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.
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Notes
When all three enzyme stock solutions were prepared at tenfold higher concentrations, and the reaction time was set to 30 min instead of 7 h, a linear dependency of A 417 on the lactose concentration between 0.25 and 1.50 mM was also found (data not shown), although the linear correlation was lower (r = 0.9932). Measurements with shorter incubation times have the disadvantages of consuming more enzyme and requiring more precise control over the incubation time, which may be a problem if a large number of samples are assayed simultaneously.
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Acknowledgments
The financial support from the Swiss National Science Foundation (200021–116205) is highly appreciated. Kenjiro Yazawa acknowledges the International Training Program (ITP) from the Japan Society for the Promotion of Science (JSPS). Martin Willeke (Department of Materials, ETH) is acknowledged for his careful and critical reading of the manuscript.
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Fornera, S., Yazawa, K. & Walde, P. Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system. Anal Bioanal Chem 401, 2307–2310 (2011). https://doi.org/10.1007/s00216-011-5312-9
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DOI: https://doi.org/10.1007/s00216-011-5312-9