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

Abstract

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 

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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

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