title: Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system.
abstract: 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.
authors.affiliation: Department of Materials, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
authors.forename: Sara
authors.initials: S
authors.lastname: Fornera
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Yazawa
authors.forename: Peter
authors.initials: P
authors.lastname: Walde
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology; D002384:Catalysis; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology; D005949:Glucose Oxidase / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007785:Lactose / Q000032:analysis* / Q000378:metabolism*; D010619:Phenazines / Q000378:metabolism; D010655:Phenylenediamines / Q000378:metabolism*; D012996:Solutions; D013053:Spectrophotometry; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D010619:Phenazines; D010655:Phenylenediamines; D012996:Solutions; C054312:2,3-diaminophenazine; C034193:1,2-diaminobenzene; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D001616:beta-Galactosidase; D007785:Lactose