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Overexpression and Purification of Aspergillus Aculeatus Beta-Mannosidase and Analysis of the Integrated Gene in Aspergillus Oryzae

Overexpression and Purification of Aspergillus Aculeatus Beta-Mannosidase and Analysis of the Integrated Gene in Aspergillus Oryzae

S Kanamasa, G Takada, T Kawaguchi, J Sumitani, M Arai

J Biosci Bioeng. 2001;92(2):131-7.

PMID: 16233072

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP67909302	4-Methylumbelliferyl β-D-mannopyranoside		67909-30-2	Price

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