Overexpression and purification of Aspergillus aculeatus beta-mannosidase and analysis of the integrated gene in Aspergillus oryzae.
An expression plasmid for the manB gene encoding Aspergillus aculeatus beta-d-mannosidase (MANB) was constructed by using an expression vector carrying an improved promoter. After transformation of A. oryzae by the plasmid, several transformants formed colonies emitting fluorescence on a plate containing 4-methylumbelliferyl beta-d-mannopyranoside (MU-Man) under UV-irradiation. The transformant that displayed the strongest fluorescence, named A. oryzae BMN1, produced about 270 mg MANB/l in liquid culture. Recombinant MANB overproduced in BMN1 was purified by two steps of column chromatography to a single protein band on SDS-polyacrylamide gel electrophoresis and had a molecular weight of 130,000. Analyses by Southern blotting and genomic PCR demonstrated that a single copy of the plasmid was integrated into the chromosome by recombination at the niaD locus.
Kanamasa
S
S
Graduate School of Agriculture and Biological Sciences and Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan.
Takada
G
G
Kawaguchi
T
T
Sumitani
J
J
Arai
M
M