Overexpression and purification of Aspergillus aculeatus β-mannosidase and analysis of the integrated gene in Aspergillus oryzae

• Published in: Journal of bioscience and bioengineering 2001, Vol.92 (2), p.131-137
• Main Authors: Kanamasa, Shin, Takada, Goro, Kawaguchi, Takashi, Sumitani, Jun-Ichi, Arai, Motoo
• Format: Article
• Language: English
• Subjects: ASPERGILLUS; Aspergillus aculeatus; Aspergillus oryzae; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; GENES; Genetic engineering; Genetic technics; HYDROLASES; Methods. Procedures. Technologies; Modification of gene expression level; overexpression; PCR; PURIFICATION; quantitative PCR; Region III; β-mannosidase
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/S1389-1723(01)80213-9

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.