Direct assay for α-amylase using fluorophore-modified cyclodextrins Original Research Article

A simple assay method for α-amylase was developed based on fluorophore-modified cyclodextrins (CDs). Four kinds of CD derivatives bearing a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-amine) moiety were prepared as artificial substrates for the assay method. The fluorescence intensity of all the NBD-amine-modified CDs decreased upon addition of Aspergillus oryzae α-amylase, indicating a reduction in hydrophobicity near the NBD-amine moiety induced by hydrolysis of the CD ring. NC4γCD, having a γ-CD and an amino-tetramethylene spacer, was the most sensitive substrate for the α-amylase assay. The initial rate of hydrolysis of NC4γCD displayed a liner correlation to the concentration of the α-amylase. NC4γCD was sensitive to the α-amylase but was not sensitive to guest compounds that were accommodated by the native CDs.