Colourimetric Plate Assays Based on Functionalized Gelatine Hydrogel Useful for Various Screening Purposes in Enzymology

Abstract

Hydrogels are intensively investigated biomaterials due to their useful physicochemical and biological properties in bioengineering. In particular, naturally occurring hydrogels are being deployed as carriers for bio-compounds. We used two approaches to develop a plate colourimetric test by immobilising (1) ABTS or (2) laccase from Trametes versicolor in the gelatine-based hydrogel. The first system (1) was applied to detect laccase in aqueous samples. We investigated the detection level of the enzyme between 0.05 and 100 µg/mL and pH ranging between 3 and 9; the stability of ABTS in the solution and the immobilised form, as well as the retention functional property of the hydrogel in 4 °C for 30 days. The test can detect laccase within 20 min in the concentration range of 2.5–100 µg/mL; is effective at pH 3–6; preserves high stability and functionality under storage and can be also successfully applied for testing samples from a microbial culture. The second system with the immobilised laccase (2) was tested in terms of substrate specificity (ABTS, syringaldazine, guaiacol) and inhibitor (NaN3) screening. ABTS appeared the most proper substrate for laccase with detection sensitivity CABTS > 0.5 mg/mL. The NaN3 tested in the range of 0.5–100 µg/mL showed a distinct inhibition effect in 20 min for 0.5 µg/mL and total inhibition for ≥75 µg/mL.