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)

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26) catalyses the stepwise hydrolysis of phytic acid (myo-inositol hexakisphosphate). Phytases are of great commercial importance due to their usage as supplement of food and animal feed, which can cater to nutrition demands and alleviate environmental problems, has been approved by many countries. Although acid phytases have been extensively studied, information regarding the phytases from Citrobacter is limited. In the work presented, a phytase was separated from Citrobacter freundii. After steps of electrophoretic homogeneity by successive ammonium sulfate between 60% and 80% saturation precipitation, DEAE-Sepharose ion-exchange chromatography and gel filtration through Superdex HR 10/30, final gel elution resulted in a 41.3-fold purification and yield of 9.3%. Gel elution is an effective method to purify the protein which contaminated with a few other proteins. The purified preparations were used in subsequent characterization studies. Based on SDS-PAGE analysis, the molecular weight of the purified phytase was calculated to be approximately 45.0kDa in monomeric form. The pure enzyme has an optimum pH of 4.0 to approximately 4.5. It was found stable between pH5.0 to approximately 7.0, about 90% of the enzyme activity was retained at 37 degrees C for 60min. The phytase has an optimum temperature of 40 degrees C which was lower than that of other phytases from Aspergillus or E. coli (average 50 to approximately 60 degrees C) and was close to the temperature of gastrointestinal tract in animals (37 to approximately 40 degrees C). Thus the enzyme is a promising candidate for animal feed applications. Activity of the purified phytase was influenced by changing the reaction temperature. Data showed that the enzyme retained its activity over a long period when stored at 4 degrees C, whereas thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 60 degrees C for 4min. The Km values of the phytase for dodecasodium phytate at 37 degrees C was 0.85nmol/L with a Vmax 0.53IU/(mg x min). Phytase activity was strongly inhibited by SDS, Zn2+ and moderately inhibited by Cu2+, Cr3+, Fe2+ and Fe3+. Activity was not significantly affected by EDTA, K+, Mg2+ and Ca2+. The phytase has excellent resistance to trypsin, but not pepsin. The N-terminal amino acids sequence of the phytase protein was determined as QCAPEGYQLQQVLMM which exhibited about 80% homology to Glucose-1-phosphatases from E. coli, Shigella flexneri and Salmonella, whereas it did not show apparent sequence similarity with any other phytase listed in the databases. Initial characterization of the purified enzyme suggested that it is a potential candidate for use as an animal feed supplement.
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PMID:[Purification and properties of Citrobacter freundii phytase]. 1657 82

A Yersinia intermedia strain producing phytase was isolated from glacier soil. The phytase gene, appA, was isolated by degenerate PCR and TAIL-PCR. The full-length fragment contained 2354bp with a 1326-bp open reading frame encoding 441 amino acids. APPA contained the active site RHGXRXP and HD sequence motifs that are typical of histidine acid phosphatases. To our knowledge, this is the first report of the detection of phytase activity and cloning of the relevant gene from Y. intermedia. The gene was overexpressed in Pichia pastoris, and the purified recombinant APPA had a specific activity for sodium phytate of 3960U/mg, which is higher than that of the Citrobacter braakii phytase (previously the highest specific activity known). Recombinant APPA had high activity from pH 2 to 6 (optimum 4.5) and optimal temperature of 55 degrees C; the enzyme was resistant to pepsin and trypsin. These characteristics suggest that APPA may be highly suitable for use in the feed industry.
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PMID:A novel phytase with preferable characteristics from Yersinia intermedia. 1703 58

A periplasmatic phytase from a bacterium isolated from Malaysian waste water was purified about 173-fold to apparent homogeneity with a recovery of 10% referred to the phytase activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 65 degrees C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be KM=0.15 mmol/l and kcat=1164 s(-1) at pH 4.5 and 37 degrees C. The purified enzyme was shown to be highly specific. Among the phosphorylated compounds tested, phytate was the only one which was significantly hydrolysed. Some properties such as considerable activity below pH 3.0, thermal stability and resistance to pepsin make the enzyme attractive for an application as a feed supplement.
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PMID:Purification and characterization of a bacterial phytase whose properties make it exceptionally useful as a feed supplement. 1751 Jul 80

A novel thermostable phytase gene was cloned from Aspergillus fumigatus WY-2. It was 1459 bp in size and encoded a polypeptide of 465 amino acids. The gene was expressed in Pichia pastoris GS115 as an extracellular enzyme. The expressed enzyme was purified to homogeneity and biochemically characterized. The purified enzyme had a specific activity of 51 U/mg with an approximate molecular mass of 88 kDa. The optimum pH and temperature for activity were pH 5.5 and 55 degrees C, respectively. After incubation at 90 degrees C for 15 min, it still remained at 43.7% of the initial activity. The enzyme showed higher affinity for sodium phytate than other phosphate conjugates, and the K(m) and K(cat) for sodium phytate were 114 microM: and 102 s(-1), respectively. Incubated with pepsin at 37 degrees C for 2 h at the ratio (pepsin/phytase, wt/wt) of 0.1, it still retained 90.1% residual activity. These exceptional properties give the newly cloned enzyme good potential in animal feed applications.
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PMID:Cloning, expression, and enzyme characterization of an acid heat-stable phytase from Aspergillus fumigatus WY-2. 1753 60

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436-amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg.mL(-1), and the enzyme activity level reached 15,000 U x mL(-1), which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U.mg(-1). The optimum pH and temperature for enzyme activity were 4.5 and 55 degrees C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.
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PMID:A novel phytase appA from Citrobacter amalonaticus CGMCC 1696: gene cloning and overexpression in Pichia pastoris. 1765 39

A gene appA encoding a novel phytase was firstly cloned from Hafnia alvei by PCR and sequenced. The gene was consisted of 1335 bp, encoding 444 amino acids. The calculated molecular weight of the mature APPA was about 45.2 kD. The gene appA was expressed in E. coli BL21 (DE3). Recombinant APPA was purified and its enzymatic properties were determined. The optimum pH for the enzyme was 4.5 and the optimum temperature was 60 degrees C. The pH stability of r-APPA is good, the relative phytase activity was above 80% after treated in buffers of pH 2.0-10.0. The specific activity of r-APPA is 356.7 U/mg, and the Km value was 0.49 mmol/L and Vmax of 238 U/mg. The enzyme showed resistance to pepsin and trypsin treatment.
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PMID:[Gene cloning, expression and characterization of a novel phytase from Hafnia alvei]. 1825 29

Two novel phytase genes belonging to the histidine acid phosphatase family were cloned from Yersinia rohdei and Y. pestis and expressed in Pichia pastoris. Both the recombinant phytases had high activity at pH 1.5-6.0 (optimum pH 4.5) with an optimum temperature of 55 degrees C. Compared with the major commercial phytases from Aspergillus niger, Escherichia coli, and a potential commercial phytase from Y. intermedia, the Y. rohdei phytase was more resistant to pepsin, retained more activity under gastric conditions, and released more inorganic phosphorus (two to ten times) from soybean meal under simulated gastric conditions. These superior properties suggest that the Y. rohdei phytase is an attractive additive to animal feed. Our study indicated that, in order to better hydrolyze the phytate and release more inorganic phosphorus in the gastric passage, phytase should have high activity and stability, simultaneously, at low pH and high protease concentration.
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PMID:A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions. 1854 46

Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris. The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL(-1), respectively. Both recombinant phytases exhibited high affinity for phytate but not for p-nitrophenyl phosphate. Optimal phytase activity was observed at 50 degrees C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 degrees C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.
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PMID:Expression and characterization of Aspergillus thermostable phytases in Pichia pastoris. 1902 60

A mature phytase cDNA, encoding 441 amino acids, from Eupenicillium parvum (BCC17694) was cloned into a Pichia pastoris expression vector, pPICZ alpha A, and was successfully expressed as active extracellular glycosylated protein. The recombinant phytase contained the active site RHGXRXP and HD sequence motifs, a large alpha/beta domain and a small alpha-domain that are typical of histidine acid phosphatase. Glycosylation was found to be important for enzyme activity which is most active at 50 degrees C and pH 5.5. The recombinant phytase displayed broad substrate specificity toward p-nitrophenyl phosphate, sodium-, calcium-, and potassium-phytate. The enzyme lost its activity after incubating at 50 degrees C for 5 min and is 50% inhibited by 5mM Cu(2+). However, the enzyme exhibits broad pH stability from 2.5 to 8.0 and is resistant to pepsin. In vitro digestibility test suggested that BCC17694 phytase is at least as effective as another recombinant phytase (r-A170) which is comparable to Natuphos, a commercial phytase, in releasing phosphate from corn-based animal feed, suggesting that BCC17694 phytase is suitable for use as phytase supplement in the animal diet.
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PMID:Biochemical characterization and in vitro digestibility assay of Eupenicillium parvum (BCC17694) phytase expressed in Pichia pastoris. 1981 56

Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris. This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50-55 degrees C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed.
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PMID:Cell-surface phytase on Pichia pastoris cell wall offers great potential as a feed supplement. 1992 69


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