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PubMed2003

Biochemical properties and substrate specificities of alkaline and histidine acid phytases.

Phytases are a special class of phosphatase that catalyze the sequential hydrolysis of phytate to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are added to animal feedstuff to reduce phosphate pollution in the environment, since monogastric animals such as pigs, poultry, and fish are unable to metabolize phytate. Based on biochemical properties and amino acid sequence alignment, phytases can be categorized into two major classes, the histidine acid phytases and the alkaline phytases. The histidine acid phosphatase class shows broad substrate specificity and hydrolyzes metal-free phytate at the acidic pH range and produces myo-inositol monophosphate as the final product. In contrast, the alkaline phytase class exhibits strict substrate specificity for the calcium-phytate complex and produces myo-inositol trisphosphate as the final product. This review describes recent findings that present novel viewpoints concerning the molecular basis of phytase classification.

6-Phytase↗
PubMed2005

Conservation of cysteine residues in fungal histidine acid phytases.

Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.

6-Phytase↗
PubMed1997

The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila.

Phytases catalyse the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. In this study genes encoding novel phytases from two different filamentous fungi, Aspergillus terreus strain 9A-1 and Myceliophthora thermophila were isolated. The encoded PhyA phytase proteins show 60% (A. terreus) and 48% (M. thermophila) identity, respectively, to the PhyA of Aspergillus niger and have 21-29% identity compared to other histidine acid phosphatases. All three PhyA proteins, in contrast to the A. niger pH 2.5-optimum acid phosphatase, prefer phytic acid as substrate and show enzyme activity at a broad range of acidic pH values. Based on their enzyme characteristics and protein sequence homology, the phytases form a novel subclass of the histidine acid phosphatase family.

6-Phytase↗
PubMed2026

In silico genome mining and characterization of putative horse feces-derived bacterial phytases as potential monogastric animal feed additive candidates.

Phytic acid exerts a significant antinutritional effect in poultry, swine, and fish, which can be mitigated by supplementing monogastric feeds with efficient microbial phytases. Accordingly, mining bacterial genomes for novel phytases represents a strategic computational approach to identifying candidates for improving monogastric animal nutrition. In this study, 162 bacterial genomes associated with horse feces were systematically mined using an in silico pipeline to identify and characterize putative phytases.A total of 69 non-redundant sequences were identified and classified as histidine acid phytase (HAPhy) or protein tyrosine phosphatase-like phytase (PTPLPhy). HAPhys were detected in the genomes of Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Acinetobacter baumannii, and Cutibacterium equinum, whereas PTPLPhys were found in K. pneumoniae, Limosilactobacillus reuteri, Pediococcus acidilactici, Bifidobacterium pseudolongum, and Prescottella equi. Principal component analysis identified glucose-1-phosphatase (CAJ1242485.1) and bifunctional acid phosphatase (NHR17779.1) as the HAPhy candidates exhibiting the most favorable predicted physicochemical properties for potential feed applications. Similarly, among the PTPLPhys, protein tyrosine phosphatase (UNQ40438.1) and a hypothetical protein (CAJ1246072.1) showed the most favorable computational profiles. Biosafety analysis identified potential virulence factors, indicating that sources should be screened prior to feed application. High-quality AlphaFold2 models were obtained for these phytases (90.9-97.2). Molecular docking analysis showed that NHR17779.1 exhibited the strongest binding to phytic acid, whereas CAJ1246072.1 demonstrated the weakest interaction. Overall, this study identifies the horse fecal microbiota as a diverse source of putative phytases that may serve as promising targets for genetic and protein engineering; however, further in vitro and in vivo studies are essential to validate the enzymatic activity and industrial efficacy of these computational candidates.

Bacterial phytase↗
PubMed2001

Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11.

The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate.

6-Phytase↗
PubMed2006

myo-Inositol phosphate isomers generated by the action of a phytate-degrading enzyme from Klebsiella terrigena on 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.

6-Phytase↗
PubMed2006

Lily pollen alkaline phytase is a histidine phosphatase similar to mammalian multiple inositol polyphosphate phosphatase (MINPP).

Phytic acid is the most abundant inositol phosphate in cells; it constitutes 1-5% of the dry weight of cereal grains and legumes. Phytases are the primary enzymes responsible for the hydrolysis of phytic acid and thus play important roles in inositol phosphate metabolism. A novel alkaline phytase in lily pollen (LlALP) was recently purified in our laboratory. In this paper, we describe the cloning and characterization of LlALP cDNA from lily pollen. Two isoforms of alkaline phytase cDNAs, LlAlp1 and LlAlp2, which are 1467 and 1533 bp long and encode proteins of 487 and 511 amino acids, respectively, were identified. The deduced amino acid sequences contains the signature heptapeptide of histidine phosphatases, -RHGXRXP-, but shares < 25% identity to fungal histidine acid phytases. Phylogenetic analysis reveals that LlALP is most closely related to multiple inositol polyphosphate phosphatase (MINPP) from humans (25%) and rats (23%). mRNA corresponding to LlAlp1 and LlAlp2 were expressed in leaves, stem, petals and pollen grains. The expression profiles of LlAlp isoforms in anthers indicated that mRNA corresponding to both isoforms were present at all stages of flower development. The expression of LlAlp2 cDNA in Escherichia coli revealed the accumulation of the active enzyme in inclusion bodies and confirmed that the cDNA encodes an alkaline phytase. In summary, plant alkaline phytase is a member of the histidine phosphatase family that includes MINPP and exhibits properties distinct from bacterial and fungal phytases.

6-Phytase↗
PubMed1998

Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis.

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55 degrees C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.

6-Phytase↗
PubMed2001

A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings.

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases.

6-Phytase↗
PubMed2000

Crystal structures of Escherichia coli phytase and its complex with phytate.

Phytases catalyze the hydrolysis of phytate and are able to improve the nutritional quality of phytate-rich diets. Escherichia coli phytase, a member of the histidine acid phosphatase family has the highest specific activity of all phytases characterized. The crystal structure of E. coli phytase has been determined by a two-wavelength anomalous diffraction method using the exceptionally strong anomalous scattering of tungsten. Despite a lack of sequence similarity, the structure closely resembles the overall fold of other histidine acid phosphatases. The structure of E. coli phytase in complex with phytate, the preferred substrate, reveals the binding mode and substrate recognition. The binding is also accompanied by conformational changes which suggest that substrate binding enhances catalysis by increasing the acidity of the general acid.

6-Phytase↗
PubMed1997

Cloning of the phytases from Emericella nidulans and the thermophilic fungus Talaromyces thermophilus.

Phytases (EC 3.1.3.8) belong to the family of histidine acid phosphatases. We have cloned the phytases of the fungi Emericella nidulans and Talaromyces thermophilus. The putative enzyme encoded by the E. nidulans sequence consists of 463 amino acids and has a Mr of 51785. The protein deduced from the T. thermophilus sequence consists of 466 amino acids corresponding to a Mr of 51450. Both predicted amino acid sequences exhibited high identity (48% to 67%) to known phytases. This high level of identity allowed the modelling of all available fungal phytases based on the three-dimensional structure coordinates of the Aspergillus niger phytase. By this approach we identified 21 amino acids which are conserved in fungal phyA phytases and are part of the residues forming the substrate pocket. Furthermore, potential glycosylation sites were identified and compared between the aforementioned phytases and the A. niger phytase.

6-Phytase↗
PubMed1998

Comparison of the thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase, and A. niger PH 2.5 acid phosphatase.

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90 degrees C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of A. fumigatus and A. niger were both denatured at temperatures between 50 and 70 degrees C. After heat denaturation at temperatures up to 90 degrees C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90 degrees C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases, A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of >/=80 degrees C. In feed pelleting experiments performed at 75 degrees C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85 degrees C, however, the recovery of enzymatic activity was considerably higher for A. fumigatus phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80 degrees C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.

6-Phytase↗
PubMed2006

Kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP.

Chemical probing of histidine residues using specific modifiers, iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted in the inactivation of phytase (phy A). The kinetic theory of the substrate reaction during the modification of enzyme activity was applied to a study of the kinetics of the course of inactivation of phytase by IAA and DEP. The results suggested that histidine residues are involved in the active site of the enzyme. They also indicated that inactivation of the enzyme by IAA was via a complexing type inhibition, while the inhibition by DEP reaction involved a conformational change step before inactivation. The dissociation constant of the enzyme-inhibitor complex of IAA, the constant of the conformational change of DEP and the microscopic rate constants of two inhibitors were obtained.

6-Phytase↗
PubMed2000

Advances in phytase research.

Since its discovery in 1907, a complex of technological developments has created a potential $500 million market for phytase as an animal feed additive. During the last 30 years, research has led to increased use of soybean meal and other plant material as protein sources in animal feed. One problem that had to be overcome was the presence of antinutritional factors, including phytate, in plant meal. Phytate phosphorus is not digested by monogastric animals (e.g., hogs and poultry), and in order to supply enough of this nutrient, additional phosphate was required in the feed ration. Rock phosphate soon proved to be a cost-effective means of supplying this additional phosphorus, and the excess phytin phosphorus could be disposed of easily with the animals' manure. However, this additional phosphorus creates a massive environmental problem when the land's ability to bind it is exceeded. Over the last decade, numerous feed studies have established the efficacy of a fungal phytase, A. niger NRRL 3135, to hydrolyze phytin phosphorus in an animal's digestive tract, which benefits the animal while reducing total phosphorus levels in manure. The gene for phytase has now been cloned and overexpressed to provide a commercial source of phytase. This monomeric enzyme, a type of histidine acid phophatase (HAP), has been characterized and extensively studied. HAPs are also found in other fungi, plants, and animals. Several microbial and plant HAPs are known to have significant phytase activity. A second A. niger phytase (phyB), a tetramer, is known and, like phyA, has had its X-ray crystal structure determined. The model provided by this crystal structure research has provided an enhanced understanding of how these molecules function. In addition to the HAP phytase, several other phytases that lack the unique HAP active site motif RHGXRXP have been studied. The best known group of the non-HAPs is phytase C (phyC) from the genus Bacillus. While a preliminary X-ray crystallographic analysis has been initiated, no enzymatic mechanism has been proposed. Perhaps the pivotal event in the last century that created the need for phytase was the development of modern fertilizers after the Second World War. This fostered a transformation in agriculture and a tremendous increase in feed-grain production. These large quantities of cereals and meal in turn led to the transition of one segment of agriculture into "animal agriculture," with their its animal production capability. The huge volumes of manure spawned by these production units in time exceeded both the capacity of their crops and crop lands to utilize or bind the increased amount of phosphorus. Nutrient runoff from this land has now been linked to a number of blooms of toxin-producing microbes. Fish kills associated with these blooms have attracted public and governmental concern, as well as greater interest in phytase as a means to reduce this phosphorus pollution. Phytase research efforts now are focused on the engineering of an improved enzyme. Improved heat tolerance to allow the enzyme to survive the brief period of elevated temperature during the pelletization process is seen as an essential step to lower its cost in animal feed. Information from the X-ray crystal structure of phytase is also relevant to improving the pH optimum, substrate specificity, and enzyme stability. Several studies on new strategies that involve synergistic interactions between phytase and other hydrolytic enzymes have shown positive results. Further reduction in the production cost of phytase is also being pursued. Several studies have already investigated the use of various yeast expression systems as an alternative to the current production method for phytase using overexpression in filamentous fungi. Expression in plants is underway as a means to commercially produce phytase, as in biofarming in which plants such as alfalfa are used as "bioreactors," and also by developing plant cultivars that would produce enough transgenic phytase so that additional supplementation of their grain or meals is not necessary. Ultimately, transgenic poultry and hogs may produce their own digestive phytase. Another active area of current phytase research is expanding its usage. One area that offers tremendous opportunity is increasing the use of phytase in aquaculture. Research is currently centered on utilizing phytase to allow producers in this industry to switch to lower-cost plant protein in their feed formulations. Development of a phytase for this application could significantly lower production costs. Other areas for expanded use range from the use of phytase as a soil amendment, to its use in a bioreactor to generate specific myo-inositol phosphate species. The transformation of phytase into a peroxidase may lead to another novel use for this enzyme. As attempts are made to widen the use of phytase, it is also important that extended exposure and breathing its dust be avoided as prudent safety measures to avoid possible allergic responses. In expanding the use of phytase, another important consideration has been achieved. Conservation of the world's deposits of rock phosphate is recognized as important for future generations. Phosphorus is a basic component of life like nitrogen, but, unlike nitrogen, phosphorus does not have a cycle to constantly replenish its supply. It is very likely that the use of phytase will expand as the need to conserve the world's phosphate reserves increases.

6-Phytase↗
PubMed1996

Phytase.

Of all the sources of phytase that have been studied (plant, animal, and microorganisms), the highest yields are produced by a wild-type strain A. niger NRRL 3135 (12.7 mg P/hr/ml = 6.8 microns P/ml/min = 113.9 nKat/ml) in a mineral salt medium in which total phosphate (4 mg %) is limiting for growth and cornstarch and glucose are the carbon sources. Synthesis of the enzyme is repressed by phosphate in the wild-type strain. Aspergillus niger NRRL 3135 produces two phytases one with pH optima at 2.5 and 5.5 (phyA) and one with an optimum at pH 2.0 (phyB). It also produces a pH 6.0 optimum phosphatase that has no phytase activity. These three glycoproteins have been purified to homogeneity, characterized, sequenced, and cloned. The sequences have been compared to each other, other phytases, and to known phosphatases. Their homology has been determined. The active sites of phytases show remarkable homology to the active site residues of the members of a particular class of acid phosphatase (histidine phosphatase). The most conserved sequence is RHGXRXP. Phytase has been covalently immobilized on Fractogel TSK HW-75 F and glutaraldehyde-activated silicate. It has been immobilized on agarose. Losses of activity have been noted on immobilization but these may be minimized by future research. It should be possible to commercially produce and recover penta-, tetra-, tri-, di-, and monoinositol phosphates using immobilized phytase if markets develop for those products. Phytase (phyA) from A. niger NRRL 3135 has been cloned into an A. niger glucoamylase producing strain CBS 513.88 using a construct that has a glucoamylae promoter and an A. niger NRRL 3135 leader sequence, and that is devoid of phosphate repression. The yield of the secreted enzyme was increased 52-fold above that of wild-type A. niger NRRL 3135. The bioengineered organism produces 270 microns P/ml/min (4500 nKat/ml) which is approximately 7.9 g/liter in the medium. The yield of the secreted enzyme was increased 1440-fold above that of wild type CBS 513.88. Commercial preparations of the cloned enzyme are available. Phytase (phyA) has been cloned into tobacco and canola. The enzyme is localized in the seed and expressed at high levels. Feeding of the seed to animals has made the phytin-P in the commercial diets available to the animals. The efficacy of feeding phytase to monogastric animals (poultry and swine) has been established. The amount of enzyme that is necessary to be added to commercial diets has been titred for broilers, layers, turkeys, ducks, and swine. The units of enzyme required are related to the phytin-P content in the diet. The use of the enzyme as a feed additive has been cleared in 22 countries. If phytase were used in the diets of all of the monogastric animals reared in the U.S., it would release phosphorus that has a value of $1.68 x 10(8) per year. The FDA has approved the enzyme preparation as GRAS. The effect of feeding phytase to animals enables assimilation of the P found in feed ingredients and diminishes the amount of phosphate in the manure and subsequently entering the environment. The effect of feeding phytase to animals on pollution has been quantitatively determined. If phytase were used in the diets of all of the monogastric animals reared in the United States, it would preclude 8.23 x 10(7) kg P from entering the environment.

6-Phytase↗
PubMed2004

Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus.

The gene phyA encoding phytase was isolated from Obesumbacterium proteus genomic library and sequenced. The cleavage site of the PhyA signal peptide was predicted and experimentally proved. The PhyA protein shows maximum identity of 53% and 47% to phosphoanhydride phosphorylase from Yersinia pestis and phytase AppA from Escherichia coli, respectively. Based on protein sequence similarity of PhyA and its homologs, the phytases form a novel subclass of the histidine acid phosphatase family. To characterize properties of the PhyA protein, we expressed the phyA gene in E. coli. The specific activity of the purified recombinant PhyA was 310 U mg(-1) of protein. Recombinant PhyA showed activity at pH values from 1.5 through 6.5 with the optimum at 4.9. The temperature optimum was 40-45 degrees C at pH 4.9. The Km value for sodium phytate was 0.34 mM with a Vmax of 435 U mg(-1).

6-Phytase↗
PubMed2004

Effect of microbial phytase on ileal digestibility of phytate phosphorus, total phosphorus, and amino acids in a low-phosphorus diet for broilers.

The study aimed to assess the effect of a commercially available microbial phytase added to a corn-soybean meal diet on phytate P and total P in terminal ileal digesta as well as on true ileal amino acid digestibility. Three low-P diets containing 0, 500, or 750 U/kg of microbial phytase were fed to 21-d-old broiler chickens. Titanium dioxide was used as an indigestible marker. Ileal contents were collected from euthanized birds and analyzed, along with the diets, for total P, phytate P, and amino acids. Endogenous P determined at the terminal ileum was 446 +/- 59 mg/kg food dry matter (mean +/- SE). Endogenous ileal amino acids ranged from 219 +/- 33 mg/kg food dry matter for tryptophan to 1,255 +/- 166 mg/kg food dry matter for glutamic acid. Supplementation with microbial phytase resulted in a significantly (P < or = 0.05) greater phytate P disappearance (11% greater disappearance vs. unsupplemented control) from the terminal ileum. Similarly, true ileal total P digestibility was (P < or = 0.05) higher (10 to 12%) when microbial phytase was added. True ileal amino acid digestibility was significantly (P < or = 0.05) greater in the presence of microbial phytase for all the amino acids examined with the exception of methionine, tyrosine, histidine, and tryptophan. The mean increase in true ileal amino acid digestibility was 3.4%. The effect of phytase on true ileal phytate P, total P, and amino acid digestibility was similar for the 2 phytase inclusion levels tested. Microbial phytase improved phytate P and total P digestibility, as well as true ileal amino acid digestibility, for a corn-soybean diet.

6-Phytase↗
PubMed2006

Molecular cloning of the phytase gene from Citrobacter braakii and its expression in Saccharomyces cerevisiae.

The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppAEc and rAppASc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppASc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppAEc.

6-Phytase↗