Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control

We have developed transgenic mouse models to determine whether endogenous expression of phytase transgenes in the digestive tract of monogastric animals can increase the bioavailability of dietary phytate, a major but indigestible form of dietary phosphorus. We constructed phytase transgenes composed of the appA phytase gene from Escherichia coli regulated for expression in salivary glands by the rat R15 proline-rich protein promoter or by the mouse parotid secretory protein promoter. Transgenic phytase is highly expressed in the parotid salivary glands and secreted in saliva as an enzymatically active 55 kDa glycosylated protein. Expression of salivary phytase reduces fecal phosphorus by 11%. These results suggest that the introduction of salivary phytase transgenes into monogastric farm animals offers a promising biological approach to relieving the requirement for dietary phosphate supplements and to reducing phosphorus pollution from animal agriculture.     

Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate

Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild-type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20-fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.     

Quantitative determination of phytate in the presence of high inorganic phosphate

The review on the determination of phytate and inositol phosphates by Oberleas (1) indicates that most methods for the determination of phytate are derived from the method of Heubner and Stadler (2). This method is based on the principle that ferric ion forms a stable complex with phytate in dilute acid solution and is the only phosphate compound, at least in significant concentration in nature, with this property. However, the phytate values were high when we applied the procedure of Oberleas (3) to samples with high inorganic phosphate content such as rat feces or semipurified rat diets. This appeared to be a result of inorganic phosphate coprecipitating with ferric phytate.     

Quantitative determination of phosphorus in soybean lipids

Black and Hammond's method for determining lipid phosphorus has been modified for use with oils containing small amounts of phosphorus. The minimum amount of P that can be determined is 3 nmoles, with a coefficient of variation sd of 2%.     

Purification and characterization of phytase from cotyledons of germinating soybean seeds

Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.     

Effect of particle size and microbial phytase on phytate degradation in incubated maize and soybean meal

The objective of the study was to evaluate the effect of screen size (1, 2 and 3 mm) and microbial phytase (0 and 1000 FTU/kg as-fed) on phytate degradation in maize (100% maize), soybean meal (100% SBM) and maize–SBM (75% maize and 25% SBM) incubated in water for 0, 2, 4, 8 and 24 h at 38°C. Samples were analysed for pH, dry matter and phytate phosphorus (P). Particle size distribution (PSD) and average particle size (APS) of samples were measured by the Laser Diffraction and Bygholm method. PSD differed between the two methods, whereas APS was similar. Decreasing screen size from 3 to 1 mm reduced APS by 48% in maize, 30% in SBM and 26% in maize–SBM. No interaction between screen size and microbial phytase on phytate degradation was observed, but the interaction between microbial phytase and incubation time was significant (P<0.001). This was because microbial phytase reduced phytate P by 88% in maize, 84% in maize–SBM and 75% in SBM after 2 h of incubation (P<0.05), whereas the reduction of phytate P was limited (<50%) in the feeds, even after 24 h when no microbial phytase was added. The exponential decay model was fitted to the feeds with microbial phytase to analyse the effect of screen size and feed on microbial phytase efficacy on phytate degradation. The interaction between screen size and feed affected the relative phytate degradation rate (R_d) of microbial phytase as well as the time to decrease 50% of the phytate P (t1/2) (P<0.001). Thus, changing from 3 to 1 mm screen size increased R_d by 22 and 10%/h and shortened t1/2 by 0.4 and 0.2 h in maize and maize–SBM, respectively (P<0.05), but not in SBM. Moreover, the screen size effect was more pronounced in maize and maize–SBM compared with SBM as a higher phytate degradation rate constant (K_d) and R_d, and a shorter t1/2 was observed in maize compared with SBM in all screen sizes (P<0.05). However, a higher amount of degraded phytate was achieved in SBM than in maize because of the higher initial phytate P content in SBM. In conclusion, reducing screen size from 3 to 1 mm increased K_d and R_d and decreased t1/2 in maize and maize–SBM with microbial phytase. The positive effect of grinding on improving microbial phytase efficacy, which was expressed as K_d, R_d and t1/2, was greater in maize than in SBM.     

Dephosphorylation of phytate by using the Aspergillus niger phytase with a high affinity for phytate.

A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 +/- 4.6 microM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a low K(m) phytase from A. niger SK-57 and a high K(m) phytase from Aspergillus ficuum was recognized.     

Ectopic expression of a soybean phytase in developing seeds of <Emphasis Type="Italic">Glycine max</Emphasis> to improve phosphorus availability

A transgenic approach was used to alter soybean seed phytate content by expressing a soybean phytase gene (GmPhy) during seed development to degrade accumulating phytic acid (IP₆). An expression vector containing the soybean phytase cDNA controlled by the seed-specific -conglycinin promoter (-subunit) was used to transform embryogenic soybean cultures. Plants from four independent transgenic lines were analyzed for transgene integration and seed IP₆ levels. The reduction in IP₆ levels in transgenic seeds compared to control Jack soybeans ranged from 12.6 to 24.8 as determined by HPLC. A low copy transformant was propagated to the T₄ generation and examined in more detail for phytase expression and enzyme activity during seed development. Expression of phytase mRNA and phytase activity increased during seed development, consistent with the use of an embryo-specific promoter. Ectopic phytase expression during seed development offers potential as an effective strategy for reducing phytate content in soybean seed.     

Heat treatment of soybean meal and rapeseed meal suppresses rumen degradation of phytate phosphorus in sheep

The effect of heat treatment on rumen degradation of phytate in soybean meal and rapeseed meal was studied on three sheep fitted with rumen cannula. Soybean meal and rapeseed meal were roasted at 133°, 143° or 153°C for 3 h and the rumen degradation of phytate phosphorus in untreated and heat treated oilseed meals was examined using the nylon-bag technique. Effective degradability of phytate phosphorus in soybean and rapeseed meals, estimated at ruminal outflow rates of 0.02, 0.05 and 0.08 h⁻¹, was significantly (p < 0.05) reduced by heat treatment. The reduction was more marked in rapeseed meal than in soybean meal. These results suggest that heat processing of oilseed meals suppresses phytate degradation in the rumen and leads to a low availability of dietary phytate phosphorus.     

Quantitative trait loci analysis of phytate and phosphate concentrations in seeds and leaves of Brassica rapa

Phytate, being the major storage form of phosphorus in plants, is considered to be an anti-nutritional substance for human, because of its ability to complex essential micronutrients. In the present study, we describe the genetic analysis of phytate and phosphate concentrations in Brassica rapa using five segregating populations, involving eight parental accessions representing different cultivar groups. A total of 25 quantitative trait loci (QTL) affecting phytate and phosphate concentrations in seeds and leaves were detected, most of them located in linkage groups R01, R03, R06 and R07. Two QTL affecting seed phytate (SPHY), two QTL affecting seed phosphate (SPHO), one QTL affecting leaf phosphate and one major QTL affecting leaf phytate (LPHY) were detected in at least two populations. Co-localization of QTL suggested single or linked loci to be involved in the accumulation of phytate or phosphate in seeds or leaves. Some co-localizing QTL for SPHY and SPHO had parental alleles with effects in the same direction suggesting that they control the total phosphorus concentration. For other QTL, the allelic effect was opposite for phosphate and phytate, suggesting that these QTL are specific for the phytate pathway.     

The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic

Phytases hydrolyse the phosphomonoesters of phytate (myo-inositol-1,2,3,4,5,6-hexakis phosphate) and thus find uses in plant and animal production through the mobilisation of phosphorus from this source. The structure of partially deglycosylated Aspergillus niger PhyA is presented in apo form and in complex with the potent inhibitor myo-inositol-1,2,3,4,5,6-hexakis sulfate, which by analogy with phytate provides a snapshot of the Michaelis complex. The structure explains the enzyme’s preference for the 3′-phosphate of phytate. The apo-and inhibitor-bound forms are similar and no induced-fit mechanism operates. Furthermore the enzyme structure is apparently unaffected by the presence of glycosides on the surface. The new structures of A. niger PhyA are discussed in the context of protein engineering studies aimed at modulating pH preference and stability.     

Quantitative trait loci analysis of individual and total isoflavone contents in soybean seeds

Soybean isoflavones play diverse roles in human health, including cancers, osteoporosis, heart disease, menopausal symptoms and pabulums. The objective of this study was to identify the quantitative trait loci (QTL) associated with the isoflavones daidzein (DC), genistein (GeC), glycitein (GlC) and total isoflavone contents (TIC) in soybean seeds. A population of 184 F_21:0 recombinant inbred lines derived from a ‘Xiaoheidou’ בGR8836’ cross was planted in pot and field conditions to evaluate soybean isoflavones. Twenty-one QTL were detected by composite interval mapping. Several QTL were associated with the traits for DC, GeC, GlC and TIC only. QDGeGlTIC4_1 and QDGlTIC12_1 are reported first in this study and were associated with the DC, GeC, GlC and TIC traits simultaneously. The QTL identified have potential value for marker-assisted selection to develop soybean varieties with desirable isoflavone content.     

Structures of Selenomonas ruminantium phytase in complex with persulfated phytate: DSP phytase fold and mechanism for sequential substrate hydrolysis

Various inositide phosphatases participate in the regulation of inositol polyphosphate signaling molecules. Plant phytases are phosphatases that hydrolyze phytate to less-phosphorylated myo-inositol derivatives and phosphate. The phytase from Selenomonas ruminantium shares no sequence homology with other microbial phytases. Its crystal structure revealed a phytase fold of the dual-specificity phosphatase type. The active site is located near a conserved cysteine-containing (Cys241) P loop. We also solved two other crystal forms in which an inhibitor, myo-inositol hexasulfate, is cocrystallized with the enzyme. In the "standby" and the "inhibited" crystal forms, the inhibitor is bound, respectively, in a pocket slightly away from Cys241 and at the substrate binding site where the phosphate group to be hydrolyzed is held close to the -SH group of Cys241. Our structural and mutagenesis studies allow us to visualize the way in which the P loop-containing phytase attracts and hydrolyzes the substrate (phytate) sequentially.     

Effect of salinity on germination,phytase activity and phytate content in lettuce seedling

Seeds of four lettuce (Lactuca sativa L.) varieties (Romaine, Augusta, Vista and Verte) differing in their salt sensitivity were sown at 0 (Control), 50, 100 and 150 mM NaCl. The final germination percentage decreased with the increasing salinity and was annulated at the highest salt concentration in Vista and Verte, the most sensitive varieties. However, in the less sensitive ones, Romaine and Augusta, it was slightly modified at 50 and 100 mM NaCl and then decreased by 50% compared with the control, at 150 mM. The effects of NaCl 100 mM on seedling growth, phytase activities, phytate and inorganic phosphorus contents were studied in Romaine and Vista showing different behaviours towards salinity. Radicle and hypocotyl length and fresh and dry weights were reduced by salt treatment in both varieties. In addition, radicle phytase activity exhibited an increase in Romaine (less sensitive) and a decrease in Vista (more sensitive). In hypocotyl, this activity showed no difference with the control in the two varieties. However, in cotyledons, and during early hours after germination, salinity decreased phytase activity in both varieties whereas in the later hours (72–96 h) this activity reached the value of the control in Romaine. The enhancement of phytase activity was concomitant with an increase in orthophosphate content and a decrease in phytate reserve. These results suggest that salt presence in the medium delays Pi remobilization from phytate stock, but stimulates assimilation of phosphorus more than its accumulation in the organs of the two lettuce varieties.     

Femur morphometry,densitometry, geometry and mechanical properties in young pigs fed a diet free of inorganic phosphorus and supplemented with phytase

The study investigated in piglets the effect of replacing dietary inorganic P by addition of microbial phytase and its impact on performance, nutrient digestibility and on the geometrical characteristics and mineralisation of the femur. Sixteen pigs on day 58 of age were divided into two groups and fed either a diet free of additional inorganic phosphorus (P) and supplemented with phytase (Diet LP, 4.23 g total P/kg diet) or a diet with a mineral source of P and not supplemented with phytase (Diet SP, 5.38 g total P/kg diet). Performance data and the apparent total tract digestibility of nutrients were estimated between days 58 and 114, and 72 and 86 of age, respectively. On day 114 of age, the pigs were slaughtered, the femur was dissected and the mineral content and mineral density, maximum strength and maximum elastic strength, cortical wall thickness, cross-sectional area and cortical index were analysed. The growth performance and digestibility of nutrient fractions (with exception of P) did not differ between treatment groups. The P-digestibility was significantly higher in Group LP. The femur of pigs in Group LP had significantly greater cortical wall thickness, cortical index, bone mineral content, bone mineral density, maximum strength and maximum elastic strength than Group SP. Femur maximum strength and maximum elastic strength were correlated with cortical wall thickness and cortical index. Resulting from the different supply of digestible P, the femur geometrical, densitometric and mechanical properties of Group LP were better than those of Group SP. The mechanical properties of the femur of pigs depended more on its geometrical characteristics than on the degree of its mineralisation.     

A method for estimating phosphate in the presence of phytate and its application to the determination of phytase

Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80°C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF₄₃₀, the prosthetic group of methylcoenzyme M reductase, F₅₆₀, an oxidation product of this compound, coenzyme F₄₂₀, F₃₄₂, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF₄₃₀, coenzyme F₄₂₀, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied.     

Possible effect of gibberellin on phytate degradation in germinating barley seeds

Changes in the contents of phytate (IP₆) and other phosphorus(P)-compoundsin germinating seeds of a huskless barley were investigatedin the embryo with scutellum (EM), the starchy endosperm (EN),and the aleurone layer with pericarp-testa (AL). More than 80%of the total P in the AL of 1-day germinated seeds was foundin acid-soluble organic P, most of which was IP₆. During germination,the IP₆ in AL decreased markedly with no accumulation of lessphosphorylated myo-inositols and Pi and acid-insoluble organicP increased in the EM. The total P in the EN of 1-day germinatedseeds was about one-third that in the AL, the greater part ofwhich was found in the acid-insoluble fraction and decreasedgradually during germination. Only a small amount of IP₆ couldbe detected in the EM and EN during the early stage of germination. IP₆ in AL of embryoless half-seeds incubated without gibberellicacid (GA₃) decreased slightly even after 6 days. Incubationwith 10 ppm GA₃ remarkably stimulated the IP₆ degradation. Thisstimulation was reduced, with no change in the Pi content, byabout 80–90% with 1 mM 6-methylpurine or 10 ppm cycloheximide.The addition of 0.1 M KH₂PO₄ caused a 4-fold increase in thePi content of AL in the presence of GA₃. In addition, it suppressedthe GA₃-dependent -amylase synthesis by about 20% and the GA₃effect on IP₆ degradation by about 50%. In light of these results, IP₆ seems to be hydrolyzed completelyinto Pi and myo-inositol within the aleurone tissue, and gibberellinseems to control this process. (Received August 24, 1979; )     

Limits to sulfur accumulation in transgenic lupin seeds expressing a foreign sulfur-rich protein

The low sulfur amino acid content of legume seeds restricts their nutritive value for animals. We have investigated the limitations to the accumulation of sulfur amino acids in the storage proteins of narrow leaf lupin (Lupinus angustifolius) seeds. Variation in sulfur supply to lupin plants affected the sulfur amino acid accumulation in the mature seed. However, when sulfur was in abundant supply, it accumulated to a large extent in oxidized form, rather than reduced form, in the seeds. At all but severely limiting sulfur supply, addition of a transgenic (Tg) sink for organic sulfur resulted in an increase in seed sulfur amino acid content. We hypothesize that demand, or sink strength for organic sulfur, which is itself responsive to environmental sulfur supply, was the first limit to the methionine (Met) and cysteine (Cys) content of wild-type lupin seed protein under most growing conditions. In Tg, soil-grown seeds expressing a foreign Met- and Cys-rich protein, decreased pools of free Met, free Cys, and glutathione indicated that the rate of synthesis of sulfur amino acids in the cotyledon had become limiting. Homeostatic mechanisms similar to those mediating the responses of plants to environmental sulfur stress resulted in an adjustment of endogenous protein composition in Tg seeds, even when grown at adequate sulfur supply. Uptake of sulfur by lupin cotyledons, as indicated by total seed sulfur at maturity, responded positively to increased sulfur supply, but not to increased demand in the Tg seeds.     

Quantitative determination of calcium oxalate and oxalate in developing seeds of soybean (Leguminosae)

Developing soybean seeds accumulate very large amounts of both soluble oxalate and insoluble crystalline calcium (Ca) oxalate. Use of two methods of detection for the determination of total, soluble, and insoluble oxalate revealed that at +16 d postfertilization, the seeds were 24% dry mass of oxalate, and three-fourths of this oxalate (18%) was bound Ca oxalate. During later seed development, the dry mass of oxalate decreased. Crystals were isolated from the seeds, and X-ray diffraction and polarizing microscopy identified them as Ca oxalate monohydrate. These crystals were a mixture of kinked and straight prismatics. Even though certain plant tissues are known to contain significant amounts of oxalate and Ca oxalate during certain periods of growth, the accumulation of oxalate during soybean seed development was surprising and raises interesting questions regarding its function.