Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system

A spectrophotometric assay was developed for the quantification of lactose in aqueous solution via a one-pot enzymatic cascade reaction at 25 °C and pH 7.2. Lactose (0.2-1.8 mM), E. coli β-galactosidase (β-Gal), Aspergillus niger glucose oxidase (GOD), horseradish peroxidase (HRP) and o-phenylenediamine (OPD) were incubated, and the increase in absorbance at 417 nm (A (417)) due to the formation of DAP (2,3-diaminophenazine), the dimeric oxidation product of OPD, was followed. The increase in A (417) was found to depend linearly on the initial lactose concentration via three consecutive but simultaneously occurring enzymatic reaction steps catalyzed by β-Gal, GOD, and HRP. No pre-incubation of lactose with β-Gal is needed with this simple lactose assay.