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

Tran, Thuy Thi, Hatti-Kaul, Rajni, Dalsgaard, Søren, Yu, Shukun
Analytical biochemistry 2011 v.410 no.2 pp. 177-184
Bacillus (bacteria), calcium chloride, grains, histidine, hydrolysis, lysozyme, pH, phosphates, phytases, phytic acid, sodium chloride, temperature, toxicity, turbidity
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.