Current Genetics

, Volume 24, Issue 6, pp 525–532

Cloning and characterization of the abfB gene coding for the major α-l-arabinofuranosidase (ABF B) of Aspergillus niger

  • Michel J. A. Flipphi
  • Margreet van Heuvel
  • Peter van der Veen
  • Jaap Visser
  • Leo H. de Graaff
Original Articles


Based on amino-acid sequence data from Aspergillus niger α-l-arabinofuranosidase B (ABF B), and cyanogen bromide fragments derived thereof, deoxyoligonucleotide mixtures were designed to the employed as primers in a polymerase chain reaction (PCR) on A. niger genomic DNA. This resulted in amplification of three related PCR products. The abfB gene encoding ABF B was isolated from a genomic library using such an amplification product as a probe. A 5.1-kb BamHI fragment was subcloned to result in plasmid pIM991. Upon introduction by co-transformation into both A. niger and A. nidulans uridine auxotrophic strains, pIM991 was shown to contain the functional gene since prototrophic transformants overproduced ABF B upon growth on the inducing carbon source sugar beet pulp. A plate assay was developed enabling quick selection of ABF B-overproducing transformants. The sequence of a 4122-bp long BamHI/SstI fragment was determined. The abfB gene does not contain introns and codes for a protein of 499 amino acids. The mature ABF B, 481 amino acids in length, has a deduced molecular weight of 50.7 kDa. A. niger abfB is the first eukaryotic gene encoding an ABF to be characterized.

Key words

Aspergillus niger α-l-Arabinofuranosidase Fungal transformation Overexpression Heterologous expression Aspergillus nidulans DNA sequence Gene structure 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Amons R (1987) FEBS Lett 212:68–72Google Scholar
  2. Andrewartha KA, Phillips DR, Stone BA (1979) Carbohydr Res 77:191–204Google Scholar
  3. Ballance DJ, Turner G (1985) Gene 36:321–331Google Scholar
  4. Beldman G, Searle-van Leeuwen MJF, Ruiter GA de, Siliha HA, Voragen AGJ (1993) Carbohydr Polymers 20:159–168Google Scholar
  5. Calzone FJ, Britten RJ, Davidson EH (1987) Methods Enzymol 152:611–632Google Scholar
  6. Dekker RFH, Richards GN (1976) Adv Carbohydr Chem Biochem 32:277–352Google Scholar
  7. Dente L, Cortese R (1987) Methods Enzymol 155:111–119Google Scholar
  8. Flipphi MJA, Panneman H, Veen P van der, Visser J, Graaff LH de (1993a) Appl Microbiol Biotechnol (in press)Google Scholar
  9. Flipphi MJA, Visser J, Veen P van der, Graaff LH de (1993 b) Appl Microbiol Biotechnol 39:335–340Google Scholar
  10. Goosen T, Bloemheuvel G, Gysler C, Bie DA de, Broek HWJ van den, Swart K (1987) Curr Genet 11:499–503Google Scholar
  11. Goosen T, Engelenburg F van, Debets F, Swart K, Bos K, Broek H van den (1989) Mol Gen Genet 219:282–288Google Scholar
  12. Graaff L de, Broek H van den, Visser J (1988) Curr Genet 13:315–321Google Scholar
  13. Gremli H, Neukom H (1968) Carbohydr Res 8:110–112Google Scholar
  14. Gunata Z, Brillouet J-M, Voirin S, Baumes R, Cordonnier R (1990) J Agric Food Chem 38:772–776Google Scholar
  15. Gurr SJ, Unkles SE, Kinghorn JR (1987) In: Kinghorn JR (ed) Gene structure in eukaryotic microbes. SGM Special Publication, vol 23. IRL Press, Oxford, UK, pp 93–139Google Scholar
  16. Harmsen JAM, Kusters-van Someren MA, Visser J (1990) Curr Genet 18:161–166Google Scholar
  17. Heijne G von (1986) Nucleic Acids Res 14:4683–4691Google Scholar
  18. Kaji A (1984) Adv Carbohydr Chem Biochem 42:383–394Google Scholar
  19. Kaji A, Tagawa K (1970) Biochim Biophys Acta 207:456–464Google Scholar
  20. Kaji A, Taki H, Shimazaki A, Shinkai T (1963) Tech Bull Fac Agr Kagawa Univ 15:34–39Google Scholar
  21. Kellett LE, Poole DM, Ferreira LMA, Durrant AJ, Hazlewood GP, Gilbert HJ (1990) Biochem J 272:369–376Google Scholar
  22. King Jr. AD, Hocking AD, Pitt JI (1979) Appl Environ Microbiol 37:959–964Google Scholar
  23. Kozak M (1991) J Biol Chem 266:19867–19870Google Scholar
  24. Kusters-van Someren MA, Harmsen JAM, Kester HCM, Visser J (1991) Curr Genet 20:293–299Google Scholar
  25. Laemmli UK (1970) Nature 227:680–685Google Scholar
  26. Lamsa M, Bloebaum P (1990) J Industr Microbiol 5:229–238Google Scholar
  27. Lerouge P, O'Neill MA, Darvill AG, Albersheim P (1993 a) Carbohydr Res 243:359–371Google Scholar
  28. Lerouge P, O'Neill MA, Darvill AG, Albersheim P (1993 b) Carbohydr Res 243:373–378Google Scholar
  29. Matsudaira P (1987) J Biol Chem 262:10035–10038Google Scholar
  30. Neukom H, Markwalder H (1975) Carbohydr Res 39:387–389Google Scholar
  31. Neukom H, Providoli L, Gremli H, Hui PA (1967) Cereal Chem 44:238–244Google Scholar
  32. Ohtsuka E, Matsuki S, Ikehara M, Takahashi Y, Matsubara K (1985) J Biol Chem 260:2605–2608Google Scholar
  33. Proudfoot N (1991) Cell 64:671–674Google Scholar
  34. Rombouts FM, Voragen AGJ, Searle-van Leeuwen MF, Geraerds CCJM, Schols HA, Pilnik W (1988) Carbohydr Polymers 9:25–47Google Scholar
  35. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  36. Sanger F, Nicklen S, Coulson AR (1977) Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  37. Schwarz WH, Adelsberger H, Jauris S, Hertel C, Funk B, Staudenbauer WL (1990) Biochem Biophys Res Commun 170:368–374Google Scholar
  38. Sewell GW, Utt EA, Hespell RB, MacKenzie KF, Ingram LO (1989) Appl Environ Microbiol 55:306–311Google Scholar
  39. Steers Jr. E, Craven GR, Anfinsen CB, Bethune JL (1965) J Biol Chem 240:2478–2484Google Scholar
  40. Tagawa K, Kaji A (1988) Methods Enzymol 160:707–712Google Scholar
  41. Unkles SE (1992) In: Kinghorn JR, Turner G (eds) Applied molecular genetics of filamentous fungi. 1st edn. Blackie Academic and Professional, London, pp 28–53Google Scholar
  42. Utt EA, Eddy CK, Keshav KF, Ingram LO (1991) Appl Environ Microbiol 57:1227–1234Google Scholar
  43. Veen P van der, Flipphi MJA, Voragen AGJ, Visser J (1991) Arch Microbiol 157: 23–28Google Scholar
  44. Vishiniac W, Santer M (1957) Bacteriol Rev 21:195–213Google Scholar
  45. Voragen AGJ, Rombouts FM, Searle-van Leeuwen MF, Schols HA, Pilnik W (1987) Food Hydrocolloids 1:423–437Google Scholar
  46. Whitaker JR (1984) Enzyme Microbiol Technol 6:341–349Google Scholar
  47. Will F, Dietrich H (1992) Carbohydr Polymers 18:109–117Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Michel J. A. Flipphi
    • 1
  • Margreet van Heuvel
    • 2
  • Peter van der Veen
    • 1
  • Jaap Visser
    • 1
  • Leo H. de Graaff
    • 1
  1. 1.Section Molecular Genetics of Industrial MicroorganismsWageningen Agricultural UniversityWageningenThe Netherlands
  2. 2.Research and Development DepartmentRoyal Gist brocades bvDelftThe Netherlands

Personalised recommendations