Molecular cloning, expression and structure of the endo-1,5-α-l-arabinase gene of Aspergillus niger

M. Flipphi, Henk Panneman, Peter van der Veen, Jaap Visser, Leo H. de Graaff

Research output: Contribution to journalArticle

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Abstract

Secretion of endo-1,5-α-l-arabinase A (ABNA) by an Aspergillus niger xylulose kinase mutant upon mycelium transfer to medium containing l-arabitol was immunochemically followed with time to monitor its induction profile. A cDNA expression library was made from polyA + RNA isolated from the induced mycelium. This library was immunochemically screened and one ABN A specific clone emerged. The corresponding abnA gene was isolated from an A. niger genomic library. Upon Southern blot analysis, a 3.1-kb HindIII fragment was identified and subcloned to result in plasmid pIM950. By means of co-tranformation using the A. niger pyrA gene as selection marker, the gene was introduced in both A. niger and A. nidulans uridine auxotrophic mutants. Prototrophic A. niger and A. nidulans transformants overproduced A. niger ABN A upon growth in medium containing sugar beet pulp as the sole carbon source, thereby establishing the identity and functionality of the cloned gene. The DNA sequence of the complete HindIII fragment was determined and the structure of the abnA gene as well as of its deduced gene product were analysed. Gene abnA contains three introns within its structural region and codes for a protein of 321 amino acids. Signal peptide processing results in a mature protein of 302 amino acids with a deduced molecular mass of 32.5 kDa. A. niger abnA is the first gene encoding an ABN to be isolated and characterized.

Original languageEnglish
Pages (from-to)318-326
Number of pages9
JournalApplied Microbiology and Biotechnology
Volume40
Issue number2-3
DOIs
Publication statusPublished - Nov 1993

Fingerprint

Aspergillus niger
Cloning
Aspergillus
Molecular Cloning
Molecular Structure
Genes
Mycelium
Amino acids
Sugar beets
Proteins
Amino Acids
Gene encoding
Genomic Library
Uridine
DNA sequences
Molecular mass
Beta vulgaris
Protein Sorting Signals
RNA
Introns

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology
  • Bioengineering
  • Biotechnology

Cite this

Molecular cloning, expression and structure of the endo-1,5-α-l-arabinase gene of Aspergillus niger. / Flipphi, M.; Panneman, Henk; van der Veen, Peter; Visser, Jaap; de Graaff, Leo H.

In: Applied Microbiology and Biotechnology, Vol. 40, No. 2-3, 11.1993, p. 318-326.

Research output: Contribution to journalArticle

Flipphi, M. ; Panneman, Henk ; van der Veen, Peter ; Visser, Jaap ; de Graaff, Leo H. / Molecular cloning, expression and structure of the endo-1,5-α-l-arabinase gene of Aspergillus niger. In: Applied Microbiology and Biotechnology. 1993 ; Vol. 40, No. 2-3. pp. 318-326.
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