Bacterial Phytase Research Articles

Functionality of a next generation biosynthetic bacterial 6-phytase in enhancing phosphorus availability to broilers fed a corn-soybean meal-based diet

The addition of phytase enzyme to broiler feed as a means of improving phosphorus (P) availability and reducing P excretion is widespread practice. New phytases with enhanced functionality continue to be developed. We tested the effects of a next generation biosynthetic bacterial 6-phytase on growth performance, tibia ash and P digestibility in broilers. Treatments included a nutritionally adequate positive control (PC) diet, a negative control (NC) diet formulated with reductions in Ca and available P (avP) of 2.0 g/kg and 1.9 g/kg (starter phase) and 2.0 g/kg and 1.8 g/kg (finisher phase), respectively, and three further diets comprising the NC supplemented with three levels of phytase (250, 500 or 1000 FTU/kg). Diets were fed in mash form to day-old Cobb 500 broilers housed in pens (9 pens for NC, 10 pens for all other treatments; 24 birds/pen), in two phases (starter, days 1–21; finisher, days 22–42). Tibias were collected from 4 birds on day 21 and from 6 birds on day 42 for determination of defatted tibia ash. Ileal digesta was collected on day 21 for determination of apparent ileal digestibility (AID) of nutrients. Compared to PC, the NC exhibited reduced tibia ash at day 21 and 42, resulting in reduced average daily gain (ADG) and average daily feed intake (ADFI) during starter, finisher, and overall phases, and increased feed conversion ratio (FCR) during finisher phase and overall (P < 0.05). Phytase at any dose-level during both phases improved tibia ash vs. NC (P < 0.05) and maintained feed intake and growth parameters equivalent to the PC. At a dose-level of 500 FTU/kg or above, phytase supplementation increased AID of P vs. NC (P < 0.05) and at 1000 FTU/kg, phytase improved the AID of P compared with PC (P < 0.05). For all measures, response values were numerically highest with 1000 FTU/kg and increased linearly or quadratically with increasing phytase dose (P < 0.05). Phytase at 1000 FTU/kg produced birds with an average day 42 body weight of 2.74 kg and overall FCR of 1.626, comparable to PC. On a grams per kilogram diet basis, phytase at 1000 FTU/kg improved ileal digestible P by 1.76 g/kg above NC (at day 21). This is equivalent to 2.07 g P from monocalcium phosphate (MCP-P), based on digestible P improvement. The results suggest the novel biosynthetic phytase has high functionality in the tested dietary setting.

Phytase Superdosing in the Diet of Lightweight Replacement Pullets: Performance, Organ Biometry and Bone Characteristics

ABSTRACT The aim of this study was to evaluate the effect of two types of phytases (the 1st produced from Escherichia coli and the 2nd from Aspergillus oryzae) with different dosages (300 and 900 Unit Phytase or FTU) on performance, organ biometry and bone quality of replacement pullets in the period of 8 to 17 weeks of age. A total of 288 Hy-Line White laying hens were used at 8 weeks of age. A completely randomized design was adopted in a 2 x 2 factorial scheme, totaling 4 treatments with 8 replicates of 9 birds each. Performance, biometric variables and bone characteristics were evaluated. There was no significant interaction between the factors for any of the evaluated variables. The conversion and the accumulated consumption were influenced (p<0.05) by the phytase dosages, with the highest results being observed for the consumption obtained with 300 FTU, and the best results for the feed conversion obtained with the dosage of 900 FTU. The relative weight of the liver was influenced by the treatments (p<0.05), presenting higher values with bacterial phytase and a dosage of 900 FTU. The sternum length and tibial deformity were influenced by fungal phytase (p<0.05). It is concluded that the use of 900 FTU superdosing and fungal phytase improves the performance and bone characteristics of light replacement pullets.

Safety evaluation of a novel variant of consensus bacterial phytase

A 90-day subchronic oral toxicity study was conducted to evaluate the safety of a consensus bacterial phytase variant 6-phytase (PhyG) for use as an animal feed additive. This phytase is produced by fermentation with a fungal (Trichoderma reesei) production strain expressing a biosynthetic variant of a consensus bacterial phytase gene assembled via ancestral reconstruction with sequence bias for the phytase from Buttiauxella sp. Rats were administered PhyG daily via oral gavage at dose-levels of 0 (distilled water), 250, 500 or 1000 mg total organic solids (TOS)/kg bodyweight (bw)/day (equivalent to 0, 112,500, 225,000 and 450,000 phytase units (FTU)/kg bw/day, respectively). No test article-related adverse effects were observed. A no-observed-adverse-effect level (NOAEL) for PhyG was established as 1000 mg TOS/kg bw/day, the highest test concentration. Based on this NOAEL and an estimate of broiler consumption determined from the proposed inclusion of the phytase in feed at the maximum recommended level (4000 FTU/kg), a margin of safety value of 1613 was calculated. Results of in vitro genotoxicity testing and in silico protein toxin evaluation further confirmed PhyG to be non-genotoxic and not likely to be a protein toxin upon consumption. These data support the safety of PhyG as an animal feed additive.

Functionality of a next generation biosynthetic bacterial 6-phytase in enhancing phosphorus availability to weaned piglets fed a corn-soybean meal-based diet without added inorganic phosphate.

The utility of a next generation biosynthetic bacterial 6-phytase (PhyG) in restoring bone ash, bone phosphorus (P) content and performance in piglets depleted in P was evaluated. A total of 9 treatments were tested as follows. Treatment 1, a negative control (NC) diet; treatments 2, 3, 4, NC supplemented with 250, 500 or 1,000 FTU/kg of PhyG; treatments 5, 6, NC supplemented with 500 or 1,000 FTU/kg of a commercial Buttiauxella sp phytase (PhyB); treatments 7, 8, 9, NC supplemented with monocalcium phosphate (MCP) to provide 0.7, 1.4 and 1.8 g/kg digestible P, equating to a digestible P content of 1.8, 2.5 and 2.9 g/kg. The latter constituting the positive control (PC) diet with adequate P and calcium (Ca). The NC was formulated without inorganic P (1.1 g digestible P/kg) and reduced in Ca (5.0 g/kg). Additional limestone was added to treatments 7 to 9 to maintain Ca-to-P ratio between 1.2 and 1.3. A total of 162 crossed Pietrain × (Large White × Landrace) 21-d-old piglets (50% males and 50% females) were fed adaptation diets until 42 d old and then assigned to pens with 2 pigs/pen and 9 pens/treatment in a completely randomized block design. Piglets were fed mash diets based on corn and soybean meal ad libitum for 28 d. At the end of the study, one piglet perpen was euthanized and the right feet collected for determination of bone strength, bone ash and mineral content. Compared with the PC, the NC group had reduced average daily gain (ADG) and increased feed conversion ratio (FCR) during all growth phases and overall, and at d 28 (70 d old) NC pigs had bones with reduced ash, Ca and P content (P < 0.05). The PhyG at 250 FTU/kg improved bone ash vs. NC. Increasing PhyG dose linearly or quadratically improved bone ash, ADG and FCR (P < 0.05). At ≥ 500 FTU/kg, both PhyG and PhyB maintained ADG and FCR equivalent to PC. Linear regression analysis was done to compare the measured response parameters to increasing digestible P from MCP. Based on this analysis it was shown that PhyG and PhyB at 1,000 FTU/kg could replace 1.83 and 1.66 g/kg digestible P from MCP in the diet, respectively, on average across metacarpi bone ash, ADG or FCR. These findings suggest that the biosynthetic phytase is highly effective in the tested dietary setting.

Characterization of Arabidopsis thaliana Plants Expressing Bacterial Phytase

Transgenic plants containing genes of bacterial phytases represent one of the promising ways to solve the problem of phosphorus deficiency in the nutrition of plants and monogastric animals. Histidine acid phytase PaPhyC from Pantoea agglomerans has a high activity and represents a promising basis for the biotechnology of plants. In this study, the analysis of morphological characteristics, phytase activity, and phosphorus content in tissues of the earlier obtained, genetically modified Arabidopsis thaliana (L.) Heynh. plants producing extracellular phytase (PaPhyC) has been carried out. According to the obtained results, modified plants are able to grow on a medium supplemented with phytate as the sole source of phosphorus. Exterior characteristics (rosette diameter and area) of phytase-expressing plants grown on media containing phytate or inorganic phosphorus do not differ, which confirms that the plants use phytate as the phosphorus source. In the case of plant cultivation on a phytate-containing medium, a high phytase activity is observed in the cell walls of modified plants. The content of inorganic phosphorus in tissues of modified plants does not change in the case of their cultivation on the medium containing phytate as the sole source of phosphorus.

Performance and bone quality of broiler chicken fed a diet with reduced nonphytate phosphorus with bacterial phytases

This study evaluated the effect of three phytases in reduced nonphytate phosphorus (NPP) diets. In total, 2100 male Cobb 500® chicks were distributed in a completely randomized design. Ten experime...

Increasing doses of phytase from Citrobacter braakii in diets with reduced inorganic phosphorus and calcium improve growth performance and lean meat of growing and finishing pigs.

The objective of this study was to evaluate the effect of increasing doses of bacterial phytase (RONOZYME HiPhos) on performance and carcass characteristics of growing and finishing pigs. The study included 120 castrated males with initial weight of 23.21 ± 1.91 kg and 68 days of age, distributed in a randomized block design with five treatments and eight replicates with three animals each. The pigs were fed five corn-soybean meal-based diets: positive control (PC), supplemented with inorganic phosphorus and calcium; negative control (NC), with 0.13% reduction in available phosphorus and 0.11% in calcium; and three NC diets supplemented with 1,000, 2,000, and 3,000 phytase units (FYT)/kg in the feed. Compared with the NC diets without phytase, diets with 1,000, 2,000, and 3,000 FYT/kg inclusion increased the daily weight gain by +12% (quadratic, p<0.05) during the growing I period; +2.9, +2.9, and +10.5% (linear, p<0.01), respectively, during the growing II period; and +4.1, +5.1, and +8.2% (linear, p<0.001), respectively, over the entire experimental period. The daily feed intake increased by 0, +2.8, and +4.3% (linear, p<0.05), respectively, considering the entire experimental period; and the final live weight increased by +3.2, +4.2, and +6.1% (linear, p<0.001), respectively. The phytase treatments did not influence feed conversion ratio, carcass weight and yield, backfat thickness, loin depth and carcass lean meat. According to the European Carcass Classification (SEUROP), however, the animals fed the PC diet and the three phytase levels had more carcasses classified as E (between 55–60% lean meat) when compared to carcasses of pigs fed the NC. Supplementing increasing levels of phytase to a corn- and soybean meal-based diet with inorganic P and Ca reduction improved daily weight gain and feed intake of growing pigs, and such effects were maintained until slaughter age.

Genetically engineered rice with appA gene enhanced phosphorus and minerals

Phytic acid is the major source of phosphorus and other mineral bound compounds in many plant tissues especially seeds and bran of cereals. During germination, phytase enzyme degrades phytic acid and bound phosphate and minerals are released. The monogastric animal cannot digest phytate due to lack of the phytase enzyme. Considering that, we have generated low phytate rice by over expressing appA gene cloned from E. coli under the aleurone-specific promoter of maize zein gene. Molecular analysis confirmed the stable integration of transgene and plants were grown up to T3 generation. The T3 seeds showed around 45% decrease in seed phytate content with a fourfold increase of inorganic phosphorus (Pi) level. The enhanced iron and zinc was twofold and threefold respectively in polished seeds of transgenic plants. There was no change in germination behaviour and other morphological traits in transgenic seeds. Thus, this result provides evidence that tissue-specific expression of bacterial phytase can lead to the reduction of phytic acid in rice seeds without hampering its other physiological processes and phenotypic cost.

Effect of supplementation with Escherichia coli phytase for broilers on performance, nutrient digestibility, minerals in the tibia and diet cost

Although phytase has been researched, new enzymes have been produced, leading to different animal responses. In this scenario, the present study proposes to evaluate the inclusion of a bacterial phytase produced by Escherichia coli in broiler diets based on corn and soybean meal, with or without nutrient reductions, on the performance, nutrient digestibility, phosphorus bioavailability, and bone minerals of those animals and on the economic viability of this practice. A total of 896 male broiler chickens were distributed into 32 experimental units, each housing 28 broilers. The experiment was set up as a completely randomized design with four treatments (Positive Control (PC)- diet meeting the nutritional requirements of the broiler chickens; Negative Control (NC) with reductions of 100kcal/kg of ME, 0.14% avP and 0.11% tCa; NC + phytase (500 FTU/kg); PC + phytase (500FTU/kg)) and 8 replicates. Phytase increased (p &lt; 0.05) feed intake and body weight gain and improved feed conversion ratio in starter (1 to 21 days) and total (1 to 42 days) phases, respectively, compared with Negative Control diet without supplementation. The Negative Control + phytase diet also led to a feed intake similar to Positive Control in the starter and total phases. The inclusion of phytase without nutrient reductions improved (p &lt; 0.05) feed conversion ratio in the starter phase, compared to Positive Control diet. There was an increase (p &lt; 0.05) in the digestibility of dry matter, crude protein and phosphorus and in apparent digestible energy in the Negative Control + phytase diet compared to the Negative Control diet. Phytase supplementation increased (p &lt; 0.05) the digestibility of crude protein, calcium and phosphorus, and apparent digestible energy compared to Positive Control diet. Ash, phosphorus, and calcium contents were higher in the Negative Control + phytase diet compared with those observed in the Negative Control diet without enzyme (p &lt; 0.05). The Positive Control + phytase diet provided higher ash contents (p &lt; 0.05), but calcium and phosphorus deposition was similar to those obtained with Positive Control diet. Phytase inclusion allows for a reduction in the diet cost per ton of produced feed. In conclusion, dietary supplementation with bacterial phytase produced from Escherichia coli for broiler chickens is recommended, as it provided increases in production performance, nutrient digestibility, and energy metabolizability and a reduction in the diet cost.

Сравнительная характеристика фитаз из Citrobacter freundii и Yersinia intermedia, экспрессированных в метилотрофных дрожжах Ogataea polymorpha и Pichia pastoris

The genes for bacterial phytases from Citrobacter freundii and Yersinia intermedia were expressed for the first time in a thermotolarant yeast Ogataea polymorpha. A comparative analysis of the properties of recombinant phytases produced by Ogataea polymorpha and Pichia pastoris yeasts was carried out. It was shown that the stability, pH and temperature profiles of the enzyme activities are the same regardless of the host strain. It was proved that O. polymorpha yeast can be used to create producers of feed enzymes and to develop a technology for their cultivation at temperatures above 37 °C. The prospects of using the O. polymorpha yeast for these purposes were evaluated. Ogataea (Hansenula) polymorpha, Pichia pastoris, methylotrophic yeast, thermal tolerance, producer, recombinant phytase The work was financially supported by the Ministry of Science and Higher Education of RF (Project Unique Identifier RFMEFI57917X0145) using the Multipurpose Scientific Installation of All-Russian National Collection of Industrial Microorganisms National Bioresource Center, NRC «Kurchatov Institute»-GosNIIgenetika.

Comparative Analysis of the Expression Efficiency of the Bacterial Phytase Genes in Pichia pastoris Yeast by the Plate Test

Comparative analysis of the efficiency of the expression of the Escherichia coli, Obessumbacterium proteus, and Citrobacter freundii phytase genes in Pichia pastoris methylotrophic yeast has been carried out. A simple model with a genetic construct based on the pPICά vector and the plate test with a modified diagnostic medium containing methanol (instead of glucose) and calcium phytate was proposed for the first time in order to assess the expression level. The optimal pH value for the phytase activity was provided by the addition of lactic acid. The highest expression in the studied heterologous genes was shown for the C. freundii phytase gene.

Heterologous Expression of Histidine Acid Phytase from Pantoea sp. 3.5.1 in Yarrowia lipolytica

The dimorphic yeasts Yarrowia lipolytica are used as effective expression system and are characterized by a high level of production of heterologous protein. In this work, we aimed to clone and express Pantoea sp. 3.5.1 agpP phytase gene in Yarrowia lipolytica. Genetic constructs containing the native phytase gene (agpP) under the control of the strong hybrid promoter hp4d and the signal peptide of the alkaline extracellular protease XPR2 gene, as well as the optimized gene (agpP-opt) of phytase under the control of two signal peptides—bacterial and yeast were obtained. Recombinant Y. lipolytica strains with integrated bacterial phytase genes were obtained and expression of phytase was analyzed.

Supplementation of fungal and/or bacterial phytase in broiler diets formulated with reduced phosphorus level and different calcium contents

ABSTRACT The objective of the current study was to evaluate the effects of three calcium:available phosphorus (Ca:aP) ratios and different supplementation with phytases from different origins on performance and ash, Ca, and P contents in the tibia of broilers. A total of 900 male broilers (22 to 35 days old) were fed ten dietary treatments in a completely randomized design. A 3 × 3 + 1 factorial scheme was used, corresponding to three Ca:aP ratios (4.5:1.0, 6.0:1.0, and 7.5:1.0) and [...]

Cloning of Phytase Genes from Pantoea Sp. 3.5.1 and Bacillus ginsengihumi M2.11 in Pichia pastoris

Phytases (myo-inositol hexakisphosphate hydrolase) catalyze the hydrolysis of phytate to inorganic phosphate and less phosphorylated myo-inositol derivatives and are widely used as feed additives in animal nutrition. Nevertheless, nowadays, there is a constant search for new phytases and new expression systems for better production of these important enzymes. In this study, we report cloning of two novel bacterial phytases belonging to the different enzyme classes and having different properties in the methylotrophic yeast Pichia pastoris. Sequences of agpP and phyC genes, encoding histidine acid phytase from Pantoea sp. 3.5.1 and β-propeller phytase from Bacillus ginsengihumi M2.11, respectively, were optimized and chemically synthesized according to the P. pastoris codon usage bias. The optimized genes were cloned into the yeast vectors pPINK-HC and pPINK-LC under the control of the inducible promoter AOX1 and two different signal peptides—signal sequence of α-amylase gene from Aspergillus niger and presequence of inulinase gene from Kluyveromyces maxianus. PCR analysis, restriction analysis, and DNA sequencing confirmed correct integration of agpP and phyC genes into P. pastoris genome. As a result, recombinant strains of P. pastoris with codon-optimized bacterial phytase genes (agpP and phyC), encoding histidine acid and β-propeller phytases, integrated into the genome under the alcohol oxidase promoter AOX1 and two different signal peptides, were obtained. Recombinant phytase AgpP was stably expressed and secreted into the culture medium of yeasts, whereas the expression of phyC gene was only confirmed at transcription level.

Directed evolution of an acid Yersinia mollaretii phytase for broadened activity at neutral pH.

Phytases are phosphohydrolases that initiate the sequential hydrolysis of phosphate from phytate, which is the main storage form of phosphorous in numerous plant seeds, especially in cereals and grains. Phytate is indigestible for most monogastric animals, such as poultry, swine, fish, and humans; therefore, microbial phytases have been widely used in plant (specially soy)-based animal feeding to improve nutrition by enhanced phosphorus, mineral, and trace element absorption, and reducing phosphorus pollution by animal waste. Most phytases used as animal feed additives have an acid pH optimum (pH 2.5 and 5.5 for Aspergillus and pH 4.5 for E. coli phytases) and show a sharp decrease in performance at neutral pH, correlating with intestinal digestion. Directed evolution of phytases has been previously reported to improve enzyme thermostability, pH, or specific activity. In this manuscript, we report a directed evolution campaign of the highly active bacterial phytase from Yersinia mollaretii (YmPh) towards a broadened pH activity spectrum. Directed evolution identified the key positions T44 and K45 for increased YmPh activity at neutral pH. Both positions are located in the active site loop of the phytase and have a synergistic effect on activity with a broadened pH spectrum. Kinetic characterization of the improved variants, YmPh-M10 and -M16, showed up to sevenfold increased specific activity and up to 2.2-fold reduced Khalf at pH 6.6 under screening conditions compared to Yersinia mollaretii phytase wild type (YmPhWT).

Constitutive expression of bacterial phytase appA in Pichia pastoris using high-density fermentation

Constitutive expression of bacterial phytase appA in Pichia pastoris using high-density fermentation

Heterologous Expression of Secreted Bacterial BPP and HAP Phytases in Plants Stimulates Arabidopsis thaliana Growth on Phytate.

Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology.

New Bacterial Phytase through Metagenomic Prospection.

Alkaline phytases from uncultured microorganisms, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives in agricultural industry. The development of metagenomics has stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. In this study, a gene encoding a phytase was cloned from red rice crop residues and castor bean cake using a metagenomics strategy. The amino acid identity between this gene and its closest published counterparts is lower than 60%. The phytase was named PhyRC001 and was biochemically characterized. This recombinant protein showed activity on sodium phytate, indicating that PhyRC001 is a hydrolase enzyme. The enzymatic activity was optimal at a pH of 7.0 and at a temperature of 35 °C. β-propeller phytases possess great potential as feed additives because they are the only type of phytase with high activity at neutral pH. Therefore, to explore and exploit the underlying mechanism for β-propeller phytase functions could be of great benefit to biotechnology.

In silico structural, functional and phylogenetic analysis of Klebsiella phytases

Phytic acid or phytate (myo-inositol hexakisphosphate) is the principal storage indigestible form of phosphorus in different crops. It is considered as an antinutrient in human as well as animal (including fish, poultry, pig, chicken etc.) diet due to its chelating behavior of certain essential divalent minerals (Fe2+, Mg2+, Zn2+, Ca2+ etc.). The unabsorbed, indigested form of phosphorus also causes phosphate pollution in the soil by animal wastes. Phytate degrading enzymes like phytases (myo-inositol hexakisphosphate phosphohydrolase) in this regard can be very useful and also economically feasible to reduce the risk of phosphate pollution and increase the nutrient value in animal feeds at the same time. The Klebsiella phytases are suitable to use in the food industries of plant origin for their excellent thermal stability and high pH tolerance. From the present in silico investigation, it was found that Klebsiella phytases were 46–47 kDa molecular weight protein of histidine phosphatase superfamily having thermostability and alkalinity nature. This thermostability can be achieved due to possession of higher percentage of α helices and β sheets at the same time; the presence of higher aliphatic indices (range in between 88 and 91) etc. Interestingly, a strong correlation was found to be pertinent from phylogenetic studies of proteins with their cDNA among both species and strain level. Hence, the present study would be beneficial for future researchers (3D model available in Protein Model Database with acc. no.: PM0080562) to meet the demand of agricultural and industrial production of bacterial phytases particularly for agricultural farming.

Progress in comprehending the phytate–phytase axis in chicken-meat production

After an extended delay, the level of acceptance of exogenous phytases by the global chicken-meat industry is now almost complete. Contemporary bacterial phytases degrade phytate primarily in the gizzard. The extent of phytate degradation determines the extent to which phytate-bound phosphorus (P) is liberated; however, studies designed to investigate phytate degradation along the digestive tract have generated some confusing outcomes. This may be related to the reactivity of the phytate moiety, coupled with problems with inert dietary markers and perhaps a lack of complete and uniform extractions of phytate from digesta due to variations in digesta pH and phytate solubility. Quite recently, phytase was shown to have profound impacts on sodium (Na) digestibility coefficients in four segments of the small intestine. This has obvious implications for intestinal uptakes of glucose and amino acids via their respective Na+-dependent transport systems and it is possible that phytate and phytase have reciprocal impacts on ‘sodium pump’ (Na+, K+-ATPase) activity. It has been recently demonstrated unequivocally that phytase has the capacity to increase amino acid digestibility coefficients to the extent that phytase may generate a ‘proximal shift’ in the sites of amino acid absorption. The impact of phytase on starch digestibility is more equivocal and phytase responses may stem more from enhanced glucose absorption rather than starch digestion. The acceptance of phytase is hardly surprising, given its capacity to increase P utilisation coupled with numerous other positive influences that are still being properly realised.