Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47phox Membrane Anchoring.

As a key component for NADPH oxidase 2 (NOX2) activation, the peripheral membrane protein p47phox translocates a cytosolic activating complex to the membrane through its PX domain. This study elucidates a potential regulatory mechanism of p47phox recruitment and NOX2 activation by inositol hexaphosphate (IP6). Through NMR, fluorescence polarization, and FRET experimental results, IP6 is shown to be capable of breaking the lipid binding and membrane anchoring events of p47phox-PX with low micromolar potency. Other phosphorylated inositol species such as IP5(1,3,4,5,6), IP4(1,3,4,5), and IP3(1,3,4) show weaker binding and no ability to inhibit lipid interactions in physiological concentration ranges. The low micromolar potency of IP6 inhibition of the p47phox membrane anchoring suggests that physiologically relevant concentrations of IP6 serve as regulators, as seen in other membrane anchoring domains. The PX domain of p47phox is known to be promiscuous to a variety of phosphatidylinositol phosphate (PIP) lipids, and this regulation may help target the domain only to the membranes most highly enriched with the highest affinity PIPs, such as the phagosomal membrane, while preventing aberrant binding to other membranes with high and heterogeneous PIP content, such as the plasma membrane. This study provides insight into a potential novel regulatory mechanism behind NOX2 activation and reveals a role for small-molecule regulation in this important NOX2 activator.

Characterization of recombinant phytase of Klebsiella sp. and the influence of novel 3-phytase on mineral solubility in broiler diets under an in vitro digestion assay.

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.

Biological and mechanical performance of calcium phosphate cements modified with phytic acid.

Calcium phosphate cements, primarily brushite cements, require the addition of setting retarders to ensure adequate processing time and processability. So far, citric acid has been the primary setting retarder used in this context. Due to the poor biocompatibility, it is crucial to explore alternative options for better processing. In recent years, the setting retarder phytic acid (IP6) has been increasingly investigated. This study investigates the biological behaviour of calcium phosphate cements with varying concentrations of IP6, in addition to their physical properties. Therefore cytocompatibility in vitro testing was performed using osteoblastic (MG-63) and osteoclastic (RAW 264.7 differentiated with RANKL) cells. We could demonstrate that the physical properties like the compressive strength of specimens formed with IP6 (brushite_IP6_5 = 11.2 MPa) were improved compared to the reference (brushite = 9.8 MPa). In osteoblast and osteoclast assays, IP6 exhibited significantly better cytocompatibility in terms of cell activity and cell number for brushite cements up to 11 times compared to the brushite reference. In contrast, the calcium-deficient hydroxyapatite (CDHA) cements produced similar results for IP6 (CDHA_IP6_0.25 = 27.0 MPa) when compared to their reference (CDHA = 21.2 MPa). Interestingly, lower doses of IP6 were found to be more effective than higher doses with up to 3 times higher. Additionally, IP6 significantly increased degradation in both passive and active resorption. For these reasons, IP6 is emerging as a strong new competitor to established setting retarders such as citric acid. These cements have potential applications in bone augmentation, the stabilisation of non-load bearing fractures (craniofacial), or the cementation of metal implants.

Effects of removing phytic acid on the bioaccessibility of Ca/Fe/Zn and protein digestion in soymilk.

BACKGROUND: Soymilk is a high-quality source of protein and minerals, such as calcium (Ca), iron (Fe), and zinc (Zn). However, phytic acid in soymilk restricts mineral and protein availability. We here investigated the effects of removing phytic acid on the physicochemical properties, mineral (Ca, Fe, and Zn) bioaccessibility, and protein digestibility of soymilk. RESULTS: Physicochemical property analysis revealed that the removal of phytic acid reduced protein accumulation at the gastric stage, thereby facilitating soymilk matrix digestion. The removal of phytic acid significantly increased Zn bioaccessibility by 18.19% in low-protein soymilk and Ca and Fe bioaccessibility by 31.20% and 30.03%, respectively, in high-protein soymilk. CONCLUSION: Removing phytic acid was beneficial for the hydrolysis of high-molecular-weight proteins and increased the soluble protein content in soymilk, which was conducive to protein digestion. This study offers a feasible guide for developing plant-based milk with high nutrient bioaccessibility. © 2024 Society of Chemical Industry.

Exploring the Biofilm Inhibition Potential of a Novel Phytic Acid-Crosslinked Chitosan Nanoparticle: In Vitro and In Vivo Investigations.

The primary factor underlying the virulence of Candida albicans is its capacity to form biofilms, which in turn leads to recurrent complications. Over-the-counter antifungal treatments have proven ineffective in eliminating fungal biofilms and the inflammatory cytokines produced during fungal infections. Chitosan nanoparticles offer broad and versatile therapeutic potential as both antifungal agents and carriers for antifungal drugs to combat biofilm-associated Candida infections. In our study, we endeavoured to develop chitosan nanoparticles utilising chitosan and the antifungal crosslinker phytic acid targeting C. albicans. Phytic acid, known for its potent antifungal and anti-inflammatory properties, efficiently crosslinks with chitosan. The nanoparticles were synthesised using the ionic gelation technique and subjected to analyses including Fourier transform infrared spectroscopy, dynamic light scattering, and zeta potential analysis. The synthesised nanoparticles exhibited dimensions with a diameter (Dh) of 103 ± 3.9 nm, polydispersity index (PDI) of 0.33, and zeta potential (ZP) of 37 ± 2.5 mV. These nanoparticles demonstrated an antifungal effect with a minimum inhibitory concentration (MIC) of 140 ± 2.2 µg/mL, maintaining cell viability at approximately 90% of the MIC value and reducing cytokine levels. Additionally, the nanoparticles reduced ergosterol content and exhibited a 62% ± 1.2 reduction in biofilm susceptibility, as supported by colony-forming unit (CFU) and XTT assays-furthermore, treatment with nanoparticles reduced exopolysaccharide production and decreased secretion of aspartyl protease by C. albicans. Our findings suggest that the synthesised nanoparticles effectively combat Candida albicans infections. In vivo studies conducted on a mouse model of vaginal candidiasis confirmed the efficacy of the nanoparticles in combating fungal infections in vivo.

Purification and biochemical characterization of phytase from Bacillus cereus isolated from gastrointestinal tract of African giant snail (Achatina fulica).

Phytases are specialized enzymes meant for phytic acid degradation. They possess ability to prevent phytic acid indigestion, including its attendant environmental pollution. This study was aimed at investigating biochemical properties of purified phytase of B. cereus isolated from Achatina fulica. Phytase produced from Bacillus cereus that exhibited optimal phytate degrading-ability of all the bacteria isolated was purified in a three-step purification. The biochemical properties of the purified enzyme were also determined. The phytase homogeny of approximately 45 kDa exhibited 12.8-purification fold and 1.6% yield with optima phytate degrading efficiency and maximum stability at pH 7 and 50 °C. Remaining activity of 52 and 47% obtained between 60 and 70 °C after 2 h further established thermostability of the purified phytase. Mg2+ and Zn2+ enhanced phytate hydrolysis by the enzyme, while Na+ showed mild inhibition but Hg2+ severely inhibited the enzymatic activity. Km and Vmax were estimated to be 0.11 mM and 55.6 µmol/min/mL, displaying enzyme-high substrate affinity and catalytic efficiency, respectively. Phytase purified from Bacillus cereus, isolated from African giant snails, has shown excellent characteristics suitable for phytic acid hydrolysis and could be employed in industrial and biotechnological applications.

Improving the color and functional properties of seabuckthorn seed protein with phytase treatment combined with alkaline solubilization and isoelectric precipitation.

BACKGROUND: Reducing anti-nutritional factors like phytates in seed protein products requires an ongoing effort. This study was the first to investigate the phytic acid content in seabuckthorn seed protein (SSP) and its reduction by an exogenous phytase during protein isolation from seabuckthorn seed meal through the common alkaline solubilization-isoelectric precipitation process. RESULTS: The additional phytase treatment could reduce the content of phytic acid from 22.46 to 13.27 g kg-1 , leading to SSP products with lighter color (lower ΔE* ), higher protein solubility, higher in vitro digestibility, but lower phenolic antioxidant content (including flavonoids and procyanidins) and some beneficial ions like Ca, Fe, Mg, and Zn. The Fourier transform infrared (FTIR) results indicated that the secondary structure of protein changed under the treatment with phytase. Correlation analysis showed that L* was significantly negatively correlated with TP, TPC and TF (P < 0.001), while a* and b* were significantly positively correlated with them (P < 0.001). CONCLUSIONS: There may be a trade-off between protein functionalities and other health-promoting components when a phytase treatment is included in SSP isolation. © 2021 Society of Chemical Industry.

Using Biotinylated myo-Inositol Hexakisphosphate to Investigate Inositol Pyrophosphate-Protein Interactions with Surface-Based Biosensors.

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated molecules that have emerged as central nutrient messengers in eukaryotic organisms. They can bind to structurally diverse target proteins to regulate biological functions, such as protein-protein interactions. PP-InsPs are strongly negatively charged and interact with highly basic surface patches in proteins, making their quantitative biochemical analysis challenging. Here, we present the synthesis of biotinylated myo-inositol hexakisphosphates and their application in surface plasmon resonance and grating-coupled interferometry assays, to enable the rapid identification, validation, and kinetic characterization of InsP- and PP-InsP-protein interactions.

Pharmacokinetic properties of a novel formulation of S-adenosyl-L-methionine phytate.

S-adenosyl-L-methionine (SAM), the main endogenous methyl donor, is the adenosyl derivative of the amino acid methionine, which displays many important roles in cellular metabolism. It is widely used as a food supplement and in some countries is also marketed as a drug. Its interesting nutraceutical and pharmacological properties prompted us to evaluate the pharmacokinetics of a new form of SAM, the phytate salt. The product was administered orally to rats and pharmacokinetic parameters were evaluated by comparing the results with that obtained by administering the SAM tosylated form (SAM PTS). It was found that phytate anion protects SAM from degradation, probably because of steric hindrance exerted by the counterion, and that the SAM phytate displayed significant better pharmacokinetic parameters compared to SAM PTS. These results open to the perspective of the use of new salts of SAM endowed with better pharmacokinetic properties.

A dansyl-derivatized phytic acid analogue as a fluorescent substrate for phytases: experimental and computational approach.

A new myo-inositol pentakisphosphate was synthesized, which featured a dansyl group at position C-5. The fluorescent tag was removed from the inositol by a 6-atom spacer to prevent detrimental steric interactions in the catalytic site of phytases. The PEG linker was used in order to enhance hydrophilicity and biocompatibility of the new artificial substrate. Computational studies showed a favorable positioning in the catalytic site of phytases. Enzymatic assays demonstrated that the tethered myo-inositol was processed by two recombinant phytases Phy-A and Phy-C, classified respectively as acid and alkaline phytases, with similar rates of phosphate release compared to their natural substrate.

Flame Retardant Functionalization of Microcrystalline Cellulose by Phosphorylation Reaction with Phytic Acid.

The functionalization of microcrystalline cellulose (MCC) is an important strategy for broadening its application fields. In the present work, MCC was functionalized by phosphorylation reaction with phytic acid (PA) for enhanced flame retardancy. The conditions of phosphorylation reaction including PA concentration, MCC/PA weight ratio and temperature were discussed, and the thermal degradation, heat release and char-forming properties of the resulting PA modified MCC were studied by thermogravimetric analysis and pyrolysis combustion flow calorimetry. The PA modified MCC, which was prepared at 90 °C, 50%PA and 1:3 weight ratio of MCC to PA, exhibited early thermal dehydration with rapid char formation as well as low heat release capability. This work suggests a novel strategy for the phosphorylation of cellulose using PA and reveals that the PA phosphorylated MCC can act as a promising flame retardant material.

Preparation of pure lower-order myo-Inositol phosphates on laboratory scale for physiological and enzymatic studies.

A simple method for the preparative production of lower-order myo-inositol phosphates was developed. Enzymatic phytate dephosphorylation was applied, because phytate-degrading enzymes generate usually predominantly one single myo-inositol phosphate isomer with five, four, three, two and one phosphate residue(s) bound to the myo-inositol ring in a regio- and stereoselective manner. The relative concentrations of the different lower-order myo-inositol phosphates in the reaction mixture were controlled by adjusting incubation time at 37 °C and a fixed phytate concentration and phytase activity. Purification of the individual lower-order myo-inositol phosphates was realized by anion-exchange chromatography on Q-Sepharose using a stepwise elution with ammonium formate:formic acid pH 2.5. Ethanol precipitation was successfully used to concentrate the pure lower-order myo-inositol phosphates. In a single approach 2-3 mg of pure myo-inositol tetrakis- or -trisphosphate isomers were obtained. About 60% of the initially applied phytate were converted into pure lower-order myo-inositol phosphates. The purified myo-inositol phosphate isomers were virtually free of other myo-inositol phosphate esters and could be used for enzymatic and physiological studies.

Limitations of an in vitro model of the poultry digestive tract on the evaluation of the catalytic performance of phytases.

BACKGROUND: The study aims to investigate the limitation of a poultry digestive tract model developed by Menezes-Blackburn et al. [J Agric Food Chem 63: 6142-6149 (2015)] on the evaluation of the bioefficacy of phytases. RESULTS: It was confirmed that the in vitro model does not mimic the in vivo situation in the birds sufficiently well to identify the best phytase product under real conditions, or to draw conclusion on the effect of phytate concentration, phytate source or feed composition on the bioefficacy of phytase. Addition of calcium ion (Ca2+ ) up to a concentration of 10 g kg-1 to the feed substrate, for example, did not affect enzymatic phytate dephosphorylation in the in vitro model in contrast to the observation in poultry. CONCLUSION: The in vitro approach was shown to be applicable as a complementary tool in the pre-selection of promising phytase candidates, resulting in a reduction in the number of feeding trials in the initial screening phase. © 2020 The Author. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Facile Preparation of Hydrophilic Mesoporous Metal-Organic Framework via Synergistic Etching and Surface Functionalization for Glycopeptides Analysis.

In view of the size and hydrophilicity of glycopeptides, materials having suitable channels (size-exclusion) and strong hydrophilic surface (hydrophilic interaction) are preferred to enrich the glycopeptides in biological samples. Metal-organic frameworks (MOFs) are good candidates. However, their smaller microporous channels and low chemical stability have limited the application. Herein, a facile strategy was established to construct hydrophilic mesoporous MOF via synergistic etching and surface functionalization by using phytic acid (PA). Besides, polyvinylpyrrolidone (PVP) was added during MOF synthesis to enhance the water stability of the MOF. Owing to the expanded hydrophilic mesoporous channels, the PA-modified Ce-MOF effectively and selectively captured 422 glycopeptides from 155 glycoproteins in tryptic digests of human serum (2 µL). The present work sheds light on the easy fabrication of hydrophilic mesoporous materials, and this established material holds unique advantages for glycopeptides analysis in biological samples.

Addition of Whole Wheat Flour During Injera Fermentation Degrades Phytic Acid and Triples Iron Absorption from Fortified Tef in Young Women.

BACKGROUND: Iron deficiency is a major public health concern in Ethiopia, where the traditional diet is based on tef injera. Iron absorption from injera is low due to its high phytic acid (PA) content. OBJECTIVES: We investigated ways to increase iron absorption from FeSO4-fortified tef injera in normal-weight healthy women (aged 21-29 y). METHODS: Study A (n = 22) investigated the influence on fractional iron absorption (FIA) from FeSO4-fortified injera of 1) replacing 10% tef flour with whole wheat flour (a source of wheat phytase), or 2) adding an isolated phytase from Aspergillus niger. Study B (n = 18) investigated the influence on FIA of replacing FeSO4 in tef injera with different amounts of NaFeEDTA. In both studies, the iron fortificants were labeled with stable isotopes and FIA was calculated from erythrocyte incorporation of stable iron isotopes 14 d after administration. RESULTS: In study A, the median (IQR) FIA from the 100% tef injera meal was 1.5% (0.7-2.8%). This increased significantly (P < 0.05) to 5.3% (2.4-7.1%) on addition of 10% whole wheat flour, and to 3.6% (1.6-6.2%) on addition of A. niger phytase. PA content of the 3 meals was 0.62, 0.20, and 0.02 g/meal, respectively. In study B, the median (IQR) FIA from the 100% tef injera meal was 3.3% (1.1-4.4%) and did not change significantly (P > 0.05) on replacing 50% or 75% of FeSO4 with NaFeEDTA. CONCLUSIONS: FIA from tef injera by young women was very low. NaFeEDTA was ineffective at increasing iron absorption, presumably due to the relatively low EDTA:Fe molar ratios. Phytate degradation, however, greatly increased during tef fermentation on addition of native or isolated phytases. Replacing 10% tef with whole wheat flour during injera fermentation tripled FIA in young women and should be considered as a potential strategy to improve iron status in Ethiopia.

Phytase catalysis of dephosphorylation studied using isothermal titration calorimetry and electrospray ionization time-of-flight mass spectroscopy.

Phytases are important commercial enzymes that catalyze the dephosphorylation of myo-inositol hexakisphosphate (phytate) to its lower inositol phosphate (IP) esters, IP6 to IP1. Digestion of phytate by Citrobacter braakii 6-phytase deviates significantly from monophasic Michaelis-Menten kinetics. Analysis of phytate digestion using isothermal titration calorimetry (ITC) using the single injection method produced a thermogram with two peaks consistent with two periods of high enzyme activity. Continuous-flow electrospray ionization time-of-flight mass spectroscopy (ESI-ToF-MS) provided real-time analysis of phytase catalysis. It was able to show that the first two cleavage steps were rapid and concurrent but the third cleavage step from IP4 to IP3 was slow. The third (IP4 to IP3), fourth (IP3 to IP2) and fifth (IP2 to IP1) cleavages were effectively sequential due to the preferred association of the more phosphorylated species with the phytase catalytic site. This created a bottleneck during the cleavage of IP4 to IP3 until the point at which IP4 was exhausted and was followed by the rapid cleavage of IP3 to IP2, which was observed as the second peak in the ITC thermogram. This work illustrates the importance of an orthogonal approach when studying non-specific or complex enzyme catalyzed reactions.

Harvesting of Arthrospira platensis by flocculation with phytic acid from rice bran.

The recovery of algal biomass is one of the critical steps involved in the commercial production of beneficial metabolites from Arthrospira platensis. Efficient and safe harvesting methods that do not sacrifice quality of final product are important for commercial application. Phytic acid (PA) is a natural non-toxic phytochemical widely distributed in plant tissues. Effect of PA from rice bran on the growth, trichome morphology such as spiral number and algal filament length, and harvesting efficiency of A. platensis were investigated. Cells aggregated into large cell flocs after the addition of PA in the medium, and algal spiral number and filament length increased. UV-vis spectra indicated the interactions between PA and algal cells. Adding PA at stationary growth phase is a good strategy for harvesting, since no adverse effect to biomass growth and harvesting efficiency. Harvesting efficiency of 95.69% at 0.5% (v/v) PA was superior to other conventional harvesting methodologies. ABBREVIATIONS: PA: Phytic acid; PUFAs: Polyunsaturated fatty acids; FAO: Food and Agriculture Organization; γ-PGA: Poly (γ-glutamic acid); CNF: Cellulose nanofibrils; NIES: National Institute for Environmental Studies; SOT: Spirulina-Ogawa-Terui; CG: Control group; pI: Isoelectric point.

Enzymatically hardened calcium phosphate biocement with phytic acid addition.

Novel enzymatically hardened tetracalcium phosphate/monetite cements were prepared applying phytic acid/phytase (PHYT/F3P) mixture as hardening liquid after dissolving in acetic acid solution (CX cement). Properties of the cements were compared with classic cement hardened with 2% NaH2PO4 (C cement) and cement hardened with acetic acid solution (CAC cement) only. In the microstructure of CX cement, columnar growth of hydroxyapatite particles was found in the form of walls around hydroxyapatite agglomerates originated from tetracalcium phosphate which were mutually separated by a material depleted low density zone. Wet compressive strengths (CS) of all cements were practically identical contrary to about 30% higher dry CS's of CX and CAC cements due to specific microstructure. It was verified noncytotoxic character of CX cement extracts and positive effect of CX cement on ALP activity and cell behavior during cultivation. The final Ca/P molar ratio and setting time of cement were effectively controlled by the amount of phytic acid and the change in PHYT/F3P mass ratio, or reaction time in hardening liquid, respectively.

Globoids and Phytase: The Mineral Storage and Release System in Seeds.

Phytate and phytases in seeds are the subjects of numerous studies, dating back as far as the early 20th century. Most of these studies concern the anti-nutritional properties of phytate, and the prospect of alleviating the effects of phytate with phytase. As reasonable as this may be, it has led to a fragmentation of knowledge, which hampers the appreciation of the physiological system at hand. In this review, we integrate the existing knowledge on the chemistry and biosynthesis of phytate, the globoid cellular structure, and recent advances on plant phytases. We highlight that these components make up a system that serves to store and-in due time-release the seed's reserves of the mineral nutrients phosphorous, potassium, magnesium, and others, as well as inositol and protein. The central component of the system, the phytate anion, is inherently rich in phosphorous and inositol. The chemical properties of phytate enable it to sequester additional cationic nutrients. Compartmentalization and membrane transport processes regulate the buildup of phytate and its associated nutrients, resulting in globoid storage structures. We suggest, based on the current evidence, that the degradation of the globoid and the mobilization of the nutrients also depend on membrane transport processes, as well as the enzymatic action of phytase.

Signalling Properties of Inositol Polyphosphates.

Several studies have identified specific signalling functions for inositol polyphosphates (IPs) in different cell types and have led to the accumulation of new information regarding their cellular roles as well as new insights into their cellular production. These studies have revealed that interaction of IPs with several proteins is critical for stabilization of protein complexes and for modulation of enzymatic activity. This has not only revealed their importance in regulation of several cellular processes but it has also highlighted the possibility of new pharmacological interventions in multiple diseases, including cancer. In this review, we describe some of the intracellular roles of IPs and we discuss the pharmacological opportunities that modulation of IPs levels can provide.