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

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Phytase enzymes are crucial for releasing bioavailable inorganic phosphate (P(i)) from phytate (IP(6)) in cereals and grains.
• Traditional phytase assays are time-consuming, cumbersome, and involve toxic reagents.
• There is a need for a more efficient and safer method to quantify phytase activity.

Purpose of the Study:

• To develop a simple, fast, nontoxic, and high-throughput kinetic method for assaying phytase activity.
• To utilize a novel substrate, IP(6)-lysozyme, for phytase detection.
• To validate the method across different classes of phytases.

Main Methods:

• A kinetic assay was developed based on the decrease in turbidity of IP(6)-lysozyme complexes upon phytate hydrolysis.
• Turbidity was monitored at 600 nm to quantify released inorganic phosphate.
• The method was tested with histidine acid phytases (3- and 6-phytases) and alkaline β-propeller phytase.

Main Results:

• The new kinetic assay demonstrated a strong correlation between turbidity decrease and released P(i).
• The method proved effective for assaying both histidine acid and alkaline phytases.
• Influences of temperature, pH, and salts were evaluated, with salts showing concentration-dependent interference.

Conclusions:

• A novel, high-throughput kinetic assay for phytase activity using IP(6)-lysozyme is presented.
• This method offers a safer and more efficient alternative to conventional phytase assays.
• The assay is adaptable for various commercial phytase types and warrants further optimization for specific applications.