Expression cloning, purification and characterization of a beta-1,4-mannanase from Aspergillus aculeatus.
A cDNA library from the filamentous fungus Aspergillus aculeatus was constructed in the yeast expression vector pYES2.0 and used to isolate 57 full length cDNA's encoding beta-1,4-mannanase by expression in S. cerevisiae. The positive clones were identified on agar plates containing 0.2% azurine dyed cross-linked mannan by the formation of blue halos around the colonies. All clones represented transcripts of the same mannanase gene (man1). The gene was sub-cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for overexpression and purification of the enzyme. The recombinant enzyme had a molecular weight of 45 kDa, an isoelectric point of pH 4.5, a pH optimum of pH 5.0 and a temperature optimum of 60-70 degrees.
Christgau
S
S
GeneExpress, Novo Nordisk A/S, Copenhagen, Denmark.
Kauppinen
S
S
Vind
J
J
Kofod
L V
LV
Dalbøge
H
H
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics*; D007526:Isoelectric Point; D007700:Kinetics; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D011993:Recombinant Fusion Proteins; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase