PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 10677002

High-performance thin-layer chromatography method for inositol phosphate analysis.

F Hatzack·S K Rasmussen
View original source ↗Export metadata ↓

Abstract

A simple and inexpensive high-performance thin-layer chromatography (HPTLC) method for the analysis of inositol mono- to hexakisphosphates on cellulose precoated plates is described. Plates were developed in 1-propanol-25% ammonia solution-water (5:4:1) and substance quantities as low as 100-200 pmol were detected by molybdate staining. Chromatographic mobilities of nucleotides and phosphorylated carbohydrates were also characterized. Charcoal treatment was employed to separate nucleotides from inositol phosphates with similar R(F) values prior to HPTLC analysis. Practical application of the HPTLC system is demonstrated by analysis of grain extracts from wild type and low-phytate mutant barley as well as phytate degradation products resulting from barley phytase activity.

Explore related subjects

6-PhytaseCharcoalChromatography, High Pressure LiquidChromatography, Thin LayerFructosediphosphatesFructosephosphatesGlucosamineGlucosephosphatesHordeumInositol PhosphatesMutationNucleotidesPhytic AcidJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

F Hatzack, S K Rasmussen. 1999-12-24. High-performance thin-layer chromatography method for inositol phosphate analysis.. https://doi.org/10.1016/s0378-4347(99)00465-x

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2002

Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis.

A novel phytase gene ( phyL) was cloned from Bacillus licheniformis by multiple steps of degenerate and inverse PCR. The coding region of the phyL gene was 1,146 bp in size and a promoter region of approximately 300 bp was identified at the upstream sequence. This gene, together with a phytase gene ( 168phyA) identified in the B. subtilis strain 168 genome by a homology search, was cloned and over-expressed in B. subtilis using a phi105MU331 prophage vector system. Up to 35 units of phytase/ml were secreted into the culture media; and mature enzymes of around 44-47 kDa were purified for characterization. Both phytases exhibited broad temperature and pH optima and showed high thermostability. Of the two, the phytase encoded by phyL exhibited higher thermostability, even at a lower calcium concentration, as it was able to recover 80% of its original activity after denaturation at 95 degrees C for 10 min. With their neutral pH optima and good temperature stabilities, these Bacillus phytases are good candidates for animal feed applications and transgenic studies.

6-Phytase↗
PubMed2002

In vitro efficacies of phosphorolytic enzymes synthesized in mycelial cells of Aspergillus niger AbZ4 grown by a liquid surface fermentation.

Activities of phytase, a pH 6.0 optimum nonspecific phosphomonoesterase and phosphodiesterase assayed toward bis(p-nitrophenyl)phosphate (phosphodiesterase I) and against p-nitrophenylphosphorylcholine (phosphodiesterase II), were partially purified from mycelial extracts of Aspergillus niger AbZ4 cultivated on a molasses medium by a liquid surface fermentation method. After elimination of phosphate from the medium, 7.3- and 3.5-fold enhancements in specific activities of phytase and phosphodiesterase II were observed. Efficacies of mycelial protein fractions in dephosphorylating a wheat-based broiler feed were determined in vitro according to a procedure that simulated digestion in the intestinal tract of poultry. The addition of 0.052 mg of protein from fractions, each of which was high in either pH 6.0 optimum phosphomonoesterase, phosphodiesterase I, phosphodiesterase II, or phytase per gram of a feed sample resulted in the enhancement of phosphorus release by 10, 11, 27, and 88%, respectively. In the presence of an excess of commercial phytase, the addition of the mycelial fraction high in phytase increased the dephosphorylation rate by 56%. The fraction high in phosphodiesterase II enhanced feed dephosphorylation by 8% in the presence of an excess of commercial phytase and commercial acid phosphatase.

6-Phytase↗
PubMed2002

Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase.

The phyA gene from Aspergillus ficuum that codes for a 441-amino-acid full-length phosphomonoesterase (phytase) was cloned and expressed in Medicago sativa (alfalfa) leaves. The expressed enzyme from alfalfa leaves was purified to homogeneity and biochemically characterized, and its catalytic properties were elucidated. The expressed phytase in alfalfa leaves retained all the biochemical properties of the benchmark A. ficuum phytase. Although the characteristic bi-hump pH optima were retained in the cloned phytase, the optimal pH shifted downward from 5.5 to 5.0. Also, the recombinant phytase was inhibited by the pseudo-substrate myo-inositol hexasulfate and also by antibody raised against a 20-mer peptide belonging to fungal phytase. The expressed phytase in alfalfa could also be modified by phenylglyoxal. Taken together, the results indicate that fungal phytase when cloned and expressed in alfalfa leaves produces stable and catalytically active phytase while retaining all the properties of the benchmark phytase. This affirms our view that "molecular biofarming" could be an alternative means of producing stable hydrolytic enzymes such as phytase.

6-Phytase↗