Phytase activity assay

Wild-type and phytase expressing cells were grown in 400 mL of selective media until the exponential growth phase (approximately 10⁶ cells). Cultures were centrifuged, washed with 1 % glycerol and centrifuged again. The resulting pellets were frozen with liquid nitrogen, thawed and frozen again previous to lyophilization in a Freezone 2.5 Benchtop Liter Lyophilizer (Labconco). The lyophilized whole-cell lysates (dry microalgae biomass) were used for phytase activity assays.

Phytase activity quantification was performed as described by Heinonen and Lahti [27] with modifications. Briefly, 25 mg of lyophilized microalgae were dissolved in 500 μL of 200 mM pH 3.5 sodium acetate – acetic acid buffer. In order to start the catalytic reaction, 100 μL of each sample were added to 500 μL of a 44.1 mM sodium phytate solution (in acetate buffer) previously incubated at 37 °C for 5 min. The mock was performed under the same conditions without the lyophilized microalgae, and a positive control was carried out adding 100 μL of a commercial phytase solution (Natuphos®). After incubation for 30 min at 37 °C, 4 mL of CRS colorimetric solution (acetone, 5 N sulphuric acid and 5 % ammonium molybdate; 2:1:1 v/v/v) was added, and absorbance (400 nm) was measured. A standard curve of absorbance was made with phosphate (0.5–2.5 μmol). The enzymatic activity (units per mg of lyophilisate) was obtained as the μmols of released phosphate per duration of assay, volume and lyophilisate mass.