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-arabi nofuranosidase B (ABF B), and cyanogen bromide fragments derived there of, deoxyoligonucleotide mixtures were designed to be employed as prim ers in a polymerase chain reaction (PCR) on A. niger genomic DNA. This resulted in amplification of three related PCR products. The abfB gen e encoding ABF B was isolated from a genomic library using such an amp lification product as a probe. A 5.1-kb BamHI fragment was subcloned t o result in plasmid pIM991. Upon introduction by co-transformation int o both A. niger and A. nidulans uridine auxotrophic strains, pIM991 wa s shown to contain the functional gene since prototrophic transformant s overproduced ABF B upon growth on the inducing carbon source sugar b eet pulp. A plate assay was developed enabling quick selection of ABF B-over-producing transformants. The sequence of a 4122-bp long BamHI/S stI fragment was determined. The abfB gene docs not contain introns an d 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.