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 carryi ng an improved promoter. After transformation of A. oryzae by the plasmid, several transformants formed colonies emitting fluorescence on a plate cont aining 4-methylumbelliferyl beta -D-mannopyranoside (MU-Man) under UV-irrad iation. 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 chromatograph y to a single protein band on SDS-polyacrylamide gel electrophoresis and ha d 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.