A simple and fast kinetic assay for phytases using phytic acid-protein complex as substrate.

Phytase (EC 3.1.3.–) hydrolyzes phytate (IP6) present in cereals and grains to release inorganic phosphate (Pi), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the Pi liberated from IP6. This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP6–lysozyme as a substrate. The assay is based on the principle that IP6 forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP6 in the complex is accompanied by a decrease in turbidity monitored at 600nm. The turbidity decrease correlates well to the released Pi from IP6. This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline β-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl2, and phosphate, showed a concentration-dependent interference.