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Cloning and expression of the alpha-L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae
- Crous, J.M., Pretorius, I.S., Van Zyl, W.H.
- Applied microbiology and biotechnology 1996 v.46 no.3 pp. 256-260
- Aspergillus niger, DNA fragmentation, Saccharomyces cerevisiae, alpha-N-arabinofuranosidase, complementary DNA, gene expression, genes, messenger RNA, nucleotide sequences, oats, phosphoglycerate kinase, plasmids, polymerase chain reaction, xylan, yeasts
- First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the alpha-L-arabinofuranosidase gene (abfB) was amplified with the polymerase chain reaction technique. The abfB DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1p) and terminator (PGK1T) sequences on a multicopy episomal plasmid. The resulting construct PGK1 p-abfB-PGK1(T) was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional alpha-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf2) that is 94% identical to the alpha-L-arabinofuranosidase B of A. niger N400. Maximum alpha-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively.