Expression cloning, purification and characterization of a beta-1,4-galactanase from Aspergillus aculeatus.
Expression cloning has been used to isolate a cDNA encoding beta-1,4-galactanase from the filamentous fungus Aspergillus aculeatus. A cDNA library was prepared from mycelia, inserted in a yeast expression vector and transformed into Saccharomyces cerevisiae. Thirteen clones secreting galactanase activity were identified from a screening of approximately 2.5 x 10(4) yeast colonies. All clones expressed transcripts of the same galactanase gene. The cDNA was re-cloned in an Aspergillus expression vector and transformed into Aspergillus oryzae. The recombinant enzyme had a molecular weight of 44,000 Da, an isoelectric point of pH 2.85, a pH optimum of pH 4.0-4.5, and a temperature optimum of 45-65 degrees C, which is similar to values obtained for a beta-1,4-galactanase purified from A. aculeatus. The enzyme degraded unsubstituted galactan to galactose and galactobiose. The deduced primary sequence of the enzyme showed no apparent homology to any known enzyme, in accordance with this being the first reported beta-1,4-galactanase cDNA. However, the deduced amino-acid sequence of a Bacillus circulans DNA sequence containing an open reading frame (ORF) with no known function, showed 36% identity and 60% similarity to the galactanase amino-acid sequence.
Christgau
S
S
GeneExpress, Novo Nordisk A/S, Copenhagen, Denmark.
Sandal
T
T
Kofod
L V
LV
Dalbøge
H
H
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D001236:Aspergillus oryzae / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000096:biosynthesis; D005685:Galactans / Q000378:metabolism; D006026:Glycoside Hydrolases; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013696:Temperature; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D005656:Fungal Proteins; D005685:Galactans; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C094192:arabinogalactan endo-1,4-beta-galactosidase