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 Ca²⁺. TAA peptide fragments were separated into two groups having no and high affinity to the substrate, soluble statch. 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.