Abstract
An expression plasmid for the manB gene encoding Aspergillus aculeatus β- 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 β- 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.
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