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)

Reaction of Aspergillus ficuum phytase with the arginine specific modifier 1,2-cyclohexanedione causes a rapid loss of activity. The inactivation can be partially reversed by 0.2 M hydroxylamine and exhibits pseudo-first order kinetics. The reaction order and second order rate constant of inactivation were 0.87 and 6.72 M-1 Min-1, respectively. Amino acid analysis of modified phytase indicates that about 7 arginine of the total 19 were modified. While the chymotryptic maps of treated and untreated phytase wer virtually identical, the tryptic maps had 4 peaks of altered mobility. An Arg containing tripeptide was identified in the phytase which is also present in other phosphohydrolases and may represent one of the labile Arg involved in the formation of the active site.
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PMID:Cyclohexanedione modification of arginine at the active site of Aspergillus ficuum phytase. 164 14

Five barrows of approximately 45 kg BW, fitted with post-valvular T-cecum cannulas at the ileo-cecal junction, were assigned randomly to five treatments (5 x 5 Latin square design) to assess the effect of microbial phytase and feeding regimen (frequency and level) on the apparent digestibilities (total tract [ATTD] and ileal [AID]) and retention of nutrients. A corn-tapioca-soybean meal diet of low intrinsic phytase activity, containing no added inorganic P, was fed either without or with microbial phytase from Aspergillus niger var. Van Tieghem (800 phytase units/kg of diet) at different feeding frequencies (once, twice, or seven times per day) and feeding levels (2.3 vs 2.8 times the maintenance requirement for ME, i.e., 418 kJ ME/BW.75). Microbial phytase enhanced significantly the ATTD of DM, OM, CP, Ca, total P, and amino acids (except for cystine and proline). Also, the AID of total P, phytic acid, methionine, and arginine was increased (P < .05 or .01). As a consequence of adding this enzyme, the retention (grams/day) of N, Ca, and P was greater (P < .01) and their daily excretion was diminished by 5.5, 2.2, and 1.9 g/d, respectively. The feeding level exerted a minor effect on the ATTD and AID (except for methionine and cystine), although the retention (grams/day) of N, Ca, and P was greater (P < .01) at the higher level of feeding. The feeding frequency influenced significantly the ATTD of Ca, tryptophan, and isoleucine and the AID of phytic acid, cystine, arginine, isoleucine, and phenylalanine. Also, N retention (grams/day) was reduced in pigs fed once daily (P < .01).
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PMID:Apparent digestibility and retention of nutrients bound to phytate complexes as influenced by microbial phytase and feeding regimen in pigs. 813 79

An amino acid deletion assay, a protein efficiency ratio (PER) assay, and a slope-ratio growth assay were used to establish the limiting order of AA, and to determine the effects of microbial phytase on protein utilization in corn gluten meal (CGM) fed to chicks during the period of 8 to 21 d posthatching. In Assay 1, a 12% CP CGM diet was fortified with AA to fulfill the digestible AA ideal profile (only Phe + Tyr, Leu, and Pro exceeded requirements) for young chicks. Amino acids were then individually deleted, and all diets were fortified to 23% CP, with Glu varying as necessary. A Met-fortified 23% CP corn-soybean meal diet served as a positive control. No weight gain or feed efficiency differences were observed between the fully fortified CGM basal diet and the corn-soybean meal positive-control diet. The limiting order of AA established in CGM was 1) Lys, 2) Trp, 3) Arg, 4) Thr, 5) Val, 6) Ile, 7) His, 8) cystine, and 9) Met. In Assay 2, diets with 10% CP furnished by CGM or casein were fed in the presence and absence of 1,200 U/kg phytase. A protein source x phytase interaction (P < 0.05) was observed for weight gain, gain:feed, and PER, indicating positive responses to phytase when casein was fed but negative responses to phytase when CGM was fed. In Assay 3, graded levels of protein (8, 16, and 24% CP) furnished by CGM were fed in the presence and absence of 1,200 U/kg phytase. Weight gain and gain:feed increased linearly (P < 0.05) as a function of protein intake, but phytase supplementation had no effect on weight gain or gain:feed slopes. These results indicate that 1,200 U/kg phytase did not increase either CP or AA utilization in CGM for young chicks.
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PMID:Limiting order of amino acids and the effects of phytase on protein quality in corn gluten meal fed to young chicks. 1094 2

The gene for Aspergillus fumigatus phytase (phyA) was cloned and expressed in Pichia pastoris. The enzyme expressed was purified to near homogeneity using sequential ion-exchange chromatography and was characterized biochemically. Although A. fumigatus phytase shows 66.2% sequence homology with A. ficuum phytase, the most widely studied enzyme, the cloned phytase showed identical molecular weight and temperature optima profile to the benchmark phytase. The pH profile of activity and kinetic parameters, however, differed from A. ficuum phytase. The cloned enzyme contains the septapeptide RHGARYP motif, which is also identical to the active site motif of A. ficuum phytase. Chemical probing of the active site Arg residues using both cyclohexanedione and phenylglyoxal resulted in the inactivation of phytase. The cloned A. fumigatus phytase, however, was more resistant to phenylglyoxal-induced inactivation. Both cloned A. fumigatus and A. ficuum phytases were identically affected by cyclohexanedione. Both the thermal characterization data and kinetic parameters of cloned and expressed A. fumigatus phytase indicate that this biocatalyst is not superior to the benchmark enzyme. The sequence difference between A. fumigatus and A. ficuum phytase may explain why the former enzyme catalyzes poorly compared to the benchmark enzyme. In addition, differential sensitivity toward the Arg modifier, phenylglyoxal, indicates a different chemical environment at the active site for each of the phytases.
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PMID:Biochemical characterization of cloned Aspergillus fumigatus phytase (phyA). 1096 58

Ileally cannulated pigs were used to assess the effects of four dietary levels of microbial phytase (Natuphos) on the apparent and true digestibility of Ca, P, CP, and AA in dehulled soybean meal. Fourteen pigs (25 kg initial BW) were surgically fitted with T-cannulas at the terminal ileum and assigned to diets in a replicated 7 x 7 Latin square design. Following a 14-d recovery, four diets consisting of 30.5% soybean meal with 0, 500, 1,000, or 1,500 units of phytase/kg of diet were fed. Diets 5 (1.05% lysine, 0.90% Ca, and 0.75% P) and 6 (1.05% lysine, 0.90% Ca, and 0.75% P) contained 35.25% soybean meal and 27.0% soy protein concentrate, respectively. Diet 7 (0.37% lysine, 0.03% Ca, and 0.05% P) was a low-CP, casein-based diet used to estimate the nonspecific endogenous losses of Ca, P, CP, and AA in order to estimate the true digestibility of these nutrients. All diets contained cornstarch and dextrose and were fortified with vitamins and minerals. Chromic oxide was used as an indigestible indicator. The diets were fed daily at 9% of metabolic BW (BW0.75). Apparent and true ileal digestibility of P increased quadratically (P < 0.01) and true digestibility of Ca increased linearly (P < 0.07) with increasing levels of phytase. Apparent digestibility of Ca was unaffected (P = 0.15) by phytase level. Apparent and true ileal digestibility of CP and most AA increased slightly with the addition of 500 units of phytase/kg of diet, but not at higher levels of phytase supplementation (in most cases, cubic effect, P < 0.05). Apparent and true ileal nutrient digestibility coefficients were unaffected by soybean meal source (Diet 1 vs Diet 5), except for arginine and Ca. The apparent and true digestibility coefficients for most of the AA tended (P < 0.10) to be lower in diets containing soy protein concentrate vs the common source of soybean meal used in Diet 5, but ileal digestibilities of Ca and P were unaffected (P = 0.15). In this study, supplemental microbial phytase did not improve the utilization of AA provided by soybean meal but was an effective means of improving Ca and P utilization by growing swine fed soybean meal-based diets.
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PMID:Effects of level of supplemental phytase on ileal digestibility of amino acids, calcium, and phosphorus in dehulled soybean meal for growing pigs. 1172 43

Fungal phyA gene from Aspergillus ficuum (niger) was cloned and expressed in potato leaves. The recombinant enzyme was stable and catalytically active. The expressed protein in the leaves of the dicotyledonous plant retained most physical and catalytic properties of the benchmark A. ficuum phytase. The expressed enzyme was, however, 15% less glycosylated than the native phytase. The usual bi-hump pH optima profile, which is characteristic of the fungal phytase, was altered; however, the pH optimum at 5.0 was unchanged for phytate and at 4.0 for synthetic substrate p-nitrophenyl phosphate. The temperature was, however, unchanged. The expressed phytase was found to be as sensitive as the native enzyme to the inhibitory action of pseudo substrate, myo-inositol hexasulfate, while losing about 90% of the activity at 20 microM inhibitor concentration. Similar to the benchmark phytase, the expressed phytase in leaves was completely inactivated by Arg modifier phenylglyoxal at 60 nM. In addition, the expressed phytase in the leaves was inhibited by antibody raised against a 20-mer internal peptide, which is present on the surface of the molecule as shown by the X-ray deduced 3D structure of fungal phytase. Taken together, the biochemical evidences indicate that fungal phytase when cloned and expressed in potato leaves produces a stable and active biocatalyst. 'Biofarming,' therefore, is an alternative way to produce functional hydrolytic enzymes as exemplified by the expression of A. ficuum (niger) phyA gene in potato leaf.
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PMID:Fungal phyA gene expressed in potato leaves produces active and stable phytase. 1280 8

In order to understand the structural basis for the high thermostability of phytase from Aspergillus fumigatus, its crystal structure was determined at 1.5 A resolution. The overall fold resembles the structure of other phytase enzymes. Aspergillus niger phytase shares 66% sequence identity, however, it is much less heat-resistant. A superimposition of these two structures reveals some significant differences. In particular, substitutions with polar residues appear to remove repulsive ion pair interactions and instead form hydrogen bond interactions, which stabilize the enzyme; the formation of a C-terminal helical capping, induced by arginine residue substitutions also appears to be critical for the enzyme's ability to refold to its active form after denaturation at high temperature. The heat-resilient property of A.fumigatus phytase could be due to the improved stability of regions that are critical for the refolding of the protein; and a heat-resistant A.niger phytase may be achieved by mutating certain critical residues with the equivalent residues in A.fumigatus phytase. Six predicted N-glycosylation sites were observed to be glycosylated from the experimental electron density. Furthermore, the enzyme's catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family. The trap of this catalytic intermediate confirms the two-step catalytic mechanism of the acid histidine phosphatase family.
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PMID:Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine. 1513 45

It has been shown recently that the twin-arginine signal peptide of Bacillus subtilis phosphodiesterase PhoD (SPPhoD) can mediate Tat dependent transport of proteins via its specific Tat-transport components. In order to test the use of Tat dependent transport signals for heterologous product synthesis, Escherichia coli phytase AppA was expressed under control of PhoD-specific export signals in B. subtilis. Induction of Tat components TatAd/TatCd was mediated by using a functionally altered PhoR/PhoP signal transduction system which regulates the expression of these components. AppA was highly susceptible to host specific extracellular proteases. Expression of appA in B. subtilis wprA strain resulted in the stable production of AppA. A fusion protein consisting of SPPhoD and mature AppA remained unprocessed, while introduction of the AppA signal peptidase cleavage site resulted in efficient processing of the fusion protein.
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PMID:Tat dependent export of E. coli phytase AppA by using the PhoD-specific transport system of Bacillus subtilis. 1537 26

Ten crossbred barrows (48.3 +/- 2.3 kg of initial BW) fitted with steered ileo-cecal valve cannulas were used to investigate the effects of supplemental microbial phytase on the apparent ileal digestibilities (AID) of AA, Ca, P, N, and DM, and the apparent total tract digestibilities of Ca, P, N, and DM. All diets were corn-soybean meal-based, and contained 0.44% Ca and 0.40% total P. Diets 1, 2, and 3 contained 12.0, 11.1, and 10.2% CP, respectively. Diets 4 and 5 had the same ingredient composition as diet 3, plus 250 and 500 U/kg phytase (Natuphos), respectively. Pigs were randomly allotted to 1 of 5 dietary treatments in a paired 5 x 5 Latin square with an extra period to test for carryover effects. Each 14-d period consisted of a 7-d adjustment followed by a 3-d total collection, a 12-h ileal digesta collection, a 3-d readjustment, and a second 12-h ileal digesta collection. Pigs were housed individually in metabolism pens (1.2 x 1.2 m). Water was supplied ad libitum, and feed was supplied at a level of 9% of the metabolic BW (BW(0.75)) per day in 2 equal daily feedings. As the dietary CP concentration increased, the AID of CP and all AA measured increased linearly (P < 0.05) with the exception of proline. In addition, the apparent total tract digestibilities (grams per day) and retention of N (grams per day) increased linearly (P < 0.01) with increasing CP levels. Supplementing diets with phytase increased the AID of Ca (P < 0.01), P (P < 0.001), CP (P = 0.07), and the AA (P < 0.10) Gly, Ala, Val, Ile, Thr, TSAA, Asp, Glu, Phe, Lys, and Arg. Protein and phytase response equations were generated for those AA affected (P < 0.10) by both CP level and phytase supplementation. Based on these equations, 500 U/kg of phytase can replace 0.52 percentage units of the dietary CP, which includes a 0.03 percentage unit improvement in Lys AID. The results of this study show that supplementing pig diets with microbial phytase improves CP and AA digestibilities in addition to Ca and P digestibilities.
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PMID:Estimating equivalency values of microbial phytase for amino acids in growing and finishing pigs fitted with steered ileo-cecal valve cannulas. 1661 14

Two studies were conducted to determine the efficacy of an Escherichia coli-derived phytase (ECP) and its equivalency relative to inorganic phosphorus (iP) from monosodium phosphate (MSP). In Exp. 1, one thousand two hundred 1-d-old male broilers were used in a 42-d trial to assess the effect of ECP and iP supplementation on growth performance and nutrient digestibility. Dietary treatments were based on corn-soybean meal basal diets (BD) containing 239 and 221 g of CP, 8.2 and 6.6 g of Ca, and 2.4 and 1.5 g of nonphytate P (nPP) per kg for the starter and grower phases, respectively. Treatments consisted of the BD; the BD + 0.6, 1.2, or 1.8 g of iP from MSP per kg; and the BD + 250, 500, 750, or 1,000 phytase units (FTU) of ECP per kg. Increasing levels of MSP improved gain, gain:feed, and tibia ash (linear, P < 0.01). Increasing levels of ECP improved gain, gain:feed, tibia ash (linear, P < 0.01), apparent ileal digestibility of P, N, Arg, His, Phe, and Trp at d 21 (linear, P < 0.05), and apparent retention of P at d 21 (linear, P < 0.05). Increasing levels of ECP decreased apparent retention of energy (linear, P < 0.01). Five hundred FTU of ECP per kg was determined to be equivalent to the addition of 0.72, 0.78, and 1.19 g of iP from MSP per kg in broiler diets based on gain, feed intake, and bone ash, respectively. In Exp. 2, forty-eight 10-kg pigs were used in a 28-d trial to assess the effect of ECP and iP supplementation on growth performance and nutrient digestibility. Dietary treatments consisted of a positive control containing 6.1 and 3.5 g of Ca and nPP, respectively, per kg; a negative control (NC) containing 4.8 and 1.7 g of Ca and nPP, respectively, per kg; the NC diet plus 0.4, 0.8, or 1.2 g of iP from MSP per kg; and the NC diet plus 500, 750, or 1,000 FTU of ECP per kg. Daily gain improved (linear, P < 0.05) with ECP addition, as did apparent digestibility of Ca and P (linear, P < 0.01). Five hundred FTU of ECP per kg was determined to be equivalent to the addition of 0.49 and 1.00 g of iP from MSP per kg in starter pigs diets, based on ADG and bone ash, respectively.
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PMID:Efficacy and equivalency of an Escherichia coli-derived phytase for replacing inorganic phosphorus in the diets of broiler chickens and young pigs. 1709 29


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