title: Isolation of Taka-amylase A Peptides Required for Substrate Binding.
abstract: An α-amylase from Aspergillus oryzae, Taka-amylase A (TAA), was cleaved into peptide fragments by an acid protease. Inactivation of TAA was greatly retarded by the addition of α-cyclodextrin or Ca(2+). TAA peptide fragments were separated into two groups having no and high affinity to the substrate, soluble starch. This separation was done by the forced affinity chromatography method by a column of epichlorohydrin cross-linked soluble starch gel. Three peptides were isolated from the high-affinity fragments, purified by the ODS-120T column, and their amino acids were sequenced. Peptides I, II, and III originated from α2-helix, α3-helix, and β2-sheet, respectively, and all of these were located in the (β/α)8 barrel of the main domain of TAA molecule. A stereo graphic view showed that Peptides I-III were at the cleft near the catalytic site. Occurrence of a Trp residue in all three peptides strongly suggested that Trp was very important in the binding of TAA to the substrate, soluble starch.
authors.affiliation: a Department of Resource Biology, Faculty of Agriculture , Ibaraki University , Ami, Ibaraki 300-03, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Sasaki
authors.affiliation: a Department of Resource Biology, Faculty of Agriculture , Ibaraki University , Ami, Ibaraki 300-03, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Takahara
authors.affiliation: b National Food Research Institute , Kannondai, Tsukuba 305 , Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kobayashi
keywords: Taka-amylase A; forced affinity chromatography; substrate binding site; α-amylase