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

Year: 2025

Type: article

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.

Source: Food Analytical Methods

DOI: 10.1007/s12161-025-02781-3

Authors: Katrin Reichenberger, Sabine Lutz-Wahl, Lucas Kettner, Lutz Fischer

Institution: University of Hohenheim

Language: English

Topic: Enzyme Production and Characterization

Subfield: Biotechnology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Zero hunger

Open Access status: hybrid

Estimated APC paid: $2,490