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S. Dalsgaard - One of the best experts on this subject based on the ideXlab platform.
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interactions of phytate and myo inositol phosphate esters ip1 5 including ip5 isomers with dietary protein and iron and inhibition of pepsin
Journal of Animal Science, 2012Co-Authors: A. Cowieson, C. Gilbert, P. Plumstead, Shi Yu, S. DalsgaardAbstract:Phytic acid (IP6) and myo-inositol phosphate esters (IP1-5), including IP5 isomers prepared chemically and enzymatically with bacterial and fungal Phytases, were examined for their effects on protein aggregation of soy protein and beta-casein, interaction with Fe3+, and pepsin activity. The results indicated that the aggregating capabilities of IP esters (IP1-6) on the 2 proteins decreased dramatically from IP6 to IP5 and became negligible with IP1-4. Among the IP5 isomers tested, InsP(5)(1,2,3,4,5) produced by 6-phytase was slightly less powerful in aggregating protein than InsP(5)(1,2,4,5,6) produced by 3-phytase (P = 0.001). For protein hydrolysis, IP esters of IP3-4 still showed inhibition of pepsin though to a lesser extent than IP5-6. The in vitro data with IP1-5 generated with microbial 3- and 6-Phytases indicate that, for complete alleviation of pepsin inhibition, IP6 needs to be broken down to IP1-2. In contrast to the aggregation with protein, the reactivity of IP1-6 toward Fe3+ decreased proportionally from IP6 to IP3. Based on the radical decrease in turbidity of IP6-protein complex observed, as a result of IP6 dephosphorylation to IP5, a novel qualitative and semi-quantitative phytase plate assay was established using IP6-protein complex incorporated into an agarose petri-dish as substrate. Phytase activity was shown as the development of clear halos on the agarose plate with time. This simple phytase plate assay method can be used at animal farms, control laboratories, and even for the screening of engineered phytase variants. The current study, thus, stresses the importance of the efficient hydrolysis of IP6 at lower pH range to alleviate the negative effect of phytic acid and its degradation products on protein and Fe3+ digestion. (Less)
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Interactions of phytate and myo-inositol phosphate esters (IP1-5) including IP5isomers with dietary protein and iron and inhibition of pepsin
Journal of Animal Science, 2012Co-Authors: Shuanghe Yu, A. Cowieson, C. Gilbert, P. Plumstead, S. DalsgaardAbstract:Phytic acid (IP(6)) and myo-inositol phosphate esters (IP(1-5)), including IP(5) isomers prepared chemically and enzymatically with bacterial and fungal Phytases, were examined for their effects on protein aggregation of soy protein and β-casein, interaction with Fe(3+), and pepsin activity. The results indicated that the aggregating capabilities of IP esters (IP(1-6)) on the 2 proteins decreased dramatically from IP(6) to IP(5) and became negligible with IP(1-4). Among the IP(5) isomers tested, InsP(5)(1,2,3,4,5) produced by 6-phytase was slightly less powerful in aggregating protein than InsP(5)(1,2,4,5,6) produced by 3-phytase (P = 0.001). For protein hydrolysis, IP esters of IP(3-4) still showed inhibition of pepsin though to a lesser extent than IP(5-6). The in vitro data with IP(1-5) generated with microbial 3- and 6-Phytases indicate that, for complete alleviation of pepsin inhibition, IP(6) needs to be broken down to IP(1-2.) In contrast to the aggregation with protein, the reactivity of IP(1-6) toward Fe(3+) decreased proportionally from IP(6) to IP(3.) Based on the radical decrease in turbidity of IP(6) -protein complex observed, as a result of IP(6) dephosphorylation to IP(5), a novel qualitative and semi-quantitative phytase plate assay was established using IP(6)-protein complex incorporated into an agarose petri-dish as substrate. Phytase activity was shown as the development of clear halos on the agarose plate with time. This simple phytase plate assay method can be used at animal farms, control laboratories, and even for the screening of engineered phytase variants. The current study, thus, stresses the importance of the efficient hydrolysis of IP(6) at lower pH range to alleviate the negative effect of phytic acid and its degradation products on protein and Fe(3+) digestion.
Markus Wyss - One of the best experts on this subject based on the ideXlab platform.
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characteristics of fungal Phytases from aspergillus fumigatus and sartorya fumigata
Applied Microbiology and Biotechnology, 2004Co-Authors: Roland Brugger, Markus Wyss, P Guggenbuhl, Simoes C Nunes, Kurt Vogel, A P G M Van Loon, F Mascarello, S Augem, Luis PasamontesAbstract:Aspergillus fumigatus phytase has previously been identified as a phytase with a series of favourable properties that may be relevant in animal and human nutrition, both for maximising phytic acid degradation and for increasing mineral and amino acid availability. To study the natural variability in amino acid sequence and its impact on the catalytic properties of the enzyme, we cloned and overexpressed the phytase genes and proteins from six new purported A. fumigatus isolates. Five of these Phytases displayed ≤2 amino acid substitutions and had virtually identical stability and catalytic properties when compared with the previously described A. fumigatus ATCC 13073 phytase. In contrast, the phytase from isolate ATCC 32239 (Sartorya fumigata, the anamorph of which was identified as A. fumigatus) was more divergent (only 86% amino acid sequence identity), had a higher specific activity with phytic acid, and displayed distinct differences in substrate specificity and pH-activity profile. Finally, comparative experiments confirmed the favourable stability and catalytic properties of A. fumigatus phytase.
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the consensus concept for thermostability engineering of proteins further proof of concept
Protein Engineering, 2002Co-Authors: Martin Lehmann, Luis Pasamontes, Anke Middendorf, Adolphus P G M Van Loon, Claudia Loch, Dominik Studer, Soren Flensted Lassen, Markus WyssAbstract:Previously, we calculated a consensus amino acid sequence from 13 homologous fungal Phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15–26°C more thermostable than all parent Phytases used in its design [Lehmann et al. (2000) Protein Eng., 13, 49–57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus Phytases-10 and -11. Since consensus phytase10, again, was 7.4°C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3°C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5°C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4°C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.
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the consensus concept for thermostability engineering of proteins
Biochimica et Biophysica Acta, 2000Co-Authors: Martin Lehmann, Luis Pasamontes, Soren Flensted Lassen, Markus WyssAbstract:Previously, sequence comparisons between a mesophilic enzyme and a more thermostable homologue were shown to be a feasible approach to successfully predict thermostabilizing amino acid substitutions. The ‘consensus approach’ described in the present paper shows that even a set of amino acid sequences of homologous, mesophilic enzymes contains sufficient information to allow rapid design of a thermostabilized, fully functional variant of this family of enzymes. A sequence alignment of homologous fungal Phytases was used to calculate a consensus phytase amino acid sequence. Upon construction of the synthetic gene, recombinant expression and purification, the first phytase obtained, termed consensus phytase-1, displayed an unfolding temperature (Tm) of 78.0°C which is 15–22°C higher than the Tm values of all parent Phytases used in its design. Refinement of the approach, combined with site-directed mutagenesis experiments, yielded optimized consensus Phytases with Tm values of up to 90.4°C. These increases in Tm are due to the combination of multiple amino acid exchanges which are distributed over the entire sequence of the protein and mainly affect surface-exposed residues; each individual substitution has a rather small thermostabilizing effect only. Remarkably, in spite of the pronounced increase in thermostability, catalytic activity at 37°C is not compromised. Thus, the design of consensus proteins is a potentially powerful and novel alternative to directed evolution and to a series of rational approaches for thermostability engineering of enzymes and other proteins.
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exchanging the active site between Phytases for altering the functional properties of the enzyme
Protein Science, 2000Co-Authors: Martin Lehmann, Markus Wyss, Claudia Loch, Rual Lopezulibarri, Celine Viarouge, Adolphus P G M Van LoonAbstract:By using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase-1, that was 15-26 degrees C more thermostable than the parent fungal Phytases used in its design (Lehmann et al., 2000). We now sought to use the backbone of consensus phytase-1 and to modify its catalytic properties. This was done by replacing a considerable part of the active site (i.e., all the divergent residues) with the corresponding residues of Aspergillus niger NRRL 3135 phytase, which displays pronounced differences in specific activity, substrate specificity, and pH-activity profile. For the new protein termed consensus phytase-7, a major - although not complete - shift in catalytic properties was observed, demonstrating that rational transfer of favorable catalytic properties from one phytase to another is possible by using this approach. Although the exchange of the active site was associated with a 7.6 degrees C decrease in unfolding temperature (Tm) as measured by differential scanning calorimetry, consensus phytase-7 still was >7 degrees C more thermostable than all wild-type ascomycete Phytases known to date. Thus, combination of the consensus approach with the selection of a "preferred" active site allows the design of a thermostabilized variant of an enzyme family of interest that (most closely) matches the most favorable catalytic properties found among its family members.
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biophysical characterization of fungal Phytases myo inositol hexakisphosphate phosphohydrolases molecular size glycosylation pattern and engineering of proteolytic resistance
Applied and Environmental Microbiology, 1999Co-Authors: Markus Wyss, Luis Pasamontes, Arno Friedlein, R Remy, Michel Tessier, Alexandra Kronenberger, Anke Middendorf, Martin Lehmann, Line Schnoebelen, Urs RothlisbergerAbstract: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.
Adolphus P G M Van Loon - One of the best experts on this subject based on the ideXlab platform.
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the consensus concept for thermostability engineering of proteins further proof of concept
Protein Engineering, 2002Co-Authors: Martin Lehmann, Luis Pasamontes, Anke Middendorf, Adolphus P G M Van Loon, Claudia Loch, Dominik Studer, Soren Flensted Lassen, Markus WyssAbstract:Previously, we calculated a consensus amino acid sequence from 13 homologous fungal Phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15–26°C more thermostable than all parent Phytases used in its design [Lehmann et al. (2000) Protein Eng., 13, 49–57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus Phytases-10 and -11. Since consensus phytase10, again, was 7.4°C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3°C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5°C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4°C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.
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exchanging the active site between Phytases for altering the functional properties of the enzyme
Protein Science, 2000Co-Authors: Martin Lehmann, Markus Wyss, Claudia Loch, Rual Lopezulibarri, Celine Viarouge, Adolphus P G M Van LoonAbstract:By using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase-1, that was 15-26 degrees C more thermostable than the parent fungal Phytases used in its design (Lehmann et al., 2000). We now sought to use the backbone of consensus phytase-1 and to modify its catalytic properties. This was done by replacing a considerable part of the active site (i.e., all the divergent residues) with the corresponding residues of Aspergillus niger NRRL 3135 phytase, which displays pronounced differences in specific activity, substrate specificity, and pH-activity profile. For the new protein termed consensus phytase-7, a major - although not complete - shift in catalytic properties was observed, demonstrating that rational transfer of favorable catalytic properties from one phytase to another is possible by using this approach. Although the exchange of the active site was associated with a 7.6 degrees C decrease in unfolding temperature (Tm) as measured by differential scanning calorimetry, consensus phytase-7 still was >7 degrees C more thermostable than all wild-type ascomycete Phytases known to date. Thus, combination of the consensus approach with the selection of a "preferred" active site allows the design of a thermostabilized variant of an enzyme family of interest that (most closely) matches the most favorable catalytic properties found among its family members.
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biochemical characterization of fungal Phytases myo inositol hexakisphosphate phosphohydrolases catalytic properties
Applied and Environmental Microbiology, 1999Co-Authors: Markus Wyss, R Remy, Alexandra Kronenberger, Martin Lehmann, Roland Brugger, Rachel Fimbel, Gottfried Oesterhelt, Adolphus P G M Van LoonAbstract:The phosphatases are a diverse class of enzymes. According to one classification, alkaline phosphatases, purple acid phosphatases, high-molecular-weight acid phosphatases, low-molecular-weight acid phosphatases, and protein phosphatases can be distinguished (13). These classes differ in their pH optima, metal ion requirements, substrate specificities, and possibly even reaction mechanisms. The Phytases (myo-inositol hexakisphosphate phosphohydrolases; EC 3.1.3.8 and 3.1.3.26) are a subfamily of the high-molecular-weight histidine acid phosphatases. The phytase reaction mechanism is a two-step mechanism which includes a covalent phosphohistidine adduct as an obligatory reaction intermediate (6). Phytases are found naturally in plants and microorganisms, particularly fungi (for a review see reference 15). They catalyze phosphate monoester hydrolysis of phytic acid (myo-inositol hexakisphosphate), which results in the stepwise formation of myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates, as well as the liberation of inorganic phosphate. Phytic acid is the major storage form of phosphorus in plant seeds and, thus, in seed-based animal feed (for reviews see references 1 and 8). Monogastric animals, such as pigs and poultry, are not able to utilize phytic acid phosphorus, since they have only low levels of phytase activity in their digestive tracts and since phytic acid cannot be resorbed. Therefore, pig and poultry feed commonly is supplemented with either inorganic phosphate or a phytase of fungal origin. Despite considerable economic interest, only limited data on the catalytic properties of fungal Phytases are available. In order to get an impression of the natural diversity of Phytases, the enzymatic properties of six fungal Phytases (Phytases from Aspergillus niger, two strains of Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila) and of Escherichia coli phytase were characterized in more detail by addressing the following questions. (i) What are the specific activities and pH optima of wild-type Phytases? (ii) What are the kinetics of phytic acid degradation, and what are the end products? (iii) Does the substrate specificity profile correlate with the results of in vitro experiments performed to determine phosphate liberation from feed samples? And (iv) what is the potential influence of modulators of enzymatic activity?
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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
Applied and Environmental Microbiology, 1998Co-Authors: Markus Wyss, Luis Pasamontes, R Remy, Josiane Kohler, Eric Kusznir, Martin Gadient, Francis Muller, Adolphus P G M Van LoonAbstract:Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90°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°C. After heat denaturation at temperatures up to 90°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°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°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°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°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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cloning of the Phytases from emericella nidulans and the thermophilic fungus talaromyces thermophilus
Biochimica et Biophysica Acta, 1997Co-Authors: Luis Pasamontes, M Haiker, Maria Henriquezhuecas, David Mitchell, Adolphus P G M Van LoonAbstract:Abstract Phytases (EC 3.1.3.8) belong to the family of histidine acid phosphatases. We have cloned the Phytases of the fungi Emericella nidulans and Talaromyces thermophilus . The putative enzyme encoded by the E. nidulans sequence consists of 463 amino acids and has a M r of 51785. The protein deduced from the T. thermophilus sequence consists of 466 amino acids corresponding to a M r of 51450. Both predicted amino acid sequences exhibited high identity (48% to 67%) to known Phytases. This high level of identity allowed the modelling of all available fungal Phytases based on the three-dimensional structure coordinates of the Aspergillus niger phytase. By this approach we identified 21 amino acids which are conserved in fungal phyA Phytases and are part of the residues forming the substrate pocket. Furthermore, potential glycosylation sites were identified and compared between the aforementioned Phytases and the A. niger phytase.
Ralf Greiner - One of the best experts on this subject based on the ideXlab platform.
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screening and characterization of Phytases from bacteria isolated from chilean hydrothermal environments
Microbial Ecology, 2018Co-Authors: Milko A Jorquera, Daniel Menezesblackburn, Stefanie Gabler, Nitza G Inostroza, Jacquelinne J Acuna, Marco Campos, Ralf GreinerAbstract:Phytases are enzymes involved in organic phosphorus cycling in nature and widely used as feed additives in animal diets. Thermal tolerance is a desired property of Phytases. The objectives of this study were to screen and characterize bacterial Phytases from Chilean hydrothermal environments. In this study, 60% (30 of 63) of screened thermophilic (60 °C) isolates showed phytase activity in crude protein extracts. The characterization of phytase from two selected isolates (9B and 15C) revealed that both isolates produce Phytases with a pH optimum at 5.0. The temperature optimum for phytate dephosphorylation was determined to be 60 and 50 °C for the Phytases from the isolates 9B and 15C, respectively. Interestingly, the phytase from the isolate 15C showed a residual activity of 46% after incubation at 90 °C for 20 min. The stepwise dephosphorylation of phytate by protein extracts of the isolates 9B and 15C was verified by HLPC analysis. Finally, the isolates 9B and 15C were identified by partial sequencing of the 16S rRNA gene as members of the genera Bacillus and Geobacillus, respectively.
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Phytases and phytase labile organic phosphorus in manures and soils
Critical Reviews in Environmental Science and Technology, 2013Co-Authors: Daniel Menezesblackburn, Milko A Jorquera, Liliana Gianfreda, Ralf Greiner, Maria De La Luz MoraAbstract:Organic phosphorus (Po) hydrolysis by microbial Phytases has extensively been considered in diverse biotechnological applications, including environmental protection and agricultural, animal, and human nutrition. The authors review the available information on the content of phytase-labile Po in manures and soils, as well as the environmental factors and enzyme properties affecting catalytic behavior of Phytases in these environments. In addition, they have critically analyzed the present and possible future biotechnological approaches for using Phytases to access phytate Po pool present in soils and manures for plant nutrition, with the concomitant reduction of runoff P in the environment.
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activity stabilization of aspergillus niger and escherichia coli Phytases immobilized on allophanic synthetic compounds and montmorillonite nanoclays
Bioresource Technology, 2011Co-Authors: Daniel Menezesblackburn, Milko A Jorquera, Liliana Gianfreda, Ralf Greiner, Elizabeth Garrido, Maria De La Luz MoraAbstract:The aim of this work was to study the stabilization of the activity of two commercial microbial Phytases (Aspergillus niger and Escherichia coli) after immobilization on nanoclays and to establish optimal conditions for their immobilization. Synthetic allophane, synthetic iron-coated allophanes and natural montmorillonite were chosen as solid supports for phytase immobilization. Phytase immobilization patterns at different pH values were strongly dependent on both enzyme and support characteristics. After immobilization, the residual activity of both Phytases was higher under acidic conditions. Immobilization of Phytases increased their thermal stability and improved resistance to proteolysis, particularly on iron-coated allophane (6% iron oxide), which showed activation energy (E(a)) and activation enthalpy (ΔH(#)) similar to free enzymes. Montmorillonite as well as allophanic synthetic compounds resulted in a good support for immobilization of E. coli phytase, but caused a severe reduction of A. niger phytase activity.
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Phytate-degrading enzyme production by bacteria isolated from Malaysian soil
World Journal of Microbiology and Biotechnology, 2007Co-Authors: Anis Shobirin Meor Hussin, Ralf Greiner, Abdelaziem Farouk, Hamzah Mohd Salleh, Ahmad Faris IsmailAbstract:Over two hundred bacteria were isolated from the halosphere, rhizosphere and endophyte of Malaysian maize plantation and screened for Phytases activity. Thirty isolates with high detectable phytase activity were chosen for media optimization study and species identification. Ten types of bacterial phytase producers have been discovered in this study, which provides opportunity for characterization of new phytase(s) and various commercial and environmental applications. The majority of the bacterial isolates with high detectable phytase activity were of endophyte origin and 1.6% of the total isolates showed phytase activity of more than 1 U/ml. Most of the strains produced extra-cellular phytase and Staphylococcus lentus ASUIA 279 showed the highest phytase activity of 1.913 U/ml. All 30 species used in media optimization study exhibit favorable enzyme production when 1% rice bran was included in the growth media.
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purification and characterization of three Phytases from germinated lupine seeds lupinus albus var amiga
Journal of Agricultural and Food Chemistry, 2002Co-Authors: Ralf GreinerAbstract:Three Phytases were purified about 14200-fold (LP11), 16000-fold (LP12), and 13100-fold (LP2) from germinated 4-day-old lupine seedlings to apparent homogeneity with recoveries of 13% (LP11), 8% (LP12), and 9% (LP2) referred to the phytase activity in the crude extract. They behave as monomeric proteins of a molecular mass of about 57 kDa (LP11 and LP12) and 64 kDa (LP2), respectively. The purified proteins belong to the acid Phytases. They exhibit a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 °C. Kinetic parameters for the hydrolysis of sodium phytate are KM = 80 μM (LP11), 300 μM (LP12), and 130 μM (LP2) and kcat = 523 s-1 (LP11), 589 s-1 (LP12), and 533 s-1 (LP2) at pH 5.0 and 35 °C. The Phytases from lupine seeds exhibit a broad affinity for various phosphorylated compounds and hydrolyze phytate in a stepwise manner. Keywords: Legume phytase; lupine; myo-inositol phosphate phosphohydrolase; phytate degradation
Shi Yu - One of the best experts on this subject based on the ideXlab platform.
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interactions of phytate and myo inositol phosphate esters ip1 5 including ip5 isomers with dietary protein and iron and inhibition of pepsin
Journal of Animal Science, 2012Co-Authors: A. Cowieson, C. Gilbert, P. Plumstead, Shi Yu, S. DalsgaardAbstract:Phytic acid (IP6) and myo-inositol phosphate esters (IP1-5), including IP5 isomers prepared chemically and enzymatically with bacterial and fungal Phytases, were examined for their effects on protein aggregation of soy protein and beta-casein, interaction with Fe3+, and pepsin activity. The results indicated that the aggregating capabilities of IP esters (IP1-6) on the 2 proteins decreased dramatically from IP6 to IP5 and became negligible with IP1-4. Among the IP5 isomers tested, InsP(5)(1,2,3,4,5) produced by 6-phytase was slightly less powerful in aggregating protein than InsP(5)(1,2,4,5,6) produced by 3-phytase (P = 0.001). For protein hydrolysis, IP esters of IP3-4 still showed inhibition of pepsin though to a lesser extent than IP5-6. The in vitro data with IP1-5 generated with microbial 3- and 6-Phytases indicate that, for complete alleviation of pepsin inhibition, IP6 needs to be broken down to IP1-2. In contrast to the aggregation with protein, the reactivity of IP1-6 toward Fe3+ decreased proportionally from IP6 to IP3. Based on the radical decrease in turbidity of IP6-protein complex observed, as a result of IP6 dephosphorylation to IP5, a novel qualitative and semi-quantitative phytase plate assay was established using IP6-protein complex incorporated into an agarose petri-dish as substrate. Phytase activity was shown as the development of clear halos on the agarose plate with time. This simple phytase plate assay method can be used at animal farms, control laboratories, and even for the screening of engineered phytase variants. The current study, thus, stresses the importance of the efficient hydrolysis of IP6 at lower pH range to alleviate the negative effect of phytic acid and its degradation products on protein and Fe3+ digestion. (Less)
