Functional assessment of AtPAP17; encoding a purple acid phosphatase involved in phosphate metabolism in Arabidopsis thaliana.

PURPOSE: Purple acid phosphatases (PAPs) includ the largest classes of non-specific plant acid phosphatases. Most characterized PAPs were found to play physiological functions in phosphorus metabolism. In this study, we investigated the function of AtPAP17 gene encoding an important purple acid phosphatase in Arabidopsis thaliana. METHODS: The full-length cDNA sequence of AtPAP17 gene under the control of CaMV-35S promoter was transferred to the A. thaliana WT plant. The generated homozygote AtPAP17-overexpressed plants were compared by the types of analyses with corresponding homozygote atpap17-mutant plant and WT in both + P (1.2 mM) and - P (0 mM) conditions. RESULTS: In the + P condition, the highest and the lowest amount of Pi was observed in AtPAP17-overexpressed plants and atpap17-mutant plants by 111% increase and 38% decrease compared with the WT plants, respectively. Furthermore, under the same condition, APase activity of AtPAP17-overexpressed plants increased by 24% compared to the WT. Inversely, atpap17-mutant plant represented a 71% fall compared to WT plants. The comparison of fresh weight and dry weight in the studied plants showed that the highest and the lowest amount of absorbed water belonged to OE plants (with 38 and 12 mg plant-1) and Mu plants (with 22 and 7 mg plant-1) in + P and - P conditions, respectively. CONCLUSION: The lack of AtPAP17 gene in the A. thaliana genome led to a remarkable reduction in the development of root biomass. Thus, AtPAP17 could have an important role in the root but not shoot developmental and structural programming. Consequently, this function enables them to absorb more water and eventually associated with more phosphate absorption.

Selectivity in Enzymatic Phosphorus Recycling from Biopolymers: Isotope Effect, Reactivity Kinetics, and Molecular Docking with Fungal and Plant Phosphatases.

Among ubiquitous phosphorus (P) reserves in environmental matrices are ribonucleic acid (RNA) and polyphosphate (polyP), which are, respectively, organic and inorganic P-containing biopolymers. Relevant to P recycling from these biopolymers, much remains unknown about the kinetics and mechanisms of different acid phosphatases (APs) secreted by plants and soil microorganisms. Here we investigated RNA and polyP dephosphorylation by two common APs, a plant purple AP (PAP) from sweet potato and a fungal phytase from Aspergillus niger. Trends of δ18O values in released orthophosphate during each enzyme-catalyzed reaction in 18O-water implied a different extent of reactivity. Subsequent enzyme kinetics experiments revealed that A. niger phytase had 10-fold higher maximum rate for polyP dephosphorylation than the sweet potato PAP, whereas the sweet potato PAP dephosphorylated RNA at a 6-fold faster rate than A. niger phytase. Both enzymes had up to 3 orders of magnitude lower reactivity for RNA than for polyP. We determined a combined phosphodiesterase-monoesterase mechanism for RNA and terminal phosphatase mechanism for polyP using high-resolution mass spectrometry and 31P nuclear magnetic resonance, respectively. Molecular modeling with eight plant and fungal AP structures predicted substrate binding interactions consistent with the relative reactivity kinetics. Our findings implied a hierarchy in enzymatic P recycling from P-polymers by phosphatases from different biological origins, thereby influencing the relatively longer residence time of RNA versus polyP in environmental matrices. This research further sheds light on engineering strategies to enhance enzymatic recycling of biopolymer-derived P, in addition to advancing environmental predictions of this P recycling by plants and microorganisms.

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris.

BACKGROUND: Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains. RESULTS: Potential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter. DISCUSSION: The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

Investigating the thermodynamic and kinetics properties of acid phosphatase extracted and purified from seedlings of Chenopodium murale.

Herein acid phosphatase isoenzyme was extracted from the C. murale seedlings. The purification was accomplished by chromatographic techniques and passing through DEAE-cellulose and Sephadex G-100 column. The specific activity of acid phosphatase 5.75 U/mg of protein was obtained with 66 purification fold 15.8% yield and molecular mass was 29 kDa with very faint bands corresponding to 18 kDa and 14 kDa. The maximal activity at pH 5.0 and 50 °C best illustrated by first order kinetics. When temperature was raised (55 °C to 75 °C), the deactivation rate constant was increased from 0.001 to 0.014 min-1, while half-life was decreased from 693 to 49 min-1. The results of activity collected at different temperature were then used to estimate, activation energy of hydrolysis reaction (Ea = 47.59 kJmol-1). A high Z-value (18.86 °C min-1) was obtained indicating a less sensitivity towards temperatures. The residual activity examinations were carried out from 55 °C to 75 °C and assessing the Deactivation Energy (Ed 116.39 kJmol-1), Enthalpy change (ΔH° 113.55kJmol-1), Entropy change (ΔS° 110.33kJmol-1) and change in Gibbs free energy (ΔG° 10.02 kJmol-1). Taken together, thermodynamic parameters confirm the high stability of enzyme and show potential commercial applicability.

A novel acid phosphatase from cactus (Opuntia megacantha Salm-Dyck) cladodes: Purification and biochemical characterization of the enzyme.

Acid phosphatase (ACP) plays an important role in regulating phosphate nutrition in plants. Herein, for the first time, a novel ACP from Opuntia megacantha Salm-Dyck cladodes was purified to homogeneity and biochemically characterized. Specific activity of 8.78 U/mg was obtained with 11.29-fold purification and 15% yield. ACP was purified as monomer with molecular weight of 44 kDa as determined by SDS-PAGE under denaturing and nondenaturing conditions. Optimum pH and temperature for ACP activity was 5.5 and 60 °C, respectively. Thermodynamic parameters (Ea, ΔH, ΔG and ΔS) were also determined. ACP activity was stimulated by Ca2+, strongly inhibited by Cu2+ and Fe3+, and moderately inhibited by Mg2+ and Zn2+. Br-, CN-, F-, I- and N3- weakly inhibited ACP activity, where more than 70% of enzyme activity was remained at 5 mM. In addition, effect of ß-ME, Cys, DTT, EDTA, H2O2, PMSF, SDS and TX-100 on ACP activity was investigated. km, Vmax, kcat and kcat/km of ACP for p-NPP were found to be 0.09 mM, 2.75 U/mL, 9.60 s-1 and 106.67 s-1 mM-1, respectively. The biochemical properties of ACP from Opuntia megacantha Salm-Dyck cladodes provide novel features with other plant ACPs and basic knowledge of ACP in Opuntia species.

Preparative-Scale Enzymatic Synthesis of rac-Glycerol-1-phosphate from Crude Glycerol Using Acid Phosphatases and Phosphate.

Glycerol is a byproduct of biodiesel production and is generated in large amounts, which has resulted in an increased interest in its valorization. In addition to its use as an energy source directly, the chemical modification of glycerol may result in value-added derivatives. Herein, acid phosphatases employed in the synthetic mode were evaluated for the enzymatic phosphorylation of glycerol. Nonspecific acid phosphatases could tolerate glycerol concentrations up to 80 wt % and pyrophosphate concentrations up to 20 wt % and led to product titers up to 167 g L-1 in a kinetic approach. In the complementary thermodynamic approach, phytases were able to condense glycerol and inorganic monophosphate directly. This unexpected behavior enabled the simple and cost-effective production of rac-glycerol-1-phosphate from crude glycerol obtained from a biodiesel plant. A preparative-scale synthesis on a 100 mL-scale resulted in the production of 16.6 g of rac-glycerol-1-phosphate with a reasonable purity (≈75 %).

Uranium bioremediation by acid phosphatase activity of Staphylococcus aureus biofilms: Can a foe turn a friend?

In this study, Staphylococcus aureus biofilms, which are considered a foe for being pathogenic, were tested for their uranium bioremediation capacity to find out if they can turn out to be a friend. Acid phosphatase activity, which is speculated to aid in bio-precipitation of U(VI) from uranyl nitrate solution, was assayed in biofilms of seven different S. aureus strains. The presence of acid phosphatase enzyme was detected in the biofilms of all S. aureus strains (in the range of 3.1 ± 0.21 to 26.90 ± 2.32 µi.u./g), and found to be higher when compared to that of their planktonic phenotypes. Among all, S. aureus V329 biofilm showed highest biofilm formation ability along with maximum phosphatase activity (26.9 ± 2.32 µi.u./g of biomass). Addition of phosphate enhanced the U(VI) remediation when treated with uranyl nitrate solution. S. aureus V329 biofilm showed significant U tolerance with only a 3-log reduction when exposed to 10 ppm U(VI) for 1 h. When treated in batch mode, V329 biofilm successfully remediated up to 47% of the 10 ppm U(VI). This new approach using the acid phosphatase from the S. aureus V329 biofilm presents an alternative method for the remediation of uranium contamination.

Nitrogen-doped carbon dots as a ratiometric fluorescent probe for determination of the activity of acid phosphatase, for inhibitor screening, and for intracellular imaging.

The author describe a method for preparation of green fluorescent nitrogen-doped carbon dots (N-CDs) through hydrothermal treatment of a mixture of lotus leaf juice and ethylenediamine (EDA). The N-CDs have uniform size, good dispersibility and water solubility. Under 316 and 366 nm photoexcitation, they show dual fluorescence with emission peaks at 415 and 509 nm, respectively. They are positively charge and display low cytotoxicity. This makes them an excellent choice for fluorometric assays and for bioimaging. A ratiometric assay was developed for the determination of the activity of acid phosphatase (ACP). It is based on the aggregation- induced quenching (AIQ) of the fluorescence of the N-CDs by sodium hexametaphosphate (NaPO3)6. Enzymatic hydrolysis of (NaPO3)6 by ACP leads to the disintegration of (NaPO3)6 and to the restoration of fluorescence. The measurement of the ratio of fluorescence at two wavelengths (415 and 509 nm), background interference and fluctuating signals can be widely eliminated. The method works in the 1-50 U·L-1 ACP activity range and has a detection limit of 0.43 U·L-1. It was successfully applied (a) to the determination of ACP in spiked serum samples, (b) to ACP inhibitor screening, and (c) to imaging of ACP in HePG2 cells. Graphical abstract Schematic presentation of the synthesis of nitrogen-doped carbon dots (N-CDs), and their application to the ratiometric fluorometric determination of acid phosphatase (ACP) based on the aggregation-induced quenching and enzymatic hydrolysis.

Contrasting effects of nitrogen and phosphorus additions on soil nitrous oxide fluxes and enzyme activities in an alpine wetland of the Tibetan Plateau.

Alpine wetlands are important ecosystems, but an increased availability of soil nutrients may affect their soil nitrous oxide (N2O) fluxes and key enzyme activities. We undertook a 3-year experiment of observing nitrogen (N) and/or phosphorus (P) addition to alpine wetland soils of the Tibetan Plateau, China, with measurements made of soil extracellular enzyme activities and soil N2O fluxes. Our study showed that soil N2O flux was significantly increased by 72% and 102% following N and N+P additions, respectively. N addition significantly increased acid phosphatase (AP) and ß-1, 4-N-acetyl-glucosaminidase (NAG) activities by 32% and 26%, respectively. P addition alone exerted a neutral effect on soil AP activities, while increasing NAG activities. We inferred that microbes produce enzymes based on 'resource allocation theory', but that a series of constitutive enzymes or the treatment duration interfere with this response. Our findings suggest that N addition increases N- and P-cycling enzyme activities and soil N2O flux, whereas P addition exerts a neutral effect on P-cycling enzyme activities and N2O flux after 3 years of nutrient applications to an alpine wetland.

Genome-wide analysis of purple acid phosphatase (PAP) family proteins in Jatropha curcas L.

Purple acid phosphatase (PAP) family genes play a crucial role in the phosphorus (P) foraging and recycling. There are 25 putative Jatropha curcas PAP genes (JcrPAP) were identified and classified into three groups based on their molecular weights. Subcellular localization prediction indicated that most of the JcrPAPs were localized to secretory pathway. Several PAPs possess signal peptide motifs and varied numbers of N-glycosylation and transmembrane helix motifs. JcrPAP proteins have 3-5 active pocket sites comprising 1 to 11 binding residues which interact with different ligands such as iron (Fe), N-acetyl l-d-Glucosamine (NAG), zinc (Zn) and manganese (Mn). The core structure of the predicted JcrPap28 was similar to the Ipomoea batatas Pap protein. Most of the JcrPAP genes showed higher expression in the root tissues compared to stem and leaf tissues. Several JcrPAP genes were upregulated under low phosphorus conditions. JcrPAP genes such as JcrPap26b, JcrPap27b, and JcrPap28 have shown multifold induction in low phosphorus treated plants which suggest that these genes might be involved in phosphorus metabolism. The present study provided the structural variations and expression regulation of JcrPAP genes in the economically viable biodiesel crop and it would be helpful for the crop improvement under phosphorus limiting conditions.

A rational design to enhance the resistance of Escherichia coli phytase appA to trypsin.

Escherichia coli phytase appA, which hydrolyzes phytate, has been widely applied as an important feed supplement, but its resistance to trypsin needs to be improved. Six putative solvent-accessible amino acid residues (K74, K75, K180, R181, K183, and K363), which could be easily attacked by trypsin, were selected to improve trypsin tolerance of Escherichia coli phytase appA. Inspection of the three-dimensional structure and computational design via hydrogen bond analysis, six optimal mutation sites of K74D/K75Q/K180N/R181N/K183S/K363N, which strengthened the hydrogen bonding, were performed to generate three mutants. Results showed that the most beneficial mutant appA-M6 had a specific activity of 3262 U/mg with molecular weight of approximately 52-55 kDa. Similar to appA-WT, the optimal pH (4.5) and temperature (60 °C) of appA-M6 were unchanged. Compared with appA-WT, appA-M6 showed a significant enhancement (p < 0.05) in resistance to trypsin and a 3.8 °C increase in melting temperature (Tm). We concluded that introduction of hydrogen bonds and N-glycosylation modification resulted in decreased enzyme flexibility and increased the enzyme stability against proteolysis and thermal denaturation. The mutant appA-M6 generated in this study could be applied for the large-scale commercial production of phytase and thus could benefit the food and feed industry.

Alumina nanoparticle-assisted enzyme refolding: A versatile methodology for proteins renaturation.

We present a high-yield method for the renaturation of negatively charged enzymes. The approach is based on the use of alumina nanoparticles, which after electrostatic interaction with denatured protein molecules, prevent their aggregation and make the process of refolding controllable. The method, demonstrated by the renaturation of several enzymes, is efficient, rapid, employs a minimal amount of reagents and even can be applied to renature mixture of the denatured enzymes.

Immobilization of Enzymes on PLGA Sub-Micrometer Particles by Crosslinked Layer-by-Layer Deposition.

Enzyme immobilization is of high interest for industrial applications. However, immobilization may compromise enzyme activity or stability due to the harsh conditions which have to be applied. The authors therefore present a new and improved crosslinked layer-by-layer (cLbL) approach. Two different model enzymes (acid phosphatase and ß-galactosidase) are immobilized under mild conditions on biocompatible, monodisperse, sub-micrometer poly(lactide-co-glycolide) (PLGA) particles. The resulting PLGA enzyme systems are characterized regarding their size, surface charge, enzyme activity, storage stability, reusability, and stability under various conditions such as changing pH and temperature. The developed and characterized cLbL protocol can be easily adapted to different enzymes. Potential future uses of the technology for biomedical applications are discussed. PLGA-enzyme particles are therefore injected into the blood circulation of zebrafish embryos in order to demonstrate the in vivo stability and activity of the designed system.

Impact of nitrogen pollution/deposition on extracellular enzyme activity, microbial abundance and carbon storage in coastal mangrove sediment.

This study applied different concentration of NaNO3 solution to simulate the effect of inorganic nitrogen (N) deposition/pollution on carbon (C) storage in coastal mangrove sediment through observing the changes of enzyme activity and microbial abundance. Sediment collected from mangrove forest (MG) and intertidal zone (IZ) were incubated with different N rates (0 (control), 5 (low-N) and 20 (high-N) µg N g-1 dry sediment, respectively). After incubation, the activities of phenol oxidase (PHO) and acid phosphatase (ACP) were enhanced, but ß-glucosidase (GLU) and N-ß-acetyl-glucosaminidase (NAG) activities were reduced by N addition. The altered enzymatic stoichiometries by N input implied that microbial phosphorus (P) limitation was increased, whereas C and N limitation were alleviated. Besides, N input decreased the bacterial abundance but increased fungal abundance in both types of sediment. The increased pH and soluble phenolics along with the exacerbated P limitation by N addition might explain these changes. Furthermore, sediment with N addition (except high-N treated MG sediment) showed a trend of C sequestration, which might be largely caused by the decrease of bacterial abundance and GLU activity. However, MG sediment with high-N suggested a trend of C loss, and the possible reason for this discrepancy might be the relatively higher increase of PHO and ACP activity. To better understand the influence of N deposition/pollution on C cycling, the long-term N effect on microorganisms, enzymes, and thus C storage should be paid more attention in the future.

Protection of enzymes from photodegradation by entrapment within alumina.

Most enzymes are highly sensitive to UV-light in all of its ranges and their activity can irreversibly drop even after a short time of exposure. Here we report a solution of this problem by using sol-gel matrices as effective protectors against this route of enzyme inactivation and denaturation. The concept presented here utilizes several modes of action: First, the entrapment within the rigid ceramic sol-gel matrix, inhibits denaturation motions, and the hydration shell around the entrapped protein provides extra protection. Second, the matrix itself - alumina in this report - absorbs UV light. And third, sol-gel materials have been shown to be quite universal in their ability to entrap small molecules, and so co-entrapment with well documented sun-screening molecules (2-hydroxybenzophenone, 2,2'-dihydroxybenzophenone, and 2,2'-dihydroxy-4-methoxybenzophenone) is an additional key protective tool. Three different enzymes as models were chosen for the experiments: carbonic anhydrase, acid phosphatase and horseradish peroxidase. All showed greatly enhanced UV (regions UV-A, UV-B, and UV-C) stabilization after entrapment within the doped sol-gel alumina matrices.

Studies on Chromatographic Fingerprint and Fingerprinting Profile-Efficacy Relationship of Saxifraga stolonifera Meerb.

This work investigated the spectrum-effect relationships between high performance liquid chromatography (HPLC) fingerprints and the anti-benign prostatic hyperplasia activities of aqueous extracts from Saxifraga stolonifera. The fingerprints of S. stolonifera from various sources were established by HPLC and evaluated by similarity analysis (SA), hierarchical clustering analysis (HCA) and principal component analysis (PCA). Nine samples were obtained from these 24 batches of different origins, according to the results of SA, HCA and the common chromatographic peaks area. A testosterone-induced mouse model of benign prostatic hyperplasia (BPH) was used to establish the anti-benign prostatic hyperplasia activities of these nine S. stolonifera samples. The model was evaluated by analyzing prostatic index (PI), serum acid phosphatase (ACP) activity, concentrations of serum dihydrotestosterone (DHT), prostatic acid phosphatase (PACP) and type II 5α-reductase (SRD5A2). The spectrum-effect relationships between HPLC fingerprints and anti-benign prostatic hyperplasia activities were investigated using Grey Correlation Analysis (GRA) and partial least squares regression (PLSR). The results showed that a close correlation existed between the fingerprints and anti-benign prostatic hyperplasia activities, and peak 14 (chlorogenic acid), peak 17 (quercetin 5-O-ß-d-glucopyranoside) and peak 18 (quercetin 3-O-ß-l-rhamno-pyranoside) in the HPLC fingerprints might be the main active components against anti-benign prostatic hyperplasia. This work provides a general model for the study of spectrum-effect relationships of S. stolonifera by combing HPLC fingerprints with a testosterone-induced mouse model of BPH, which can be employed to discover the principle components of anti-benign prostatic hyperplasia bioactivity.

Evaluation of the antioxidant and anti-osteoporosis activities of chemical constituents of the fruits of Prunus mume.

The present study investigated the antioxidant and anti-osteoporosis activities of phytochemicals in the fruits of Prunus mume. From the methanol extract, three new acylated sucroses, mumeoses P-R (1-3), were isolated together with 20 known compounds (4-23). Compounds 1-3 showed potent peroxyl radical-scavenging activities and 12-19 showed both potent peroxyl radical-scavenging and reducing activities. The anti-osteoporosis activity was evaluated using murine pre-osteoblastic MC3T3-E1 cells and pre-osteoclastic RAW 264.7 cells. Compounds 2 and 3 (cis-trans isomers), 5, 7, 8, and 10 significantly stimulated the differentiation of pre-osteoblastic MC3T3-E1 cells to increase collagen synthesis or mineralization functions of osteoblasts, while compounds 5, 6, 9, 10, 12, 14-16, 18, 20, and 22 significantly suppressed tartrate-resistant acid phosphatase activity in receptor activator of nuclear factor-κB ligand-induced osteoclastic RAW 264.7 cells. These results indicated that the fruits of P. mume are an excellent source of antioxidant and anti-osteoporosis phytochemicals.

GmPAP4, a novel purple acid phosphatase gene isolated from soybean (Glycine max), enhanced extracellular phytate utilization in Arabidopsis thaliana.

KEY MESSAGE: GmPAP4 , a novel plant PAP gene in soybean, has phytase activity. Over-expressing GmPAP4 can enhance Arabidopsis growth when phytate is the sole P source in culture. Phosphorus (P) is an important macronutrient for plant growth and development. However, most of the total P in soils is fixed into organic phosphate (Po). Purple acid phosphatase (PAP) can hydrolyze Po in the soil to liberate inorganic phosphate and enhance plant P utilization. We isolated a novel PAP gene, GmPAP4, from soybean (Glycine max). It had an open reading frame of 1,329 bp, encoding 442 amino acid residues. Sequence alignment and phylogenetics analysis indicated that GmPAP4 was similar to other plant PAPs with large molecular masses. Quantitative real-time PCR analysis showed that the induced expression of GmPAP4 was greater in P-efficient genotype Zhonghuang15 (ZH15) than in P-inefficient genotype Niumaohuang (NMH) during the periods of flowering (28-35 days post phytate stress; DPP) and pod formation (49-63 DPP). Moreover, peak expression, at 63 DPP, was about 3-fold higher in 'ZH15' than in 'NMH'. Sub-cellular localization showed that GmPAP4 might be on plasma membrane or in cytoplasm. Over-expressing GmPAP4 in Arabidopsis resulted in significant rises in P acquisition and utilization compared with the wild-type (WT). Under phytate condition, transgenic Arabidopsis plants showed increases of approximately 132.7 % in dry weight and 162.6 % in shoot P content compared with the WT. Furthermore, when phytate was added as the sole P source in cultures, the activity of acid phosphatase was significantly higher in transgenic plants. Therefore, GmPAP4 is a novel PAP gene that functions in plant's utilization of organic phosphate especially under phytate condition.

The physical characteristics and emulsification properties of partially dephosphorylated bovine ß-casein.

Bovine ß-casein was purified from phosphocasein by rennet coagulation and cold solubilisation from the resultant curd. ß-Casein was then dephosphorylated using potato acid phosphatase. Urea-polyacrylamide gel electrophoresis (PAGE) of partially dephosphorylated ß-casein showed a number of bands, depending on the final level of phosphorylation. Dephosphorylating ß-casein increased its pH of minimum solubility from ∼pH 5 to 5.5 and reduced its net negative charge from -30.8 to -27.0 mV. During the acidification of ß-casein solutions, partially dephosphorylated ß-casein failed to form a gel, unlike the phosphorylated (i.e., control) ß-casein. Use of partially dephosphorylated ß-casein to stabilise oil-in-water emulsions resulted in larger fat globules compared to control ß-casein, but such globules were less susceptible to aggregation in the presence of 15 or 30 mM CaCl(2). Overall, the dephosphorylation of ß-casein resulted in a protein similar to human ß-casein in terms of physicochemical functionality, with increased stability against calcium-induced aggregation.

18O enrichment in phosphorus pools extracted from soybean leaves.

The objective of this study was to investigate the isotopic composition of oxygen bound to phosphate (δ(18)O-PO(4)) in different phosphorus (P) pools in plant leaves. As a model plant we used soybean (Glycine max cv Toliman) grown in the presence of ample P in hydroponic cultures. The leaf blades were extracted with 0.3 M trichloroacetic acid (TCA) and with 10 M nitric acid. These extractions allowed measurement of the TCA-soluble reactive P (TCA P) that is rapidly cycled within the cell and the total leaf P. The difference between total leaf P and TCA P yielded the structural P which includes organic P compounds not extractable by TCA. P uptake and its translocation and transformation within the soybean plants lead to an (18)O enrichment of TCA P (δ(18)O-PO(4) between 16.9 and 27.5‰) and structural P (δ(18)O-PO(4) between 42.6 and 68.0 ‰) compared with 12.4‰ in the phosphate in the nutrient solution. δ(18)O values of phosphate extracted from soybean leaves grown under optimal conditions are greater than the δ(18)O-PO(4) values of the provided P source. Furthermore, the δ(18)O-PO(4) of TCA P seems to be controlled by the δ(18)O of leaf water and the activity of inorganic pyrophosphatase or other pyrophosphatases.