Development and characterization of a polyampholyte-based reactor immobilizing soybean seed coat peroxidase for analytical applications in a flow system

The aim of this work was the development of a reusable enzymatic reactor based on soybean peroxidase for analytical purposes in a flow system. The innovation consisted in the application of a polyampholyte as a support for the immobilization of the enzyme extracted from soybean seed coat, without previous purification.A polyampholyte with high loading capacity for proteins was tested as a sorbent, reaching a maximum adsorption capacity (q0) of 0.10 mg g−1 and an equilibrium dissociation constant (Kd) of 1.3 × 10−8 M.The catalytic activities of free and immobilized enzyme were first compared in batch. The Michaelis constant (Km) for phenol at saturating H2O2 decreased upon immobilization.The reactor was then coupled to an amperometric detector in a flow system. The effects of the immobilization on kinetics and equilibrium parameters were evaluated using catechol and H2O2 as substrates. The values for the apparent Michaelis constant for catechol (K′mK′m) were higher than the intrinsic parameter of the immobilized enzyme (Km, 0.08 mM) due to external mass-transport limitations. Internal diffusional restrictions were absent since the normalized Eadie–Hofstee plots did not exhibit sigmoidal curves.The reactor was tested as an analytical tool for food analysis. The enzymatic activity was stable over two months.

► A polyampholyte was tested as support for a non-purified enzyme.
► The polyampholyte can be obtained by a one-step synthetic strategy.
► Immobilized soybean seed coat peroxidase was used for analytical purposes.
► Kinetic parameters and diffusional restrictions were evaluated for the flow reactor.
► The reactor based on peroxidase was used for food discrimination.