 33:917–925Google Scholar
  4. Christgau S, Sandal T, Kofod LV, Dalbøge H (1995) Expression cloning, purification and characterization of a β-1,4-galactanase fromAspergillus aculeatus. Curr Genet 27:135–141Google Scholar
  5. Cohen E (1993) Chitin synthesis and degradation as targets for pesticide action. Arch Insect Biochem Physiol 22:245–261Google Scholar
  6. Dalboge H, Heldt-Hansen H (1994) A novel method for efficient expression cloning of fungal enzyme genes. Mol Gen Genet 243:253–260Google Scholar
  7. De La Cruz J, Hildago-Gallego A, Lora JM, Benitez T, Pintor-Toro J, Llobell A (1992) Isolation and characterization of three ehitinases fromTrichoderma harzianum. Eur J Biochem 206:859–867Google Scholar
  8. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395Google Scholar
  9. Di Pietro A, Lorito M, Hayes CK, Broadway RM, Harman GE (1993) Endochitinase fromGliocladium virens: isolation, characterization, and synergistic antifungal activity in combination with gliotoxin. Mol Plant Pathol 83:308–313Google Scholar
  10. Draborg H, Kauppinen S, Dalboge H, Christgau S (1995) Molecular cloning and expression inS. cerevisiae of two exochitinases fromTrichoderma harzianum. Biochem Mol Biol Int 36:781–791Google Scholar
  11. Elad Y, Chet I, Hems Y (1982) Degradation of plant pathogenic fungi byTrichoderma harzianum. Can J Microbiol 28:719–725Google Scholar
  12. Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13Google Scholar
  13. Fuhrman JA, Lane WS, Smith RF, Piessens WF, Perller FB (1992) Transmission-blocking antibodies recognize microfilarial chitinase in brugian lymphatic filariasis. Proc Natl Acad Sci USA 89:1548–1552Google Scholar
  14. Fuller RS, Brake AJ, Thorner J (1989) Intracellular targeting and structural conservation of a prohormone endoprotease. Science 246:482–486Google Scholar
  15. Garcia I, Lora JM, De La Cruz J, Benitez T, Llobell A, Pintor-Toro JA (1994) Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungusTrichoderma harzianum. Curr Genet 27:83–89Google Scholar
  16. Harman GE, Hayes CK, Lorito M, Broadway RM, Di Pietro A, Peterbauer C, Tronsmo A (1993) Chitinolytic enzymes ofTrichoderma harzianum: purification of chitobiosidase and endochitinase. Mol Plant Pathol 83:313–318Google Scholar
  17. Hayes CK, Klemsdal S, Lorito M, Di Pietro A, Peterbauer C, Nakas JP, Tronsmo A, Harman GE (1994) Isolation and sequence of an endochitinase gene from a cDNA library ofTrichoderma harzianum. Gene 135:143–148Google Scholar
  18. Hazel HB van den, Kielland-Brandt MC, Winther JR (1992) Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens. Eur J Biochem 207:277–283Google Scholar
  19. Heijne G von (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690Google Scholar
  20. Kalyanmay D, Bagchi P (1959) Studies on the ultraviolet absorption spectra of coumarins and chromones II hydroxy derivatives. J Organic Chem 24:3–16Google Scholar
  21. Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP, Dörreich K, Dalbøge H (1994) Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases fromAspergillus aculeatus. J Biol Chem 269:29182–29189Google Scholar
  22. Meissner PS, Sisk WP, Berman ML (1987) Bacteriophage A cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci USA 84:4171–4176Google Scholar
  23. Penttilä M, Lehtovaara P, Nevalainen H, Bhikhabhai R, Knowles J (1986) Homology between cellulase genes ofTrichoderma reesei: complete nucleotide sequence of the endoglucanase-I gene. Gene 45:253–263Google Scholar
  24. Penttilä M, André L, Lehtovaara P, Bailey M, Teeri TT, Knowles JKC (1988) Efficient secretion of two fungal cellobiohydrolases bySaccharomyces cerevisiae. Gene 63:103–112Google Scholar
  25. Roberts GAF (1992) Chitin chemistry, 1st edn. Macmillan, LondonGoogle Scholar
  26. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  27. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467Google Scholar
  28. Sherman F (1991) Getting started with yeast. Methods Enzymol 194:3–21Google Scholar
  29. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  30. Ulhoa CJ, Peberdy IF (1992) Purification and some properties of the extracellular chitinase produced byTrichoderma harzianum. Enzyme Microbiol Technol 14:236–240Google Scholar
  31. Watanabe T, Suzuki K, Oyanagi W, Ohnishi K, Tanaka H (1990) Gene cloning of chitinase A1 fromBacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type-III homology units of fibronectin. J Biol Chem 265:15659–15665Google Scholar
  32. Yelton MM, Hamer JE, Timberlake WE (1984) Transformation ofAspergillus nidulans by using atrpC plasmid. Proc Natl Acad Sci USA 81:1470–1474Google Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • Henriette Draborg
    • 1
  • Stephan Christgau
    • 1
  • Torben Halkier
    • 1
  • Grethe Rasmussen
    • 1
  • Henrik Dalbøge
    • 1
  • Sakari Kauminen
    • 1
  1. 1.GeneSearchNovo Nordisk A/SBagsværdDenmark

Personalised recommendations