Cloning and characterization of the abfB gene coding for the major alpha-L-arabinofuranosidase (ABF B) of Aspergillus niger.
Based on amino-acid sequence data from Aspergillus niger alpha-L-arabinofuranosidase B (ABF B), and cyanogen bromide fragments derived thereof, deoxyoligonucleotide mixtures were designed to be 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.
Flipphi
M J
MJ
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, The Netherlands.
van Heuvel
M
M
van der Veen
P
P
Visser
J
J
de Graaff
L H
LH
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping
D017931:DNA Primers; D004271:DNA, Fungal; D010446:Peptide Fragments; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase