Phytase turbidity assay

The phytase turbidity assay developed in this study for P. tricornutum cell extracts is based on a modified version of the protocol previously described by Tran et al.⁴³, Assays were carried out in 96-well microtiter plate (MTP) format, in 120 µL final reaction volume consisting of 60 µL of substrate suspension (50 mM glycine pH 2.5, 0.4 mM phytic acid, 1.7 mg/mL lysozyme) and 60 µL of clarified cell lysate. Cell lysates were tested in serial dilutions. For positive control reactions, a water solution of a commercially-supplied phytase (EC: 3.1.3.26, Xi’an Rongsheng Biotechnology Co., Ltd. Shaanxi, China, 200 mg/mL) was used. To conduct the assay, 60 µL of cell lysate (or its dilution) was firstly add to the well, followed by 60 µL of substrate suspension. The final reaction was mixed thoroughly by pipetting and transferred to a microtiter plate reader pre-heated at 37 °C (FLUOstar Omega, BMG Labtech; Offenburg, Germany). The change in turbidity was monitored at 600 nm every 30 seconds for 20–60 minutes with shaking (double orbital) at 700 rpm for 5 seconds prior to each reading. Turbidity data was exported to R for analysis. Phytase activity (FTU/mg soluble protein) was calculated from the linear portion of each assay curve using a model of FTU regressed against change in OD₆₀₀ nm. One phytase activity unit (FTU) is defined as the amount of enzyme which liberates 1 µmol inorganic phosphorus from an excess of substrate (here IP6-lysozyme) in 1 minute at 37 °C and pH 2.5. Total soluble protein content was determined by direct absorbance at 280 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific).