An Extremely Sensitive Amylase Activity Assay and its Application for the Determination of the Residual Amylase Activity in Bread

Abstract An extremely sensitive amylase activity assay was developed using the natural substrate starch and two ancillary enzymes: a glucose oxidase (GOD) and a peroxidase, to measure the residual activity of the α -amylase from Bacillus subtilis in white bread. Firstly, the concentrations of the assay components: electron acceptor DA-67 (50 μM), horseradish peroxidase (681 nkat mL −1 ), a GOD from Aspergillus niger (1550 nkat mL −1 ) and the natural substrate starch (0.01% (w/v)), were optimized to achieve high sensitivity. The linearity of the assay was then tested with both an endo- ( α -amylase from B. subtilis ) and exo-acting amylase (maltogenic amylase from Geobacillus stearothermophilus ), and the effect of the incubation time on the assay sensitivity was investigated and optimized. The optimized assay was capable of determining a minimal amylase activity of 0.33 pkat mL −1 for both amylases tested with an assay run time of 7.5 h. This new DA-67 amylase assay demonstrated 4.7- and 4.2-times higher sensitivity, respectively, compared to optimized versions of the commercial Ceralpha (determination of endo-amylase activities) and Betamyl3 (determination of exo-amylase activities) assays. The new DA-67 amylase assay was used to determine the residual activity of α -amylase from B. subtilis in white bread. A consistent residual activity of 2.26 ± 0.15% was reliably determined.