Добірка наукової літератури з теми "Myo-inositol hexakisphosphate (phytate)"

For the first time a dual pathway for dephosphorylation of myo-inositol hexakisphosphate by a histidine acid phytase was established. The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 and alternatively via D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, D-Ins(2,4)P2 to finally Ins(2)P. It was estimated that more than 98% of phytate hydrolysis occurs via D-Ins(1,2,4,5,6)P5. Therefore, the phytate-degrading enzyme from K. terrigena has to be considered a 3-phytase (EC 3.1.3.8). A second dual pathway of minor importance could be proposed that is in accordance with the results obtained by analysis of the dephosphorylation products formed by the action of the phytate-degrading enzyme of K. terrigena on myo-inositol hexakisphosphate. It proceeds preferably via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, D-Ins(2,3)P2 and alternatively via D-Ins(1,2,3,5,6)P5, D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, D-Ins(2,3)P2 to finally Ins(2)P. D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, and D-Ins(2,4)P2 were reported for the first time as intermediates of enzymatic phytate dephosphorylation. A role of the phytate-degrading enzyme from K. terrigena in phytate breakdown could not be ruled out. Because of its cytoplasmatic localization and the suggestions for substrate recognition, D-Ins(1,3,4,5,6)P5 might be the natural substrate of this enzyme and, therefore, may play a role in microbial pathogenesis or cellular myo-inositol phosphate metabolism.Key words: myo-inositol phosphate isomers, phytate-degrading enzyme, phytate, phytase, Klebsiella terrigena.

The pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytate-degrading enzymes of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and Bacillus amyloliquefaciens 45 was established using a combination of high-performance ion chromatography analysis and kinetic studies. The data demonstrate that all the Bacillus phytate-degrading enzymes under investigation dephosphorylate myo-inositol hexakisphosphate by sequential removal of phosphate groups via two independent routes; the routes proceed via D-Ins(1,2,4,5,6)P5 to Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3 or D-Ins(2,5,6)P3 and via D-Ins(1,2,4,5,6)P5 to D-Ins(1,2,5,6)P4 to finally D-Ins(1,2,6)P3. The resulting myo-inositol trisphosphate D-Ins(1,2,6)P3 was degraded via D-Ins(2,6)P2 to finally Ins(2)P after prolonged incubation times in combination with increased enzyme concentration. Key words: Bacillus spp., myo-inositol phosphate isomers, phytase, phytate degradation.

ABSTRACT Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed inAspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U · mg, of protein−1 and were capable of hydrolyzing phytate down tomyo-inositol monophosphate. Surprisingly, 1H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26 ).

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by the β-propeller phytase of Shewanella oneidensis was established, which was then compared with that of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and B. amyloliquefaciens 45 β-propeller phytases. The data demonstrate that all of these β-propeller phytases dephosphorylate myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via d-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3. Thus, the β-propeller phytases prefer the hydrolysis of every second phosphate over that of adjacent ones. This finding does not support previous phytate degradation models proposed by J. Kerovuo, J. Rouvinen, and F. Hatzack (2000. Biochem. J. 352: 623–628) and R. Greiner, A. Farouk, M. Larsson Alminger, and N.G. Carlsson (2002. Can. J. Microbiol. 48: 986–994) , but seems to fit with the structural model given by S. Shin, N.C. Ha, B.C. Oh, T.K. Oh, and B.H. Oh (2001. Structure, 9: 851–858) .

Phytase (myo-inositol-hexakisphosphate phosphohydrolase, EC 3.1.3.8) has been purified from 5-7-day-old maize (Zea mays) seedlings, using a four-step purification procedure. The native protein has a molecular mass of about 76 kDa and is built up from two 38 kDa subunits. The pH and temperature optima of the purified enzyme were respectively 4.8 and 55 degrees C. The apparent Km for phytate was estimated to be 117 microM. Like other acidic phytases, the maize seedling enzyme exhibited a broad affinity for various phosphorylated substrates and especially for penta- and tri-phosphate esters of myo-inositol. The amino acid composition of the h.p.l.c.-purified protein indicated a high hydrophobicity (44% non-polar amino acids). Rabbit antibodies were produced in response to maize seedling phytase. Western-blot analyses clearly demonstrate that the increase of phytase activity observed during the first 7 days of germination corresponded to an accumulation of the protein in maize seedlings. Phytase accumulated essentially in the shoots (mesocotyl plus coleoptiles.

Abstract: Phytic acid (myo-inositol hexakisphosphate) and its salts (phytates) are the major storage form of phosphorus in plants. Monogastric animals including hogs, poultry, and fish cannot utilize phytates as a source of phosphorus unless they are enzymatically destroyed with exogenous enzyme—phytase. Phytases are added to fodder in ever increasing dosage to improve utilization of plant-derived phosphorus because this reduces dependence of farms on inorganic fodder phosphates. Because of technological considerations, feed phytases have to withstand elevated temperatures (60-80°C), which are used during preparation of fodder. Enzymatic feed additives are becominutesg of high demand in Kazakhstan, and development of domestic technologies for production of agricultural enzymes is an ongoing challenge to the country’s biotechnology.Objectives: To develop a system for recombinant expression of industrially important thermotolerantphytase and confirm activity and thermal stability of the recombinantly expressed enzyme.Methods: De novo gene synthesis, expression of 6xHis-tagged protein in E.coli, immobilized metal affinity chouromatography, biochemical tests for activities of phosphatase and phytase.Results: Thermotolerantphytase was produced in E.coli using recombinant expression system. The obtained enzyme had phosphatise activity (hydrolyzed p-nitrophenyl phosphate) and phytase activity (hydrolyzed sodium phytate). The recombinant phytase tolerated increase of incubation temperature up to 70°C and demonstrated increase in activity towards phytate with increase in the reaction temperature in the range 30°C-70°C.Conclusion: Described gene and expression system have prospects of utilization in development of pilot industrial production of phytase in the country.

Includes bibliographical references.
Previous studies have shown that caJclum oxalate (CaOx) stone-formers have lower urinary concentrations of myo-inositol hexakisphosphate (phytate or IPe) than healthy individuals, that dietary intake of this substance leads to its increased urinary excretion and that it is an inhibitor of CaOx nucleation and growth In South Africa it has been reported that the black population has a higher dietary phytate intake than whites. The present study was undertaken to test the hypothesis that South African black subjects have higher urinary phytate levels than their white cOLlflterparts and that this contributes to the relative rarity of caOx kidney stone disease in this population group A modified indirect extraction/photometry method to measure urinary IPe was designed, developed and tested in the present study. This assay was then used to measure IPo in the urine of rural black and urban white subjects while on their free unrestricted diets In addition, urban black and white subjects each followed IPo-restricted followed by lPG-rich dietary protocols for a period of three days Urines were collected after administration of each protocol and were again analysed for IPe using the newly developed assay. Urines were then used in several crystallization experiments to measure the CaOx metastable limit, "C-oxalate deposition kinetics and inhiOition of CaOx crystal aggregation. The results showed that while on their free diets, rural blacks excreted significantly less IPs than urban whites despite their previously reported higher dietary intake of this substance This suggests that the renal handling of dietary IP

A novel bacterial protein tyrosine phosphatase (PTP)-like enzyme has recently been isolated that has a PTP-like active site and fold and the ability to dephosphorylate myo-inositol hexakisphosphate. In order to expand our knowledge of this novel class of enzyme, four new representative genes were cloned from 3 different anaerobic bacteria related to clostridia and the recombinant gene products were examined. A combination of site-directed mutagenesis, kinetic, and high-performance ion-pair chromatography studies were used to elucidate the mechanism of hydrolysis, substrate specificity, and pathways of Ins P6 dephosphorylation. The data indicate that these enzymes follow a classical PTP mechanism of hydrolysis and have a general specificity for polyphosphorylated myo-inositol substrates. These enzymes dephosphorylate Ins P6 in a distributive manner, and have the most highly ordered pathways of sequential dephosphorylation of InsP6 characterized to date. Bioinformatic analyses have indicated homologues that are involved in the regulation of cellular function.
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Organic phosphorus is potentially an important source of phosphorus (P) for agriculture, although it is not directly available for plant or microbial uptake. However, organic P can be converted into available inorganic P though hydrolysis or mineralisation. The rate of P release from organic P forms depends partly on the specific organic P compounds present in the soil. Until recently characterising soil organic P has been limited by the lack of appropriate analytic techniques. Consequently, organic P dynamics remains poorly understood. In this thesis, the focus was on improving techniques for the characterisation of soil organic P using solution ³¹P nuclear magnetic resonance (NMR) spectroscopy, applying these techniques to characterise a range of Australian soils and developing a better understanding of the cycling and potential bioavailability of soil organic P. The characterisation of soil organic P relies on the correct identification of resonances. Orthophosphate monoester peaks were identified by spiking model organic P compounds into NaOH- EDTA soil extracts. In this way, seven major resonances that were common to most of the NMR spectra were assigned to adenosine-monophosphate (AMP), scyllo-inositol hexakisphosphate, α- and β-glycerophosphate and myo-inositol hexakisphosphate (phytate). More importantly, spiking highlighted the similarly in appearance and chemical shift of some of the orthophosphate monoester resonances, particularly those of phytate and α- and β-glycerophosphate. This may have resulted in the misidentification and over-estimation of the concentrations of these species in previous studies. To provide a detailed quantitative assessment of soil organic P using ³¹P NMR spectroscopy, a modified method of spectral deconvolution, which included using an internal standard (methylenediphosphonic acid; MDP), was developed. The method of deconvolution implemented in this thesis considered P contained in larger humic molecules. A broad signal, in addition to the routinely fitted sharp peaks, was fitted to the orthophosphate monoester region of the NMR spectrum. A large proportion of monoester P (32–78%) could be assigned to this signal. When the broad signal was not taken into account phytate concentrations were over-estimated by 54%. It is likely that the concentrations of other specific orthophosphate monoester compounds were also over-estimated. The potential over-estimation of phytate concentrations has implication for the understanding of phytate stability in soils. High phytate concentrations in soils are usually explained by the stability of phytate in soils or the limited presence or activity of specialised enzymes (phytase). Lower phytate concentrations suggest phytate maybe less stable in soils than previously supposed. Therefore, the rate of phytate degradation in a calcareous soil was investigated. Phytate was applied to a calcareous soil at four different concentrations (ranging from 58–730 mg kg⁻¹) and the effect of wheat straw as an additional source of carbon was also examined. Regardless of treatment, phytate concentrations decreased over the 13-week incubation period and were adequately fitted to a first order decay model. There was no clear trend in the rate of phytate loss with treatment and the half life of phytate ranged from 4 to 8 weeks. The loss of phytate coincided with an increase in orthophosphate concentration, that in some cases more than doubled the native soil P concentrations, and there was very little variation in extraction efficiency. This result provided evidence for the microbial degradation of phytate. It demonstrated that in the calcareous soil examined, phytate was not highly stable, but a bioavailable source of organic P. The composition of soil P in 18 diverse Australian soils was also examined. Across all NaOH-EDTA soil extracts analysed, phytate comprised up to 9%, but averaged only 3% of total extractable P. Two other resonances that were also prominent in all the ³¹P NMR spectra and comprised a similar proportion of total organic P were due to α- and β-glycerophosphate. By examining the alkaline hydrolysis of a phospholipid (phosphatidlycholine), the potential source of α- and β-glycerophosphate was identified. Although α- and β-glycerophosphate and phyate gave rise to the most intense peaks, the broad signal, which was attributed to humic P, represented the most abundant form of soil organic P (27–72% of total extractable organic P). Therefore, it was suggested that the development of methods that aim to increase the availability of stabilised forms of organic P should give preference to increasing the availability of P contained in humic P complexes. Understanding P cycling not only relies on analytical methods that enable the accurate identification and quantification of soil organic P but also requires methods that can gauge the susceptibility of different organic P species to enzymatic hydrolysis. Therefore, enzymatic hydrolysis was combined with ³¹P NMR spectroscopy to identify and compare the specific organic P species in the enzyme labile and non-enzyme labile fractions of a range of NaOH-EDTA soil extracts. Phosphorus-31 NMR analysis of NaOH-EDTA soil extracts treated with active and inactivated phytase enzyme preparations showed that phytase hydrolysed the majority of the small, orthophosphate monoester compounds (α- and β-glycerophosphate, phytate, scyllo-inositol hexakisphosphate) and pyrophosphate, but orthophosphate diesters (DNA) and humic P were generally unaffected. The ³¹P NMR spectra revealed that not only was organic P hydrolysed but new orthophosphate monoester species were formed, possibly as a result of enzymatic phosphorylation. Although combining enzymatic hydrolysis and ³¹P NMR spectroscopy enabled the identification of individual organic P species that were susceptible or resistant to enzyme hydrolysis, there is still a need for further improvement and refinement of the technique in order to provide an accurate estimate of the potentially available fraction of soil organic P.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2010