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

Current Genetics - Tập 24 - Trang 525-532 - 1993
Michel J. A. Flipphi1, Margreet van Heuvel2, Peter van der Veen1, Jaap Visser1, Leo H. de Graaff1
1Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Wageningen, The Netherlands
2Research and Development Department, Royal Gist brocades bv, Delft, The Netherlands

Tóm tắt

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.

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