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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)

The wild-type phytases from the Aspergillus niger strains NRRL 3135 and T213 display a three-fold difference in specific activity (103 versus 32 U/mg protein), despite only 12 amino acid differences that are distributed all over the sequence of the protein. Of the 12 divergent positions, three are located in or close to the substrate binding site. Site-directed mutagenesis of these residues in A. niger T213 phytase showed that the R297Q mutation (R in T213, Q in NRRL 3135) fully accounts for the differences in catalytic properties observed. Molecular modelling revealed that R297 may directly interact with a phosphate group of phytic acid. The fact that this presumed ionic interaction - causing stronger binding of substrates and products - correlates with a lower specific activity indicates that product (myo-inositol pentakisphosphate) release is the rate-limiting step of the reaction.
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PMID:Active site residue 297 of Aspergillus niger phytase critically affects the catalytic properties. 1078 5

Phytase enzymes can increase the nutritional value of food and feed by liberating inorganic phosphate from phytate, the major storage form of phosphorus in plants. The phytase (phyC) from Bacillus subtilis VTT E-68013 was expressed in Lactobacillus plantarum strain 755 using Lact. amylovorus alpha-amylase secretion signals. In an overnight cultivation in MRS medium containing cellobiose for induction of the alpha-amylase promoter, catalytically active phytase was secreted as a predominant extracellular protein. However, Western blot analysis revealed unprocessed and processed phytase in the cell fraction. Pulse chase experiments showed that the recombinant phytase was secreted at a slower rate in comparison to the native proteins of Lact. plantarum 755.
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PMID:Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755. 1079 56

The enzyme phytase dephosphorylates phytin (inositol hexaphosphate), a major phosphate reserve in plants. We found that a large number of yeast species secreted a phytase. Several species were identified as high phytase producers. The yeast enzymes had an optimal activity at pH 4-5 and generally a very high optimal temperature, ranging from 60 degrees C to 80 degrees C.
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PMID:Secreted phytase activities of yeasts. 1083 May 2

Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate (InsP6). The pathway of hydrolysis of InsP6 by phytase from wheat bran of Triticum aestivum L. cv. Nourin #61 is proved in this study. Structures of the intermediates were established by a variety of nuclear magnetic resonance techniques (1H-, two-dimensional 1H-1H coupling-correlation spectra and two-dimensional 31P-1H correlation spectra), gas chromatography, and bioassay. On the basis of the structures identified, initial hydrolysis of the phosphate ester occurs at the D/L-4 position of InsP6 to yield D/L-Ins (1, 2, 3, 5, 6) P5. After the dephosphorylation, the pathway of dephosphorylation is divided into two routes. The main route proceeds via D/L-Ins (1, 2, 5, 6) P4, D/L-Ins (1, 2, 6) P3 and D/L-Ins (1, 2) P2, while the minor route proceeds via D/L-Ins (1, 2, 3, 6) P4, Ins (1, 2, 3) P3 and D/L-Ins (1, 2) P2. D/L-Ins (1, 2) P2 is hydrolyzed at the D/L-1 or 2-position, and finally myo-inositol is produced.
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PMID:The pathway of dephosphorylation of myo-inositol hexakisphosphate by phytases from wheat bran of Triticum aestivum L. cv. Nourin #61. 1087 69

Approaches to the rational design of vanadium-based semi-synthetic enzymes and biomimetic models as catalysts for enantioselective oxidations are reviewed. Incorporation of vanadate ion into the active site of phytase (E.C. 3.1.3.8), which in vivo mediates the hydrolysis of phosphate esters, afforded a semi-synthetic peroxidase. It catalyzed the enantioselective oxidation of prochiral sulfides with H2O2 affording the S-sulfoxide, e.g. in 66% ee at quantitative conversion of thioanisole. Under the reaction conditions the semi-synthetic vanadium peroxidase was stable for more than 3 days with only a slight decrease in turnover frequency. Amongst the transition-metal oxoanions that are known to be potent inhibitors of phosphatases, only vanadate resulted in a semi-synthetic peroxidase when incorporated into phytase. In a biomimetic approach, vanadium complexes of chiral Schiff base complexes were encapsulated in the super cages of a hydrophobic zeolite Y. Unfortunately, these ship-in-a-bottle complexes afforded only racemic sulfoxide in the catalytic oxidation of thioanisole with H2O2.
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PMID:Biocatalytic and biomimetic oxidations with vanadium. 1088 67

Five strains of lactic bacteria have been isolated from sour doughs and examined for their ability to degrade phytic acid. In white flour medium in which phytic acid was the only source of phosphorus, the disappearance of phytate and an elevation of inorganic phosphate were observed after only 2 h of incubation in all strains tested (-30 and +60%, respectively). Both phenomena correspond to phytate breakdown. No difference was observed in the levels of phytic acid hydrolysis among strains, suggesting that phytase enzymes are similar among these bacteria. Using whole wheat flour medium naturally rich in phytic acid in the presence of Leuconostoc mesenteroides strain 38, a 9 h fermentation established that the degradation of PA and the production of lactic acid lead to greater Ca and Mg solubility than in control medium.
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PMID:Strains of lactic acid bacteria isolated from sour doughs degrade phytic acid and improve calcium and magnesium solubility from whole wheat flour. 1088 37

Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U x [mg protein](-1) at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site-directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U x (mg protein)(-1), and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme-substrate complexes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate-limiting step of the A. fumigatus wild-type phytase reaction.
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PMID:Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure. 1093 95

A total of four enzymatic steps were combined, in a one-pot reaction, to synthesize carbohydrates starting from glycerol. First, phosphorylation of glycerol by reaction with pyrophosphate in the presence of phytase at pH 4.0 in 95% glycerol afforded racemic glycerol-3-phosphate in 100% yield. The L-enantiomer of the latter underwent selective aerobic oxidation to dihydroxyacetone phosphate (DHAP) at pH 7.5 in the presence of glycerolphosphate oxidase (GPO) and catalase. Subsequently, fructose-1,6-bisphosphate aldolase catalyzed the aldol reaction of DHAP with butanal. Finally, dephosphorylation of the aldol adduct was mediated by phytase at pH 4 affording 5-deoxy-5-ethyl-D-xylulose in 57% yield from L-glycerol-3-phosphate. The phytase on/off-switch by pH was the key to controlling phosphorylation and dephosphorylation.
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PMID:A four-step enzymatic cascade for the one-pot synthesis of non-natural carbohydrates from glycerol. 1103 Oct 13

Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P(5), D/L-Ins(2,3,4,5)P(4), D/L-Ins(2,4,5)P(3) or D/L-Ins(1,2,4)P(3), D/L-Ins(1,2)P(2) or Ins(2, 5)P(2) or D/L-Ins(4,5)P(2) to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D-Ins(1, 2,3,5,6)P(5) isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F(1) and F(2) from wheat bran and the myo-inositol phosphates produced by fraction F(2). Biochim. Biophys. Acta 302, 326-328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P(5), D-Ins(2,3,4,5)P(4), D-Ins(2,4,5)P(3), Ins(2,5)P(2) to finally Ins(2)P (notation 6/1/3/4/5).
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PMID:Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli. 1103 87

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


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