Molecular cloning and characterization of the fructooligosaccharide-producing beta-fructofuranosidase gene from Aspergillus niger ATCC 20611.
The fopA gene encoding a fructooligosaccharide-producing beta-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other beta-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the beta-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any beta-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-beta-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.
Yanai
K
K
Bio Science Laboratories, Meiji Seika Kaisha, Ltd., Sakado-shi, Saitama, Japan. koji_yanai@meiji.co.jp
Nakane
A
A
Kawate
A
A
Hirayama
M
M
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003470:Culture Media; D015723:Gene Library; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D043324:beta-Fructofuranosidase
D003470:Culture Media; D009844:Oligosaccharides; C116580:fructooligosaccharide; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase