Cycloheptaamylose as an affinity ligand of cereal alpha amylase. Characteristics and a possible mechanism of the interaction

The characteristics of alpha amylase purification on a column of cycloheptaamylose-substituted, epoxy-activated Sepharose 6B were investigated. The enzyme was recovered in high yield from crude triticale and wheat extracts. Enzyme activity assessed after elution from the column was 132% of that measured prior to chromatography. There was no evidence of beta amylase isozymes in the purified alpha amylase. Neither barley beta amylase nor sweet-potato beta amylase was retained by the column. Cycloheptaamylose did not inhibit triticale or wheat alpha amylase activity, but did inhibit barley beta amylase activity, yielding a K₁ of 4.5mm. Equilibriumdialysis experiments showed that alpha amylase did interact with cycloheptaamylose. The dissociation constant for the enzyme-ligand was 19μm. It was concluded that cycloheptaamylose bound at a non-catalytic site on the alpha amylase molecule.