Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.8 (phytase)
 1,997 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two periplasmatic phytases, called P1 and P2, were purified about 16,500-fold to an apparent homogeneity with a recovery of 7 and 18%, respectively. The enzymes behave as monomeric proteins with molecular masses of about 42 kDa. Because of the limited amounts recovered, the amino terminal sequence of only one of the phytases was determined. Both enzymes are very specific for phytate and have little or no activity on other phosphate esters tested. The kinetic parameters for the hydrolysis of Na-phytate and p-nitrophenyl phosphate are kcat/KM 478 x 10(5) s-1 M-1 and 0.6 x 10(5) s-1 M-1 at pH 4.5. The hydrolysis pathway for phytate was elucidated for P2; consequently, this enzyme is a 6-phytase. The chemical and kinetic properties of the purified phytase P2 points to an identity with an enzyme described by Dassa et al. (1982, J. Biol. Chem. 257, 6669-6676) as a pH 2.5 acid phosphatase. Because of the kinetic parameters it would be better to denote this enzyme as a phytase.
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PMID:Purification and characterization of two phytases from Escherichia coli. 838 49

A study was conducted to determine the efficacy of phytase, an enzymic cocktail, and a waste Aspergillus niger mycelium to hydrolyze phytate present in corn-soybean meal diets. One hundred turkey poults were assigned to dietary treatments for 2 wk (days 7 to 21). Dietary treatments included: 1) NRC (1994) diet (NRC), with recommended concentration of 0.6% available P (aP) and 1.2% Ca; 2) Phytase diet (PHYT), 1,000 units phytase/kg diet, 0.16% aP, and 0.84% Ca; 3) cocktail diet (COC), 1,000 units of phytase/kg diet plus acid phosphatase (100 units/g of diet), acid protease (42 units/g of diet), pectinase (2.94%), 0.16% aP, and 0.84% Ca; 4) Fungal mycelium diet (MYC), 5% mycelium, 0.16% aP, and 0.84% Ca; and 5) a positive control diet (CTRL+), 0.42% aP, and 0.84% Ca. Turkeys fed the PHYT diet consumed less feed and gained less weight but retained more P than poults fed the CTRL+ or NRC diets. Poults fed the COC diet performed as well as poults fed CTRL+ or NRC diets but retained more P (77%) and Ca (68%). Poults fed the MYC diet retained 79% P, gained the most weight, and were more efficient than poults fed any other dietary treatment. In vitro P release from experimental diets correlated well (R = 0.906) with P retention as observed in the feeding trial. Compared with the diet containing phytase as the sole supplemental enzyme, both the enzymic cocktail and fungal mycelium enhanced performance, bone mineralization, and retention of P and Ca in growing turkeys.
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PMID:The efficacy of an enzymic cocktail and a fungal mycelium in dephosphorylating corn-soybean meal-based feeds fed to growing turkeys. 877 33

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.
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PMID:Phytase. 886 87

Obtaining high quality protein crystals remains a rate-limiting step in the determination of three-dimensional X-ray structures. A frequently encountered problem in this respect is the high or heterogeneous carbohydrate content of many eukaryotic proteins. A number of reports have demonstrated the use of enzymatic deglycosylation in the crystallization of certain glycoproteins. Although this is an attractive tool, there are some problems that hinder the more widespread use of glycosidases in crystallization. First, commercially available glycosidases are relatively expensive, which virtually prohibits their use on a large scale. Second, the glycosidase must be removed from the glycoprotein of interest following deglycosylation, which is not always straightforward. To circumvent these problems we have cloned the two most generally useful glycosidases, peptide-N-glycosidase F and endoglycosidase F1 from Flavobacterium meningosepticum, as fusion proteins with glutathione S-transferase. The fusion not only allows rapid purification of these enzymes from Escherichia coli cell extracts, but also permits rapid removal from target proteins following deglycosylation. We have used these enzymes to obtain crystals of phytase from Aspergillus ficuum and acid phosphatase from Aspergillus niger and to obtain a new crystal form of recombinant human renin.
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PMID:Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases. 897 70

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.
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PMID:The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila. 902 98

The kinetics, mineral dependency, and pH dependency of phytate hydrolysis by preparations of chicken small intestinal brush border membrane vesicles were determined. Substantial phytate hydrolysis occurred over the pH range from 5 to 6.5 with a maximum hydrolysis at pH of 6. Inclusion of 25 mM MgCl2 in the media doubled the rate of phytate hydrolysis. The brush border was shown to have no nonspecific acid phosphatase activity and excess phytate had no effect on alkaline phosphatase activity at pH 11. Under optimal conditions of pH 6 plus 25 mM MgCl2, a kinetic model of a single Michaelis-Menten type of enzymatic activity with a Km of 0.160 +/- 0.008 mM and a Vmax of 42.5 +/- 1.0 nmol/mg vesicle protein per min plus a small unsaturable component converged to the data (P < 0.05). The specific and total activities of intestinal brush border phytase were highest in the duodenum (P < 0.05) and decreased progressively down the length of the gut. Intestinal brush border vesicles prepared from broiler chicks and mature laying hens had comparable specific phytase activity. However, the total activity of brush border phytase was 35% higher in the small intestine of laying hens (P < 0.05). Intestinal brush border phytase could contribute to phytate-phosphorus digestibility and may be subject to regulation in response to the dietary phosphorus and vitamin D status of the chicken.
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PMID:Phytase activity in the small intestinal brush border membrane of the chicken. 956 39

A close examination of the protein sequence encoded by the Arabidopsis thaliana gene F21M12.26 reveals the gene product to be a phosphomonoesterase, acid optimum (EC 3.1.3.2). A subclass of this broad acid phosphatase is also known as 'histidine acid phosphatase. ' This is the first sequence-based evidence for a 'histidine acid phosphatase' in a dicotyledon. One important member of this class of enzymes is Aspergillus niger (ficuum) phytase, which came into prominence for its commercial application as a feed additive. The putative protein from A. thaliana gene F21M12.26 shares many important features of Aspergillus phytase, namely, size, active-site sequence, catalytic dipeptide and ten cysteine residues located in the key areas of the molecule, but lacks all nine N-glycosylation sites.
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PMID:Identification of a histidine acid phosphatase (phyA)-like gene in Arabidopsis thaliana. 979 Sep 41

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.
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PMID: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. 979 5

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37 degreesC ranged from 23 to 196 U. (mg of protein)-1, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, alpha- and beta-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.
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PMID:Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. 992 55

Bacterial strains were isolated from the pig colon to screen for phytase and acid phosphatase activities. Among 93 colonies, Colony 88 had the highest activities for both enzymes and was identified as an Escherichia coli strain. Using primers derived from the E. coli pH 2.5 acid phosphatase appA sequence (Dassa et al. (1990), J. Bacteriol. 172, 5497-5500), we cloned a 1482 bp DNA fragment from the isolate. In spite of 95% homology between the sequenced gene and the appA, 7 amino acids were different in their deduced polypeptides. To characterize the properties and functions of the encoded protein, we expressed the coding region of the isolated DNA fragment and appA in Pichia pastoris, respectively, as r-appA2 and r-appA. The recombinant protein r-appA2, like r-appA and the r-phyA phytase expressed in Aspergillus niger, was able to hydrolyze phosphorus from sodium phytate and p-nitrophenyl phosphate. However, there were distinct differences in their pH profiles, Km and Vmax for the substrates, specific activities of the purified enzymes, and abilities to release phytate phosphorus in soybean meal. In conclusion, the DNA fragment isolated from E. coli in pig colon seems to encode for a new acid phosphatase/phytase and is designated as E. coli appA2.
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PMID:Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon. 1009 20


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