Enzyme-assisted HPTLC method for the simultaneous analysis of inositol phosphates and phosphate

Abstract

Background

The analysis of myo-inositol phosphates (InsP_x) released by phytases during phytic acid degradation is challenging and time-consuming, particularly in terms of sample preparation, isomer separation, and detection. However, a fast and robust analysis method is crucial when screening for phytases during protein engineering approaches, which result in a large number of samples, to ensure reliable identification of promising novel enzymes or target variants with improved characteristics, for example, pH range, thermal stability, and phosphate release kinetics.

Results

The simultaneous analysis of several InsP_x (InsP₁-InsP₄ and InsP_5 + 6) as well as free phosphate was established on cellulose HPTLC plates using a buffered mobile phase. Inositol phosphates were subsequently stained using a novel enzyme-assisted staining procedure. Immobilized InsP_x were hydrolyzed by a phytase solution of Quantum® Blue_liquid 5G followed by a molybdate reagent derivatization. Resulting blue zones were captured by DAD scan. The method shows good repeatability (intra-day and intra-lab) with maximum deviations of the Rf value of 0.01. The HPTLC method was applied to three commercially available phytases at two pH levels relevant to the gastrointestinal tract of poultry (pH 5.5 and pH 3.6) to observe their phytate degradation pattern and thus visualize their InsP_x fingerprint.

Conclusion

This HPTLC method presents a semi-high-throughput analysis for the simultaneous analysis of phytic acid and the resulting lower inositol phosphates after its enzymatic hydrolysis and is also an effective tool to visualize the InsP_x fingerprints and possible accumulations of inositol phosphates.