Multi-wavelength colorimetric determination of large-ring cyclodextrin content for the cyclization activity of 4-α-glucanotransferase

Large-ring cyclodextrins (LR-CDs) have a number of intriguing properties for potential use in pharmaceutical and food industry. To date, no colorimetric method has been reported for LR-CD content quantification. In this study, triple wavelength colorimetry (TWC) and orthogonal-function spectrophotometry (OFS) have been successfully applied to determine ingredient concentrations in a mixture of amylose and LR-CDs. Both TWC and OFS yielded precise amylose content data in good agreement with expected values. For quantification of LR-CD content, OFS provided a higher accuracy than TWC, which resulted in a slight over-determination. As a comparison, single-wavelength colorimetry performed at the corresponding absorption maximum led to a significant over-determination of both amylose and LR-CD contents. The validity of TWC and OFS allowed their application for discriminative detection of the cyclization and total activity of a 4-α-glucanotransferase (4αGTase) from Thermus aquaticus regarding the synthesis of LR-CDs and the conversion of amylose to small molecules, respectively. High pressure size exclusion chromatography analysis of the post-reaction mixtures following 4αGTase-catalyzed conversion of amylose revealed the presence of linear malto-oligosaccharides in the LR-CD fraction. By introduction of a correction factor, the interference caused by linear malto-oligosaccharides was eliminated for a more accurate determination of LR-CD cyclization activity.