Pathway of dephosphorylation of myo-inositol hexakisphosphate by phytases of legume seeds

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytases purified from faba bean and lupine seeds, respectively, was established. The data demonstrate that the legume seed phytases under investigation dephosphorylate myo-inositol hexakisphosphate in a stereospecific way. The phytase from faba bean seeds and the phytase LP2 from lupine seeds degrade phytate by sequential removal of phosphate groups via D-Ins(1,2,3,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), and D-Ins(1,2)P(2) to finally Ins(2)P, whereas the phytases LP11 and LP12 from lupine seeds generate the final degradation product Ins(2)P via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), and D-Ins(1,2)P(2).     

Quantitative analysis of phytate globoids isolated from wheat bran and characterization of their sequential dephosphorylation by wheat phytase

Wheat phytase was purified to investigate the action of the enzyme toward its pure substrate (phytic acid - myo-inositol hexakisphosphate) and its naturally occurring substrate (phytate globoids). Phytate globoids were purified to homogeneity from wheat bran, and their nutritionally relevant parameters were quantified by ICP-MS. The main components of the globoids were phytic acid (40% w/w), protein (46% w/w), and several minerals, in particular, K > Mg > Ca > Fe (in concentration order). Investigation of enzyme kinetics revealed that K(m) and V(max) decreased by 29 and 37%, respectively, when pure phytic acid was replaced with phytate globoids as substrate. Time course degradation of phytic acid or phytate globoids using purified wheat phytase was followed by HPIC identification of inositol phosphates appearing and disappearing as products. In both cases, enzymatic degradation initiated at both the 3- and 6-positions of phytic acid and end products were inositol and phosphate.     

Inositol hexaphosphate hydrolysis by Baker's yeast. Capacity, kinetics, and degradation products

Phytases hydrolyze myo-inositol 1,2,3,4,5,6-hexaphosphate (IP(6)), yielding lower inositol phosphates and inorganic orthophosphate. Two commercial strains of baker's yeast (Saccharomyces cerevisiae), Y(1) and Y(2), were able to express phytase activity. This was determined by the capacity to grow in a synthetic medium with IP(6) as the sole phosphorus source. IP(6) hydrolysis was rapid for both strains, and after 24 h, all IP(6) was degraded. Control cultures contained inorganic orthophosphate (P(i)) and no IP(6). Growth rate in IP(6) medium was for both strains essentially identical to growth in P(i) medium, indicating a well-adapted metabolism for utilization of phosphorus from IP(6). There was some difference in growth yield (milligrams of biomass per milligram of glucose) between the two strains: 0.95 (Y(1)) and 1.35 (Y(2)) in IP(6) medium and 1.03 and 1. 35, respectively, in P(i) medium. The phytases were of the 3-phytase type, forming mainly DL-Ins(1,2,4,5,6)P(5), DL-Ins(1,2,5,6)P(4), and DL-Ins(1,2,6)P(3).     

Generation of thiols by biotransformation of cysteine-aldehyde conjugates with baker's yeast

Baker's yeast was shown to catalyze the transformation of cysteine-furfural conjugate into 2-furfurylthiol. The biotransformation's yield and kinetics were influenced by the reaction parameters such as pH, incubation mode (aerobic and anaerobic), and substrate concentration. 2-Furfurylthiol was obtained in an optimal 37% yield when cysteine-furfural conjugate at a 20 mM concentration was anaerobically incubated with whole cell baker's yeast at pH 8.0 and 30 degrees C. Similarly to 2-furfurylthiol, 5-methyl-2-furfurylthiol (11%), benzylthiol (8%), 2-thiophenemethanethiol (22%), 3-methyl-2-thiophenemethanethiol (3%), and 2-pyrrolemethanethiol (6%) were obtained from the corresponding cysteine-aldehyde conjugates by incubation with baker's yeast. This work indicates the versatile bioconversion capacity of baker's yeast for the generation of thiols from cysteine-aldehyde conjugates. Thanks to its food-grade character, baker's yeast provides a biochemical tool to produce thiols, which can be used as flavorings in foods and beverages.     

Kinetic and equilibrium constants of phytic acid and ferric and ferrous phytate derived from nuclear magnetic resonance spectroscopy

Inositol phosphates are metabolically derived organic phosphates (P) that increasingly appear to be an important sink and source of P in the environment. Salts of myo-inositol hexakisdihydrogen phosphate (IHP) or more commonly phytate are the most common inositol phosphates in the environment. IHP resists acidic dephosphorylation and enzymatic dephosphorylation as ferric or ferrous IHP. Mobility of IHP iron complexes is potentially pH and redox responsive, making the time scale and environmental fate and transport of the P associated with the IHP of interest to the mass balance of phosphorus. Ferric and ferrous complexes of IHP were investigated by proton nuclear magnetic resonance spectroscopy ( (1)H NMR) and enzymatic dephosphorylation. Ferrous IHP was found to form quickly and persist for a longer period then ferric IHP. Dissociation constants derived from (1)H NMR experiments of chemically exchanging systems at equilibrium were 1.11 and 1.19 and formation constants were 0.90 and 0.84 for ferric and ferrous IHP, respectively. The recovery of P from enzymatic dephosphorylation of ferric and ferrous IHP was consistent with the magnitude of the kinetic and equilibrium rate constants.     

不同种类解磷微生物的溶磷效果及其磷酸酶活性的变化

比较了不同种类的微生物菌株对不同种类难溶性磷酸盐及磷矿粉的溶解能力。结果发现,细菌、酵母、霉菌在解磷方面均有一定作用,发挥着不同优势。磷酸钙、磷酸铝、磷酸铁等难溶性磷酸盐容易被酵母菌、霉菌溶解,而磷矿粉容易被巨大芽孢杆菌溶解,显示不同微生物与不同磷源的亲和溶解能力不同。不同种类磷酸盐或磷矿粉对微生物磷酸酶活力的影响不同,贫磷条件可以促进酸性和碱性磷酸酶活性的增加     

Purification and characterization of phytase induced in tomato roots under phosphorus-deficient conditions

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from roots of tomato plants grown under phosphorus-deficient conditions using five purification schemes. The phytase was successfully separated from the major acid phosphatase to an electrophoretic homogeneity. The native molecular weight of this enzyme was estimated to be about 164 kD by Bio-Gel P-200 gel filtration. The molecular weight of the subunit on SDS-PAGE was approximately 82 kD, indicating that the native form of the enzyme was a homodimer. The isoelectric point of tomato phytase was about 5.5. The enzyme exhibited a high affinity for phytic acid (K _m = 38 μM), and was strongly inhibited by phosphate, molybdate and fluoride. Among other characteristics of tomato phytase, the pH and temperature optima were 4.3 and 45°C, respectively. Tomato phytase contained a fairly high concentration of aspartic, glutamic acid and glycine residues.     

Phytate degradation in a mixture of ground wheat and ground defatted soybeans during feed processing: effects of temperature, moisture level, and retention time in small- and medium-scale incubation systems

The optimal conditions for degradation of phytate (IP6, myo-inositol hexaphosphate) in a mixture of ground wheat and ground defatted soybeans (1:2, w/w) with added exogenous E. coli phytase were investigated at different temperatures (45, 60, 75, and 95 degrees C), moisture levels (25%, 35%, and 45%), and retention times (2-45 min). All treatment combinations were investigated in a small-scale mixer conditioner (experiment 1). The combined 45 degrees C and 45% moisture treatment was most efficient and reduced the content of IP6 by 86% during 45 min of incubation. This treatment combination was applied in a medium-scale mixer conditioner (experiment 2), and 76% reduction of IP6 at 45 min was obtained. During incubation, the content of lower groups of inositol phosphates, such as IP4 (myo-inositol tetraphosphate) and IP3 (myo-inositol triphosphate), increased significantly as the content of IP6 decreased. The major isomer formed was Ins(1,2,5,6)P(4).     

改性磷矿粉在石灰性土壤上的生物有效性及其机理研究

为缓解磷资源危机,充分利用不适于化学加工的磷矿资源,对磷矿采用活化技术开发利用,由此生产的改性磷肥已被证明在酸性土壤上有很高的生物有效性,但在石灰性土壤上的研究还比较少。本文选择4种不同产地(云南昆明、四川德阳、四川绵竹和贵州开阳)的磷矿粉,分别用有机活化剂和无机活化剂进行活化处理,制备改性磷矿粉,并就其在石灰性土壤对苗期春小麦的生物有效性进行了盆栽试验。结果表明,4种磷矿粉经活化处理后有效磷和水溶性磷含量均明显增加,无机活化剂提高了有效磷的含量,而有机活化剂对水溶性磷的提高幅度较大。改性磷矿粉处理均不同程度提高了石灰性土壤上春小麦的生物量干重、植株吸磷量和磷利用效率,有机活化剂处理制备的改性磷矿粉对春小麦生长的促进作用更为明显。运用红外光谱谱学技术对4种磷矿粉及相应的8种改性磷矿粉结构分析结果表明,磷矿粉经改性后其结构发生了明显变化,H2PO4和HPO42的特征谱带明显增加,增加的程度因磷矿粉的产地和活化剂种类不同而有差异。对磷矿粉化学成分分析结果表明,不同产地的磷矿粉其磷以及钙、铁、铝、镁等化学成分的含量差异较大,活化剂对磷矿粉的活化效果与磷矿粉本身的氧化物含量有关。在本试验条件下,磷矿粉Ⅲ(四川绵竹)的活化效果相对最好,与其氧化镁含量最高、总氧化物含量最低有关。不同类型活化剂对磷矿粉的活化效果不同,红外光谱分析结果表明,无机活化剂活化效果较好,而土培试验结果表明有机活化剂的活化效果较好,这一结果有待进一步的研究。     

<Emphasis Type="Italic">Urochloa ruziziensis</Emphasis> cover crop increases the cycling of soil inositol phosphates

Ruzigrass (Urochloa ruziziensis) is a cover crop that is commonly used in Brazil and exudes high concentrations of organic acids from its roots, and is therefore expected to mobilize soil organic P such as inositol phosphates. However, it is not known if this can occur only under P deficient conditions. Specifically, we aimed to test the hypothesis that the degradation of inositol phosphates is increased by growing ruzigrass at two different P levels. To investigate this, we studied soil organic P in a 9-year-old field experiment, with treatments consisting of ruzigrass or fallow during the soybean (Glycine max) off-season, with or without P addition. Organic P was extracted in NaOH-EDTA, followed by colorimetric quantification of organic P hydrolysable by phytase, and myo-inositol hexakisphosphate by hypobromite oxidation and HPLC separation. Ruzigrass dry matter yield increased by about 80% with P application. Ruzigrass reduced the concentration of phytase labile P and myo-inositol hexakisphosphate, but only in soil receiving P. A corresponding increase in unidentified inositol phosphates, presumably representing lower-order esters, was also observed after ruzigrass in soil with P application. We deduce that the degradation of inositol phosphates under ruzigrass with P application is due to greater ruzigrass productivity in the more fertile treatment, increasing the release of root exudates that solubilize inositol phosphates and promote their decomposition by phytase. We conclude that ruzigrass cover cropping can promote the cycling of recalcitrant soil organic P, but only when fertility is raised to a sufficient level to ensure a productive crop.     

Determination of the activity of acidic phytate-degrading enzymes in cereal seeds

Five different methods were compared to elucidate the total activity of the acidic phytate-degrading enzymes present in the seeds of rye, wheat, and barley. Phytate-degrading activity was studied at pH 5.0 by quantifying the liberated phosphate. Rye showed the highest acid phytate-degrading activity among the cereals studied. Using an aqueous extract, only 30-50% of the activity was found (rye, 3443 mU g(-1) of grain; wheat, 1026 mU g(-1) of grain; barley, 1032 mU g(-1) of grain) compared to that found by the direct incubation of the dry-milled cereal grains in a buffered phytate-containing solution (rye, 6752 mU g(-1) of grain; wheat, 2931 mU g(-1) of grain; barley, 2093 mU g(-1) of grain). Extending the extraction time resulted in an increase in extractable phytate-degrading activity by, at maximum, 10-15%. Extraction of phytate-degrading activity is strongly enhanced in the presence of Triton X-100 and the protease inhibitor phenylmethylsulfonyl fluoride (rye, 6536 mU g(-1) of grain; wheat, 2873 mU g(-1) of grain; barley, 2023 mU g(-1) of grain), suggesting at least a partial association with membrane structures and a degradation by proteolytic activity during extraction. In addition, it was shown that determining phytate-degrading activity by quantification of the liberated inorganic phosphate is more robust and precise than determining phytate-degrading activity by quantification of the residual phytate.     

Phosphorus speciation in temperate basaltic grassland soils by solution 31P NMR spectroscopy

Phosphorus (P) speciation in 21 basaltic and four non-basaltic Irish grassland soils was determined by NaOH–EDTA extraction and ³¹P NMR spectroscopy. Organic P in basaltic soils ranged between 30 and 697 mg P kg⁻¹ and consisted of phosphate monoesters (84–100%), DNA (0–16%) and phosphonates (0–5%). Inorganic P was mainly phosphate (83–100%) with small concentrations of pyrophosphate (0–17%). Phosphate monoesters were more important as a proportion of extracted P in basaltic soils, probably because of their greater oxalate-extractable Fe and Al contents. Phosphate monoesters appeared to be strongly associated with non-crystalline Al and increased with total soil P concentration, indicating that they do accumulate in grassland soils. In non-basaltic soils myo -inositol hexakisphosphate constituted between 20 and 52% of organic P, while scyllo -inositol hexakisphosphate constituted between 12 and 17%. These compounds were not quantified separately in basaltic soils because of poor NMR resolution in the phosphate monoester region, but appeared to represent a considerable proportion of the organic P in most samples. DNA concentrations were greater in basaltic soils compared with non-basaltic soils and were associated with acidic pH and large total C contents. The inability of the Olsen P test to assess effectively the P status of basaltic soils may result from strong phosphate sorption to Fe and Al oxides, inducing plant utilization of soil organic P. Phosphorus nutrient management should account for this to avoid over-application of P and associated financial and environmental costs.     

ORGANIC PHOSPHATE COMPOUNDS IN CALCIUM CHLORIDE EXTRACTS OF SOILS: IDENTIFICATION AND AVAILABILITY TO PLANTS

The organic phosphate in CaCl₂ extracts of dried soils was concentrated and purified by the use of ion exchange resins. Graded elution with formic acid separated three phosphate fractions (P1, P2, and P3), all of which contained inositol. By paper chromatography and assay of inositol, the phosphate in two of these fractions (P2 and P3) was found to be present mainly as myo-inositol monophosphate. One form (P2) was resistant to hydrolysis and had a low availability to plants grown under aseptic conditions. The other (P3) was not pure, but was more easily hydrolysed and was readily used by plants. The phosphate in the remaining fraction (P1) was also available to plants and may also have been present as myo-inositol monophosphate.     

Effectiveness of different precipitated phosphates as phosphorus sources for plants

Abstract. Eleven precipitated phosphates were evaluated as sources of phosphorus (P) for plant growth by comparing their effectiveness with that of monocalcium phosphate, a source of water soluble P that is generally considered to be fully plant available. The precipitated phosphates comprised struvites recovered from waste water discharges (mainly magnesium ammonium phosphate), laboratory synthesised struvites, a synthetic iron phosphate and a recovered calcium phosphate. Precipitating phosphates in these forms could be a way for removing P from waste water before it is discharged to rivers, so reducing the risk of eutrophication. Application to agricultural land would be one potential use for such phosphates. Evaluation was by pot experiments with a sandy loam soil and with a sandy clay loam soil using perennial ryegrass ( Lolium perenne) as the test crop. The soils differed in pH (6.6 and 7.1) and in Olsen P (28 and 11 mg L⁻¹). Measured variables were grass dry matter (DM) yield and grass P concentration which were used to calculate offtake of P in the harvested grass. DM yields of ryegrass and P offtakes given by the synthetic and recovered struvites were not significantly different statistically either between themselves or to MCP applied at the same rate. On this basis these struvites could be used to recycle P to similar soils and the effect of the P on crop yield should be similar to that of MCP     

三种溶磷真菌对不同磷源溶解效果的比较研究

报道了溶磷菌株P39(Aspergilusspp.)和P66、P2.3(Penicilliumspp.)在液体培养条件下对5种难溶性磷源的溶解效果。试验结果表明,3种溶磷菌株在所有磷源培养基中生长良好,但它们之间的溶磷量相差很大,菌株P39对不同磷源的溶磷效果为磷酸铁>磷酸铝>磷酸钙>宜昌磷矿粉>摩洛哥磷矿粉,其溶磷率分别达到96.60%、84.00%、66.05%、61.43%和41.52%;P66菌株对不同磷源的溶磷效果为磷酸铝>磷酸钙>摩洛哥磷矿粉>宜昌磷矿粉>磷酸铁,溶磷率分别为78.18%、58.45%、41.09%、33.00%和14.27%;而菌株P2.3对不同磷源的溶磷效果要小于菌株P39和P66,研究结果表明P2.3菌株对磷酸铁不具有溶磷活性,对其它4种不同磷源的溶磷效果为磷酸钙>磷酸铝>摩洛哥磷矿粉>宜昌磷矿粉。对不同菌株培养滤液可滴定酸含量和pH测定结果发现,菌株P39的培养滤液可滴定酸含量与pH值之间存在直线相关,菌株P66和P2.3处理的二者相关性不显著,表明不同的溶磷菌株在不同磷源条件下,可能从有机酸组成和含量两方面发生了变化而影响对难溶性磷的溶解。     

Validation of antifreeze properties of glutathione based on its thermodynamic characteristics and protection of baker's yeast during cryopreservation

The antifreeze ability of glutathione was evaluated on the basis of its thermodynamic characteristics and protection of baker's yeast during cryopreservation at -30 degrees C. The thermodynamic characteristics and protection of baker's yeast of glutathione were similar to those of known antifreeze proteins, such as carrot antifreeze protein and holly antifreeze protein. These properties included lowering the freezing point at about 0.20 degrees C non-colligatively, decreasing freezable water content, controlling the movement of free water for its strong hydrophilicity, and improving baker's yeast survival during the simulated processing of frozen dough. Therefore, glutathione was viewed to be an antifreeze protein like substance on the basis of its unique thermodynamic characteristics and protection of baker's yeast. The method combining thermodynamic characteristic analysis and protection evaluation is a new and simple way to screen new antifreeze proteins.     

Phosphorus Solubilization from Low-Grade Rock Phosphates in the Presence of Decomposing Soybean Leaf Litter

Abstract

The natural phenomenon of defoliation of mature soybean leaves onto the soil surface provides a large quantity of easily decomposable organic matter in the form of leaf litter. The potential of decomposing soybean leaf litter (SLL) to solubilize phosphorus (P) from two low‐grade rock phosphates, Jhabua rock phosphate (JRP) and Hirapur rock phosphate (HRP), alone or amended with pyrite, was assessed in an incubation study. Decomposing SLL solubilized P both from JRP and HRP and concurrently increased water‐soluble as well as organic P contents. Amending rock phosphates with pyrite (in a 1∶2 P to S ratio) promoted P solubilization. Approximately 71 to 92% of the total solubilized P was converted to organic P. The rate of P solubilization increased with SLL decomposition time, reaching its peak at 60 days with rock phosphates alone and at 90 days with pyrite‐blended rock phosphates. The maximum P solubilization (as a percentage of total P added) with different rock phosphates and their mixtures with pyrite followed this order: HRP (11.4%)<HRP+pyrite (16.5%)<JRP (20.2%)<JRP+pyrite (26.5%). These findings clearly suggest that the decomposing SLL has the potential to solubilize P from the otherwise insoluble low‐grade rock phosphates and can offer a natural opportunity for direct use of rock phosphates in the cropping systems that have soybean as a component crop.     

EFFECT OF pH ON PHOSPHATE ADSORPTION AND ISOTOPIC EXCHANGE IN ACID SOILS AT LOW AND HIGH ADDITIONS OF SOLUBLE PHOSPHATE

Measurements of equilibrium phosphate concentrations, adsorbed phosphate and isotopically exchangeable phosphate (E_t) were made on suspensions of acid soils at initial P concentrations ranging from 1–1000 μM and pH values between 4.2 and 6.8. At low P concentrations (1–100 μM), the affinity of the adsorption reaction was greatest at pH 5.2–5.5. This result, which was inconsistent with the ligand-exchange hypothesis of Kingston et al. (1967, 1972, 1974), was interpreted in terms of the formation of basic aluminium phosphates of varying composition that depended on the OH/P ratio in solution. At the highest P concentrations (1000 μM), when the affinity of adsorption was least at pH 5.2–5.5, it appeared that basic calcium phosphates were precipitating at pH values > 5.5. In suspensions containing 1–100 μM P initially, values of E_t did not change consonantly with values of adsorbed P over the pH range 4.8 to 5.5. It is suggested that the E_t values were underestimated due to the presence of labelled complexes in solution which were detected by scintillation counting but not by the analysis for orthophos-phate. Evidence from solution studies (White et al., 1976) suggested that as much as one-third of the phosphate could be complexed with aluminium in solutions between pH 4.7 and 5.4 at P/A1 mole ratios of 0.075 to 7.5. It was apparent that the interpretation of the physical significance of E values in acid soils is complex and requires careful appraisal of the experimental techniques used.