Enzyme‐assisted HPTLC method for the simultaneous analysis of inositol phosphates and phosphate
- Background The analysis of myo‐inositol phosphates (InsPx) 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 ensureBackground The analysis of myo‐inositol phosphates (InsPx) 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 InsPx (InsP1‐InsP4 and InsP5 + 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 InsPx were hydrolyzed by a phytase solution of Quantum® Blueliquid 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 InsPx 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 InsPx fingerprints and possible accumulations of inositol phosphates.…
Document Type: | Article (reviewed) |
---|---|
Zitierlink: | https://opus.hs-offenburg.de/7117 | Bibliografische Angaben |
Title (English): | Enzyme‐assisted HPTLC method for the simultaneous analysis of inositol phosphates and phosphate |
Author: | Corinna HenningerStaff MemberGND, Bernd SpangenbergStaff MemberORCiDGND, Mario Schmidt, Katrin OchsenreitherORCiD, Thomas EiseleStaff MemberORCiDGND |
Year of Publication: | 2023 |
Date of first Publication: | 2023/04/05 |
Place of publication: | Chichester, UK |
Publisher: | Blackwell Publishing Ltd |
First Page: | 170 |
Last Page: | 179 |
Parent Title (English): | JSFA Reports |
Volume: | 3 |
Issue: | 4 |
ISSN: | 2573-5098 |
DOI: | https://doi.org/10.1002/jsf2.109 |
URN: | https://urn:nbn:de:bsz:ofb1-opus4-71176 |
Language: | English | Inhaltliche Informationen |
Institutes: | Fakultät Maschinenbau und Verfahrenstechnik (M+V) |
Institutes: | Bibliografie |
Tag: | HPTLC; animal feed; inositol phosphates; phosphate release; phytase; phytate dephosphorylation | Formale Angaben |
Relevance: | Wiss. Zeitschriftenartikel reviewed: Sonstiger Nachweis des Review-Verfahrens |
Open Access: | Open Access |
Gold | |
Licence (German): | Creative Commons - CC BY - Namensnennung 4.0 International |