Starch-binding domain of Aspergillus glucoamylase-I. Interaction with beta-cyclodextrin and maltoheptaose.
The characterization is reported of two peptide fragments (SBD106 and SBD122) containing the starch-binding domain (SBD) of Aspergillus sp. glucoamylase I. The starch-binding peptides were produced in Escherichia coli as fusion proteins of the maltose-binding protein (MBP). SBD106 (11.9 kDa) and SBD122 (13.8 kDa) were purified from the factor Xa digest of MBP fusion proteins. The amino acid compositions were similar to those deduced from their amino acid sequences. The interactions of beta-cyclodextrin and maltoheptaose with purified SBD peptides were investigated by UV difference spectroscopy. SBD106 and SBD122 bound specifically beta-cyclodextrin with a dissociation constant (Kd) of 34 microM and 23.5 microM, respectively. Maltoheptaose binding to SBD106 and SBD122 was weaker than that of beta-cyclodextrin; dissociation constants were 0.57 and 0.50 mM, respectively. The results indicate that the intramolecular disulfide bonding is not required for the domain functioning and that O-glycosylation is not critical for the functioning of the starch-binding domain, but may affect its conformation and dynamics.
Kusnadi
A R
AR
Department of Food Science and Human Nutrition, Iowa State University, Ames 50011.
Chang
H Y
HY
Nikolov
Z L
ZL
Metzler
D E
DE
Metzler
C M
CM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002236:Carbohydrate Conformation; D003505:Cyclodextrins / Q000737:chemistry / Q000378:metabolism; D005800:Genes, Fungal; D005818:Genetic Engineering; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism*; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D047392:beta-Cyclodextrins
D000596:Amino Acids; D003505:Cyclodextrins; D005936:Glucans; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D047392:beta-Cyclodextrins; C031146:maltoheptaose; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase; C031215:betadex