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1.
Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties 总被引:15,自引:0,他引:15
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Markus Wyss Roland Brugger Alexandra Kronenberger Roland Rmy Rachel Fimbel Gottfried Oesterhelt Martin Lehmann Adolphus P. G. M. van Loon 《Applied microbiology》1999,65(2):367-373
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°C 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, α- and β-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. 相似文献
2.
The secretion of the bacterial phytase PHY‐US417 by Arabidopsis roots reveals its potential for increasing phosphate acquisition and biomass production during co‐growth
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Nibras Belgaroui Pierre Berthomieu Hatem Rouached Moez Hanin 《Plant biotechnology journal》2016,14(9):1914-1924
Phytic acid (PA) is a major source of inorganic phosphate (Pi) in the soil; however, the plant lacks the capacity to utilize it for Pi nutrition and growth. Microbial phytases constitute a group of enzymes that are able to remobilize Pi from PA. Thus, the use of these phytases to increase the capacity of higher plants to remobilize Pi from PA is of agronomical interest. In the current study, we generate transgenic Arabidopsis lines (ePHY) overexpressing an extracellular form of the phytase PHY‐US417 of Bacillus subtilis, which are characterized by high levels of secreted phytase activity. In the presence of PA as sole source of Pi, while the wild‐type plants show hallmark of Pi deficiency phenotypes, including the induction of the expression of Pi starvation‐induced genes (PSI, e.g. PHT1;4) and the inhibition of growth capacity, the ePHY overexpressing lines show a higher biomass production and no PSI induction. Interestingly, when co‐cultured with ePHY overexpressors, wild‐type Arabidopsis plants (or tobacco) show repression of the PSI genes, improvement of Pi content and increases in biomass production. In line with these results, mutants in the high‐affinity Pi transporters, namely pht1;1 and pht1;1‐1;4, both fail to accumulate Pi and to grow when co‐cultured with ePHY overexpressors. Taken together, these data demonstrate the potential of secreted phytases in improving the Pi content and enhancing growth of not only the transgenic lines but also the neighbouring plants. 相似文献
3.
This study had the following objectives: (i) to evaluate the thermoregulatory and behavioral responses of light laying hens supplemented with different types and dosages of phytases in the two day shifts; and (ii) to integrate the thermoregulatory and behavioral responses with performance of these birds raised in a hot environment. 270 light laying hens of the Hy-Line White lineage, with a body weight of 1.60 ± 0.092 kg were distributed in a completely randomized design in a 2 × 2 + 1 factorial model with two types of phytases (bacterial and fungal) and two dosages (450 and 900 FTU), and a control diet. The day shift (morning and afternoon) was considered as a fixed effect in the factorial arrangement. Principal component analysis (PCA), correspondence analysis (CA) and canonical discriminant analysis (CDA) were used. There was no interaction (P > 0.05) between phytases and dosages for thermoregulatory responses. Respiratory rate (RR), cloacal temperature (CT), and surface temperature with feathers (STWF) and featherless (STF) were higher (P < 0.001) in the afternoon. Birds show different thermoregulatory and behavioral responses in the two shifts of the day. We also observed that birds supplemented with bacterial and fungal phytase showed similar thermoregulatory and behavioral responses to the control group in both day shifts. Expression of the “eating” activity was greater in the morning, while the birds remained sitting longer in the afternoon. Egg production was higher (P < 0.001) in birds supplemented with bacterial phytase. The phytase dosages had no effect on thermoregulatory, behavioral or performance responses. Egg production, feed conversion per dozen eggs corresponded to 81.1% of the differences between bacterial and fungal phytase supplementation and group control. Thus, we conclude that: (i) phytase dietary supplementation has no effect on the thermoregulatory responses of laying hens reared in a hot environment; (ii) birds supplemented with bacterial phytase showed higher egg production; and (iii) phytases (450 and 900 FTU) do not interfere with productive, behavioral and thermoregulatory responses. 相似文献
4.
A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture.
In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection
of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg−1. The optimum pH was 7.0, and the optimum temperature was 45°C. The K
m, V
max, and k
cat values were 1.28 mM, 71.9 μmol min−1 mg−1, and 45.1 s−1, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that
r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry. 相似文献
5.
A. Atkinson A.D. Gatenby A.G. Lowe 《Biochimica et Biophysica Acta (BBA)/General Subjects》1973,320(1):195-204
A simple and rapid method is described for determining Pi by spectrophotometric measurement of a soluble complex of phosphomolybdic acid and Cirrasol ALN-WF, a non-ionic detergent formerly known as Lubrol W. The measured complex has a molar extinction coefficient of 4.59 · 103 at 390 nm and little interference is found with relatively high concentrations of chelating agents, salts, and other compounds which interfere with most other Pi assays. Linearity is observed in the range 0–1.2 μmoles Pi and developed assay samples are stable for 8 h at 20 °C or 24 h at 4 °C. The method is suitable for use in the presence of moderate concentrations of protein or ATP.After suitable modification the assay can be used at pH 4.0. Sensitivity is reduced at this pH (εM, 390nm = 2.79 · 103) but linearity is maintained up to 1 μmole Pi and the coloured complex is stable for 4 h at 20 °C. The pH-4 procedure is suitable for measurement of Pi in the presence of very labile phosphate esters such as creatine phosphate.The phosphomolybdic acid-Cirrasol complex can be reduced at ambient temperature in both the above systems. A blue complex results with εM, 820nm of 9.9 · 103 at pH 4.0, and 1.8 · 104 under more acidic conditions. 相似文献
6.
Bovine heart mitochondria which have been allowed to swell in isotonic NH
4
+
phosphate contract in response to initiation of oxidative phosphorylation. The contraction occurs optimally at pH 6.0 and appears from inhibition studies to result from Pi uptake being slower than removal of internal Pi via phosphorylation of external ADP. Similar results are obtained when K+ + nigericin is substituted for NH
4
+
. Mersalyl inhibition of Pi transport in respiring, nonphosphorylating mitochondria which have been allowed to swell in NH
4
+
phosphate reveals a contractile process having an alkaline pH optimum. This contraction resembles closely the contraction observed in salts of strong acids and presumably occurs by electrophoretic ejection of Pi anions driven by electrogenic H+ ejection. 相似文献
7.
Ragon M Aumelas A Chemardin P Galvez S Moulin G Boze H 《Applied microbiology and biotechnology》2008,78(1):47-53
Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa
with 28.8% glycosylation. Its activity was optimal at 60°C and pH 4.0. The K
m value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase
fully hydrolyzed myo-inositol hexakisphosphate (or phytic acid, Ins P6) to inositol and inorganic phosphate. The sequence of Ins P6 hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance.
D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins (1,2,4,5,6) P5, Ins (1,2,5,6) P4, Ins (1,2,6) P3, Ins (1,2) P2, Ins (1 or 2) P1, and inositol (notation 3/4/5/6/1 or 2). 相似文献
8.
Phytase activity in rabbit cecal bacteria 总被引:1,自引:0,他引:1
The presence of phytase activity was demonstrated in 26 strains of rabbit cecal bacteria. In 25 strains a low phytase activity,
0.10–0.62 μmol phosphate released per min per mg protein, was found. High activity (2.61 μmol/min per mg protein) was found
in the strain PP2 identified as Enterococcus hirae. Phytase activity was cell-associated, being higher in the cell extract than in the cell walls. Extracellular phytase activity
and cell-associated phosphatase activity were not detected. Phytase activity was optimal around pH 5.0, which is below the
physiological cecal pH range. The K
m determined using the Lineweaver-Burk plot was 0.19 μmol/mL. Cations Fe3+, Cu2+ and Zn2+ at 0.5 mmol/L decreased phytase activity in sonicated cells of E. hirae by 99.4, 90.7 and 96.5 %, respectively. In contrast, Mg2+ increased activity by 11.0 %. Characteristics of E. hirae phytase (pH optimum, K
m, cation sensitivity) were similar to those of other bacterial phytases reported in the literature. Other bacteria with a
high phytase activity may be present in the rabbit cecum but remain to be identified. 相似文献
9.
《Archives of animal nutrition》2013,67(4):353-373
The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70°C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40°C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives. 相似文献
10.
Plant phytases (myoinositol hexaphosphate phosphohydrolase, EC 3.1.3.8) were inhibited by phloroglucinol (1,3,5-benzenetriol) in vitro. The inhibition of the Cucurbita maxima phytase was found to be non-competitive and pH dependent with an apparent inhibition constant (Ki) value of 2.3 × 10?1 M at optimum pH (4.8) and temperature (50°). The apparent number of inhibitor molecules (n) bound per enzyme molecule was found to be 2.2 suggesting that the positive cooperativity phenomenon may be present. 相似文献
11.
Biochemical properties and substrate specificities of alkaline and histidine acid phytases 总被引:8,自引:0,他引:8
Phytases are a special class of phosphatase that catalyze the sequential hydrolysis of phytate to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are added to animal feedstuff to reduce phosphate pollution in the environment, since monogastric animals such as pigs, poultry, and fish are unable to metabolize phytate. Based on biochemical properties and amino acid sequence alignment, phytases can be categorized into two major classes, the histidine acid phytases and the alkaline phytases. The histidine acid phosphatase class shows broad substrate specificity and hydrolyzes metal-free phytate at the acidic pH range and produces myo-inositol monophosphate as the final product. In contrast, the alkaline phytase class exhibits strict substrate specificity for the calcium–phytate complex and produces myo-inositol trisphosphate as the final product. This review describes recent findings that present novel viewpoints concerning the molecular basis of phytase classification. 相似文献
12.
Biophysical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Molecular Size, Glycosylation Pattern, and Engineering of Proteolytic Resistance 总被引:9,自引:0,他引:9
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Markus Wyss Luis Pasamontes Arno Friedlein Roland Rmy Michel Tessier Alexandra Kronenberger Anke Middendorf Martin Lehmann Line Schnoebelen Urs Rthlisberger Eric Kusznir Guido Wahl Francis Müller Hans-Werner Lahm Kurt Vogel Adolphus P. G. M. van Loon 《Applied microbiology》1999,65(2):359-366
Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract. 相似文献
13.
Phytases catalyze the hydrolysis of phytic acid (InsP6, myo-inositol hexakisphosphate), the most abundant inositol phosphate in cells. In cereal grains and legumes, it constitutes 3-5% of the dry weight of seeds. The inability of humans and monogastric animals such as swine and poultry to absorb complexed InsP6 has led to nutritional and environmental problems. The efficacy of supplemental phytases to address these issues is well established; thus, there is a need for phytases with a range of biochemical and biophysical properties for numerous applications. An alkaline phytase that shows unique catalytic properties was isolated from plant tissues. In this paper, we report on the biochemical properties of an alkaline phytase from pollen grains of Lilium longiflorum. The enzyme exhibits narrow substrate specificity, it hydrolyzed InsP6 and para-nitrophenyl phosphate (pNPP). Alkaline phytase followed Michaelis-Menten kinetics with a K(m) of 81 microM and V(max) of 217 nmol Pi/min/mg with InsP6 and a K(m) of 372 microM and V(max) of 1272 nmol Pi/min/mg with pNPP. The pH optimum was 8.0 with InsP6 as the substrate and 7.0 with pNPP. Alkaline phytase was activated by calcium and inactivated by ethylenediaminetetraacetic acid; however, the enzyme retained a low level of activity even in Ca2+-free medium. Fluoride as well as myo-inositol hexasulfate did not have any inhibitory affect, whereas vanadate inhibited the enzyme. The enzyme was activated by sodium chloride and potassium chloride and inactivated by magnesium chloride; the activation by salts followed the Hofmeister series. The temperature optimum for hydrolysis is 55 degrees C; the enzyme was stable at 55 degrees C for about 30 min. The enzyme has unique properties that suggest the potential to be useful as a feed supplement. 相似文献
14.
Kavita Bhavsar Priyanka Buddhiwant Sarvesh K. Soni Dilip Depan Sampa Sarkar Jayant M. Khire 《Process Biochemistry》2013,48(11):1618-1625
Aspergillus niger NCIM 563 produces dissimilar phytase isozymes under solid state and submerged fermentation conditions. Biochemical characterization and applications of phytase Phy III and Phy IV in SSF and their comparison with submerged fermentation Phy I and Phy III were studied. SSF phytases have a higher metabolic potential as compared to SmF. Phy I is tetramer and Phy II, III and IV are monomers. Phy I and IV have pH optima of 2.5 and Phy II and III have pH optima of 5.0 and 5.6, respectively. Phy I, III and IV exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. SSF phytase is less thermostable as compared to SmF phytase. Phy I and II show homology with other known phytases while Phy III and IV show no homology with SmF phytases and any other known phytases from the literature suggesting their unique nature. This is the first report about differences among phytase produced under SSF and SmF by A. niger and this study provides basis for explanation of the stability and catalytic differences observed for these enzymes. Exclusive biochemical characteristics and multilevel application of SSF native phytases determine their efficacy and is exceptional. 相似文献
15.
G E Edwards S P Robinson N J Tyler D A Walker 《Archives of biochemistry and biophysics》1978,190(2):421-433
In studying conditions for obtaining photosynthetically functional chloroplasts from mesophyll protoplasts of sunflower and wheat, a strong requirement for chelation was found. The concentration of chelator, either EDTA or pyrophosphate (PPi), required for maximum activation depended on the pH, the concentration of orthophosphate (Pi) in the assay, and the chelator used. Studies with EDTA indicate that including the chelator in the isolation, resuspension, and assay media, in the absence of divalent cations, was most effective. Increased concentration of EDTA from 1 to 10 mm broadened the pH response curve for photosynthesis, inasmuch as a higher concentration of chelator was required for activation of photosynthesis at lower pH.Either EDTA, PPi, or citrate could activate photosynthesis of sunflower chloroplasts isolated and assayed at pH 8.4. At pH 7.6, PPi and EDTA were equally effective at low Pi concentrations but PPi was particularly effective in shortening the induction period at high concentrations of Pi (2.5 mm) in the assay medium. Including 1 mm 3-phosphoglycerate in the assay medium with or without Pi could not replace the need for chelation. However, 3-phosphoglycerate + EDTA in the assay medium with 0.5 mm Pi, pH 7.6, gave a short induction period and rates of photosynthesis similar to those with 10 mm PPi. The results suggest that PPi can have a dual effect at the lower pH through chelation and inhibition of the phosphate transporter.Photosynthesis by sunflower chloroplasts isolated and assayed at pH 8.4 with 0.2 mm EDTA (+ 0.5 mm Pi in the assays) was severely inhibited by 2 mM CaCl2, MgCl2, or MnCl2. Wheat chloroplasts isolated and assayed at pH 8.4 without chelation, and assayed with 0.2 mm Pi, had low rates of photosynthesis (25 μmol O2 evolved mg?1 chlorophyll h?1) which were strongly inhibited by 2 to 4 mm MgCl2, MnCl2, or CaCl2. With inclusion of EDTA and Pi at optimum levels, isolated chloroplasts of sunflower and wheat have high rates of photosynthesis and PPi or divalent cations are not of benefit. 相似文献
16.
Summary Two phytases from lily pollen (Lilium longiflorum Thunb.) were partially purified and characterized. The first (pH optimum 5.0) was purified 40-fold from ungerminated pollen. The second (pH optimum 6.5) appeared during germination and was purified 68-fold from pollen germinated 2 h. Molecular weight of the first was 72 kD, and the second was 36 kD as determined by gel filtration. Both were active against phosphate esters other than phytate, although purification of the first reduced its activity against AMP and myo-inositol 2-P to 10% of activity against phytate. Phytase from germinated pollen (but not ungerminated) was inhibited by the sulfhydryl agent parahydroxy mercuribenzoate; P
i
inhibited phytase from ungerminated but not germinated pollen. Such different catalytic and physical properties may reflect different biochemical functions.Abbreviations HPLC
High performance liquid chromatography
- DEAE
diethyl aminoethyl
-
P
i
orthophosphate
- PP
i
pyrophosphate
-
p-NPP
para-nitrophenyl phosphate
- pNP
para-nitrophenol
- MI
myo-inositol
- MI 2-P
myo-inositol 2-P
- MI penta P
myo-inositol pentakisphosphate
- PHMB
para-hydroxy mercuribenzoate
- PMSF
phenyl methyl sulfonyl fluoride
- AMP
adenosine monophosphate
- GMP
guanosine monophosphate
- EGTA
ethylene glycol-bis (-aminoethyl ether) N, N, N, N-tetraacetic acid 相似文献
17.
Zeng YF Ko TP Lai HL Cheng YS Wu TH Ma Y Chen CC Yang CS Cheng KJ Huang CH Guo RT Liu JR 《Journal of molecular biology》2011,409(2):214-224
Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general. 相似文献
18.
Using a combination of High-Performance Ion Chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by a phytase from a Malaysian waste-water bacterium was established. The data
demonstrate that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-I(1,2,3,4,5)P5, D-I(2,3,4,5)P4, D-I(2,3,4)P3, D-I(2,3)P2 to finally I(2)P. It was estimated that more than 90% of phytate hydrolysis occurs via D-I(1,2,3,4,5)P5. Thus, the phytase from the Malaysian waste-water bacterium has to be considered a 6-phytase (E.C. 3.1.3.26). A second pathway
of minor importance could be proposed which is in accordance with the results obtained from analysis of the dephosphorylation
products formed by the action of the phytase under investigation on myo-inositol hexakisphosphate. It proceeds via D/L-I(1,2,4,5,6)P5, D/L-I(1,2,4,5)P4, D/L-I(1,2,4)P3, D/L-I(2,4)P2 to finally I(2)P. 相似文献
19.
In mammals the type IIb Na/Pi-cotransporter is expressed in various tissues such as intestine, brain, lung and testis. The type IIb cotransporter shows 51% homology with the renal type IIa Na/Pi-cotransporter, for which a detailed model of the secondary structure has emerged based on recent structure/function studies. To make the type IIb Na/Pi-cotransporter available for future structural studies, we have expressed this cotransporter in Sf9 cells. Sf9 cells were infected with recombinant baculovirus containing 6His NaPi-IIb. Infected cells expressed a polypeptide of ~90 kDa, corresponding to a partially glycosylated form of the type IIb cotransporter. Transport studies demonstrated that the type IIb protein expressed in Sf9 cells mediates transport of phosphate in a Na-dependent manner with similar kinetic characteristics (apparent K
ms for sodium and phosphate and pH dependence) as previously described. Solubilization experiments demonstrated that, in contrast to the type IIa cotransporter, the type IIb can be solubilized by nonionic detergents and that solubilized type IIb Na/Pi-cotransporter can be purified by Ni-NTA chromatography. 相似文献
20.
Yufeng Yao Yi Xie Donna Perace Yi Zhong Jie Lu Jing Tao Xiaokui Guo & Kwang Sik Kim 《FEMS microbiology letters》2009,300(1):18-24
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 °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 °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. 相似文献