Zein-in-agarose microplate turbidimetric assay for screening and quantifying protease activity

Proteolytic (zeinase) activity is most often screened on substrate-containing agar plates, where activity is scored from the diameter of a cleared halo after the residual protein has been precipitated or stained. This format is slow, semi-quantitative, subjective and low throughput. Here, we describe a miniaturised turbidimetric assay that transfers the substrate into a 96-well microplate. A zein-agarose gel is cast in each well; after the sample is added and incubated, dilute acetic acid precipitates the unhydrolysed zein, and activity is read as the increase in well transmittance at 510 nm relative to a substrate blank. The detection wavelength was selected from spectral scans, and the three gel parameters governing the signal window (zein concentration, agarose concentration and gel volume per well) were optimised by a Box-Behnken response-surface design. The optimised assay is quantitative over 1-10 mU, and its screening performance was confirmed by the Z'-factor, intra- and inter-day coefficients of variation, and limits of detection and quantification.•96 samples are quantified in parallel from a single plate-reader measurement.•A continuous, objective transmittance signal replaces manual halo-diameter scoring.•The assay is validated to high-throughput screening standards (Z'-factor, %CV, LOD/LOQ).