Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

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

Proteolysis of two purified recombinant enzymes, namely, the Aspergillus niger phytase (r-PhyA) and the Escherichia coli pH 2.5 acid phosphatase (r-AppA), by pepsin and trypsin was investigated in this study. After r-PhyA and r-AppA were incubated with different concentrations of pepsin or trypsin, their residual phytase activities and amounts of inorganic phosphorus released from soybean meal were determined. Both enzymes retained more than 85% of their original activities at the trypsin/phytase ratios (w/w) 0.001 and 0. 005, while r-AppA and r-PhyA lost 60 and 20% of the original activity at the ratio of 0.01 or 0.025, respectively. In contrast, there was a 30% increase in phytase activity after r-AppA was incubated with pepsin at the ratios of 0.005 or 0.01. Meanwhile, r-PhyA lost 58 to 77% of its original activity under the same conditions. Trypsin and pepsin affected the hydrolysis of phytate phosphorus from soybean meal by r-AppA and r-PhyA in a similar way to their residual phytase activities. All of these in vitro proteolyses were confirmed by SDS-PAGE analysis. Our results demonstrate different sensitivities of r-AppA and r-PhyA to trypsin and pepsin, suggesting active trypsin resistant r-PhyA and pepsin resistant r-AppA polypeptides.
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PMID:Different sensitivity of recombinant Aspergillus niger phytase (r-PhyA) and Escherichia coli pH 2.5 acid phosphatase (r-AppA) to trypsin and pepsin in vitro. 1032 21

The efficacy of Aspergillus niger (APhy) phytase, Trichoderma reesei (TPhy) phytase and acid phosphatase (TAcPh) preparations in improving the utilization of phytin-phosphorus in the maize-soybean meal (SBM) or barley-SBM (800:200 g kg-1) diets was studied in two separate digestibility and balance trials with ten growing pigs using 5 x 5 Latin square designs. The positive control diet contained a total phosphorus (P) of 6.5 g kg-1, while the negative control as well as the APhy, TPhy and TAcPh supplemented diets which did not contain additional inorganic-P, had a total P of 4.1 g kg-1. The APhy and TPhy supplements provided phytase activity of 1000 PU g-1 together with AcPh of 8000 HFU g-1. TAcPh at a level of 8000 HFU g-1 was the only addition to one diet. The intrinsic phytase activity of barley was 355 PU g-1 while maize and soybean meal showed no phytase activity. Phytase supplements of the APhy and TPhy sources increased ash digestibility in both diets but had only a minor effect on nitrogen utilization. The addition of phytase improved absorption of P by 21%-units in barley-SBM diet and 29%-units in maize-SBM diet, without any difference between the two phytase sources. The retained P in diets with phytase was higher than in diets without phytase, 4.4 (APhy), 4.5 (TPhy) vs. 2.9 g d-1 in barley-SBM-diets and 3.7 (APhy), 4.0 (TPhy) vs. 1.8 g d-1 in maize-SBM-diets. No difference was found between the two sources of phytase. TAcPh without additional phytase did not show any effect on P absorption or retention. Ca absorption and retention were improved due to the phytase treatments. Supplementing pig diets with either APhy or TPhy sources seems to be equally effective in enhancing the availability of phytate-P. Consequently, these supplements can reduce the P-excretion of pigs by 32-40% as compared with the diet supplemented with inorganic-P.
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PMID:Comparison of Aspergillus niger phytase and Trichoderma reesei phytase and acid phosphatase on phytate phosphorus availability in pigs fed on maize-soybean meal or barley-soybean meal diets. 1054 73

A study was conducted to determine the cumulative effects of phosphorolytic enzymes, cell wall-degrading enzymes, and citric acid and Ca levels on feed intake, BW gain (BWG), feed conversion, intestinal viscosity, and toe ash of broilers (d 1 to 21) fed wheat-based diets. Broilers were fed the following six diets at either 0.59, 0.69, or 0.79% Ca: 1) a negative control (NC) diet, 0.17% available P; 2) NC + 750 phytase units/kg diet; (3) phytase + 3,156 units of acid phosphatase/kg diet; 4) phytase + acid phosphatase + 1,900 units of pectinase/g diet; 5) phytase + acid phosphatase + pectinase + 3% citric acid; and (6) NC plus 0.24% available P. The 18 dietary treatments were fed to four pen replicates of eight birds each. Phytase addition at the low Ca level increased BWG, improved feed intake and conversion and toe ash, and reduced intestinal viscosity and ileal length. Subsequent addition of acid phosphatase, at 0.69% Ca, resulted in increases in BWG, 42%; feed intake 32%; feed conversion 7.5%; and toe ash, 63% over the NC diet. Pectinase addition produced further improvements in 21-d BWG and feed intake at 0.59 and 0.79% Ca, increased toe ash in chicks fed 0.79% Ca, and reduced intestinal viscosity. Supplementation of wheat-based 0.17% available P diets with phytase and acid phosphatase and with appropriate concentrations of pectinase, citric acid, and Ca significantly improved BWG, feed intake and conversion and intestinal viscosity over the 0.41% available P diets. Bone mineralization of chicks fed phytase + acid phosphatase and 0.69% Ca and those fed phytase + acid phosphatase + pectinase + citric acid and 0.59% Ca was similar to that of chicks fed the 0.41% available P diets.
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PMID:Effects of phosphorolytic and cell wall-degrading enzymes on the performance of growing broilers fed wheat-based diets containing different calcium levels. 1068 91

The appA gene that was previously shown to code for an acid phosphatase instead codes for a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The purified enzyme with a molecular mass of 44,708 Da was further separated by chromatofocusing into two isoforms of identical size with isoelectric points of 6.5 and 6.3. The isoforms had identical pH optima of 4.5 and were stable at pH values from 2 to 10. The temperature optimum for both phytase isoforms was 60 degrees C. When heated at different pH values the enzyme showed the greatest thermal resistance at pH 3. The pH 6.5 isoform exhibited K(m) and Vmax values of 0.79 mM and 3165 U.mg-1 of protein for phytase activity and 5.5 mM and 712 U.mg-1 of protein for acid phosphatase, respectively. The pH 6.3 isoform exhibited slightly lower K(m) and Vmax values. The enzyme exhibited similar properties to the phytase purified by Greiner et al. (1993), except the specific activity of the enzyme was at least 3.5-fold less than that previously reported, and the N-terminal amino acid sequence was different. The Bradford assay, which was used by Greiner et al. (1993) for determination of enzyme concentration was, in our hands, underestimating protein concentration by a factor of 14. Phytase production using the T7 polymerase expression system was enhanced by selection of a mutant able to grow in a chemically defined medium with lactose as the carbon source and inducer. Using this strain in fed-batch fermentation, phytase production was increased to over 600 U.mL-1. The properties of the phytase including the low pH optimum, protease resistance, and high activity, demonstrates that the enzyme is a good candidate for industrial production as a feed enzyme.
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PMID:Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. 1069 72

A total of 183 samples representing 24 feedstuffs were analyzed for total phosphorus, phytate phosphorus content, phytase (Phy), and acid phosphatase (AcPh) activities with the objective to predict the capacity to hydrolyze phytic acid and to contribute to formulating environmentally adequate diets for monogastric animals. Of the cereals and cereal byproducts analyzed, only rye (5147 U kg(-)(1); 21 955 U g(-)(1)), wheat (1637 U kg(-)(1); 10 252 U g(-)(1)), rye bran (7339 U kg(-)(1); 56 722 U g(-)(1)), and wheat bran (4624 U kg(-)(1); 14 106 U g(-)(1)) were rich in Phy and AcPh activities. Legume seeds and oilseeds contained negligible Phy activity and a moderate amount of AcPh activity, except for kidney bean (33 433 U g(-)(1)) and full-fat linseed meal (13 263 U g(-)(1)). On the other hand, a significant linear regression between phytate phosphorus (y) and total phosphorus (x) was observed in cereal byproducts (R(2) = 0. 95; y = 0.8458x - 0.0367; P < 0.001) and oil seeds (R(2) = 0.95; y = 0.945x - 0.20; P < 0.001). Phy and AcPh were positively correlated with respect to phytate phosphorus in cereals, cereal byproducts, and other byproducts and negatively correlated in legume seeds and oilseeds. Except for cereals, the highest correlation between enzyme activities and phytate phosphorus was found for phytase. It is not possible to predict Phy and AcPh activities from phytate phosphorus content by linear and quadratic regressions. Finally, only highly significant and positive correlation was found between Phy and AcPh activities for cereals, cereal byproducts, and oilseeds.
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PMID:Phytase and acid phosphatase activities in plant feedstuffs. 1099 5

Escherichia coli pH 2.5 acid phosphatase gene (appA) and three mutants were expressed in Pichia pastoris to assess the effect of strategic mutations or deletion on the enzyme (EcAP) biochemical properties. Mutants A131N/ V134N/D207N/S211N, C200N/D207N/S211N, and A131N/ V134N/C200N/D207N/S211N had four, two, and four additional potential N-glycosylation sites, respectively. Extracellular phytase and acid phosphatase activities were produced by these mutants and the intact enzyme r-AppA. The N-glycosylation level was higher in mutants A131N/V134N/D207N/S211N (48%) and A131N/V134N/ C200N/D207N/S211N (89%) than that in r-AppA (14%). Despite no enhancement of glycosylation, mutant C200N/ D207N/S211N was different from r-AppA in the following properties. First, it was more active at pH 3.5-5.5. Second, it retained more (P < 0.01) phytase activity than that of r-AppA. Third, its specific activity of phytase was 54% higher. Lastly, its apparent catalytic efficiency kcat/Km for either p-nitrophenyl phosphate (5.8 x 10(5) vs 2.0 x 10(5) min(-1) M(-1)) or sodium phytate (6.9 x 10(6) vs 1.1 x 10(6) min(-1) M(-1)) was improved by factors of 1.9- and 5.3-fold, respectively. Based on the recently published E. coli phytase crystal structure, substitution of C200N in mutant C200N/D207N/S211N seems to eliminate the disulfide bond between the G helix and the GH loop in the alpha-domain of the protein. This change may modulate the domain flexibility and thereby the catalytic efficiency and thermostability of the enzyme.
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PMID:Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris. 1105 Nov 3

Efficacies of phytase, phosphorolytic enzymes (phytase + acid phosphatase), an enzymic "cocktail" (phytase + acid phosphatase + pectinase + citric acid), a novel Aspergillus niger (fungal) mycelium (FM), and FM enriched in phytase and antioxidants were investigated in growing broilers (Days 1 to 21) fed wheat-based diets. Broilers were fed the following seven diets at 0.69% Ca: 1) a negative control diet, 0.17% nonphytate P (NPP); 2) Diet 1 + 750 phytase units/kg diet; 3) Diet 1 + 750 phytase units + 3,156 units acid phosphatase/kg diet; 4) Diet 1 + 750 phytase units + 3,156 acid phosphatase units + 1,900 units of pectinase/g diet + 3% citric acid; 5) Diet 1 + 4% FM; 6) Diet 1 + 4% FM + 1,300 phytase units + 2% ascorbic acid and 1% of glucose oxidase; and 7) a positive control diet (Diet 1 + 0.24% NPP from dicalcium phosphate). The dietary treatments were fed to four pen replicates of eight birds each. Prior to feed formulation, mycelium and antioxidants dosages were optimized on Diet 1 by an in vitro technique and an experimental design module of a statistical software package. Phytase addition increased BW gain (BWG), feed intake, and P retention. Subsequent addition of acid phosphatase resulted in further increases in BWG, feed intake, and toe ash and reduced digesta viscosity; however, neither P nor Ca retention were improved. Body weight gain and feed intakes superior to those found in chicks fed Diet 7 were observed in birds receiving the cocktail of enzymes (Diet 4) or FM. Chicken fed Diet 6 had the highest percentage of toe ash and retained 76 and 51% of P and Ca, respectively. Supplementation of wheat-based 0.17% NPP diets with FM increased bursa of Fabricius weights and reduced the intestinal surface covered by Peyer's patches.
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PMID:Comparison of the efficacies of a novel aspergillus niger mycelium with separate and combined effectiveness of phytase, acid phosphatase, and pectinase in dephosphorylation of wheat-based feeds fed to growing broilers. 1105 50


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