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Ralf Greiner - One of the best experts on this subject based on the ideXlab platform.
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novel glucose 1 phosphatase with high phytase activity and unusual metal ion activation from soil bacterium pantoea sp strain 3 5 1
Applied and Environmental Microbiology, 2015Co-Authors: Aliya D Suleimanova, Inna B Chastukhina, Eugene V Shakirov, Ralf Greiner, Astrid Beinhauer, Liia R Valeeva, N P Balaban, M R SharipovaAbstract:Phosphorus is an important macronutrient, but it's availability in soil is limited. Many soil microorganisms improve bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes and urban landscape. Bacterial isolate Pantoea sp. 3.5.1 with the highest level of phytase activity was isolated from forest soil type and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy and biochemically characterized. AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values towards phytate, glucose-6-phosphate and glucose-1-phosphate. Unexpectedly, a stimulation of enzymatic activity by several divalent metal ions was observed for AgpP enzyme. HPLC and HPIC analyses of phytate hydrolysis products identify D/L-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.
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performance of seven commercial phytases in an in vitro simulation of poultry digestive tract
Journal of Agricultural and Food Chemistry, 2015Co-Authors: Daniel Menezesblackburn, Stefanie Gabler, Ralf GreinerAbstract:The aim of this study was to compare the biochemical properties of seven commercially available phytase products as well as their catalytic performance in an in vitro simulation of the digestive tract of poultry. Their enzymatic properties relevant with respect to phytate dephosphorylation in the digestive tract of birds were determined under identical assay conditions. All phytase products included in the study showed an acid pH optimum of activity and were capable of releasing the organically bound phosphate from phytate during the in vitro simulation. However, their overall biochemical properties and relative catalytic performances were remarkably different. The in vitro degradation system was considered as a simple and useful tool to evaluate the suitability of a phytase to be used as a feed supplement. Although relevant factors such as dietary P levels, intestinal phytase, and P absorption are not implemented in the system, this approach might help to reduce the number of feeding trials necessary in the search for a better suited phytase for animal feeding application.
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performance of seven commercial phytases in an in vitro simulation of poultry digestive tract
Journal of Agricultural and Food Chemistry, 2015Co-Authors: Daniel Menezesblackburn, Stefanie Gabler, Ralf GreinerAbstract:The aim of this study was to compare the biochemical properties of seven commercially available phytase products as well as their catalytic performance in an in vitro simulation of the digestive tract of poultry. Their enzymatic properties relevant with respect to phytate dephosphorylation in the digestive tract of birds were determined under identical assay conditions. All phytase products included in the study showed an acid pH optimum of activity and were capable of releasing the organically bound phosphate from phytate during the in vitro simulation. However, their overall biochemical properties and relative catalytic performances were remarkably different. The in vitro degradation system was considered as a simple and useful tool to evaluate the suitability of a phytase to be used as a feed supplement. Although relevant factors such as dietary P levels, intestinal phytase, and P absorption are not implemented in the system, this approach might help to reduce the number of feeding trials necessary in ...
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identification of β propeller phytase encoding genes in culturable paenibacillus and bacillus spp from the rhizosphere of pasture plants on volcanic soils
FEMS Microbiology Ecology, 2011Co-Authors: Milko A Jorquera, Maria Teresa Fernandez, Daniela Romero, Daniel Menezesblackburn, David E Crowley, Petra Marschner, Ralf Greiner, Maria MoraAbstract:Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.
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identification of β propeller phytase encoding genes in culturable paenibacillus and bacillus spp from the rhizosphere of pasture plants on volcanic soils
FEMS Microbiology Ecology, 2011Co-Authors: Milko A Jorquera, Maria Teresa Fernandez, Daniela Romero, Daniel Menezesblackburn, David E Crowley, Petra Marschner, Ralf Greiner, Maria MoraAbstract:Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.
Eugene V Shakirov - One of the best experts on this subject based on the ideXlab platform.
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heterologous expression of secreted bacterial bpp and hap phytases in plants stimulates arabidopsis thaliana growth on phytate
Frontiers in Plant Science, 2018Co-Authors: Lia R. Valeeva, Chuluuntsetseg Nyamsuren, Eugene V Shakirov, Margarita R. SharipovaAbstract:Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits towards improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology.
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microbial phytases and phytate exploring opportunities for sustainable phosphorus management in agriculture
American Journal of Molecular Biology, 2017Co-Authors: Nelly P. Balaban, Aliya D Suleimanova, Inna B Chastukhina, N. L. Rudakova, Lia R. Valeeva, Margarita R. Sharipova, Eugene V ShakirovAbstract:Myo-inositol phosphates (phytates) are important biological molecules produced largely by plants to store phosphorus. Phytate is very abundant in many different soils making up a large portion of all soil phosphorus. This review assesses current phytase science from the perspective of its substrate, phytate, by examining the intricate relationship between the phytate-hydrolyzing enzymes and phytate as their substrate. Specifically, we examine available data on phytate’s structural features, distribution in nature and functional roles. The role of phytases and their localization in soil and plant tissues are evaluated. We provide a summary of the current biotechnological advances in using industrial or recombinant phytases to improve plant growth and animal nutrition. The prospects of future discovery of novel phytases with improved biochemical properties and bioengineering of existing enzymes are also discussed. Two alternative but complementary directions to increase phosphorus bioavailability through the more efficient utilization of soil phytate are currently being developed. These approaches take advantage of microbial phytases secreted into rhizosphere either by phytase-producing microbes (biofertilizers) or by genetically engineered plants. More research on phytate metabolism in soils and plants is needed to promote environmentally friendly, more productive and sustainable agriculture.
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novel glucose 1 phosphatase with high phytase activity and unusual metal ion activation from soil bacterium pantoea sp strain 3 5 1
Applied and Environmental Microbiology, 2015Co-Authors: Aliya D Suleimanova, Inna B Chastukhina, Eugene V Shakirov, Ralf Greiner, Astrid Beinhauer, Liia R Valeeva, N P Balaban, M R SharipovaAbstract:Phosphorus is an important macronutrient, but it's availability in soil is limited. Many soil microorganisms improve bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes and urban landscape. Bacterial isolate Pantoea sp. 3.5.1 with the highest level of phytase activity was isolated from forest soil type and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy and biochemically characterized. AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values towards phytate, glucose-6-phosphate and glucose-1-phosphate. Unexpectedly, a stimulation of enzymatic activity by several divalent metal ions was observed for AgpP enzyme. HPLC and HPIC analyses of phytate hydrolysis products identify D/L-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.
Maria Mora - One of the best experts on this subject based on the ideXlab platform.
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identification of β propeller phytase encoding genes in culturable paenibacillus and bacillus spp from the rhizosphere of pasture plants on volcanic soils
FEMS Microbiology Ecology, 2011Co-Authors: Milko A Jorquera, Maria Teresa Fernandez, Daniela Romero, Daniel Menezesblackburn, David E Crowley, Petra Marschner, Ralf Greiner, Maria MoraAbstract:Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.
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identification of β propeller phytase encoding genes in culturable paenibacillus and bacillus spp from the rhizosphere of pasture plants on volcanic soils
FEMS Microbiology Ecology, 2011Co-Authors: Milko A Jorquera, Maria Teresa Fernandez, Daniela Romero, Daniel Menezesblackburn, David E Crowley, Petra Marschner, Ralf Greiner, Maria MoraAbstract:Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.
Richard J Simpson - One of the best experts on this subject based on the ideXlab platform.
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expression of a fungal phytase gene in nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils
Plant Biotechnology Journal, 2005Co-Authors: Timothy S George, Richard J Simpson, P A Hadobas, Alan RichardsonAbstract:Transgenic Nicotiana tabacum plants expressing a chimeric phytase gene (ex::phyA) from the soil fungus Aspergillus niger were generated. Three independently transformed lines showed increased extracellular phytase activity compared with a vector control and wild-type plants, both of which had no detectable extracellular phytase. Transgenic N. tabacum plants grown in sterile agar supplied with phosphorus (P) as phytate accumulated 3.7-fold more P than vector control plants. Despite this, the expression of ex::phyA in plants did not lead to an improved accumulation of P from two unamended P-deficient soils. However, when soils were amended with either phytate or phosphate and lime, transgenic plants accumulated up to 52% more P than controls. Positive responses by transgenic plants were, in some instances, coincident with a putative increase in soil phytate. We conclude that the development of plants that exude phytase to the soil may not ensure improved plant P nutrition, as the availability of phytate in the soil also appears to be critical. Nevertheless, if plants that express ex::phyA are combined with soil amendments that promote the availability of phytate, there is the potential to enhance the P nutrition of crop plants and to improve the efficiency of P fertilizer use in agricultural systems.
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characterization of transgenic trifolium subterraneum l which expresses phya and releases extracellular phytase growth and p nutrition in laboratory media and soil
Plant Cell and Environment, 2004Co-Authors: Timothy S George, P A Hadobas, Alan Richardson, Richard J SimpsonAbstract:Transgenic Trifolium subterraneum expressing a phytase gene (phyA) from Aspergillus niger were generated. Five independently transformed lines showed an average 77-fold increase in exuded phytase activity in comparison with null segregant and wild-type controls. Unlike other phosphatases, exuded phytase activity was unaffected by P supply, verifying the constitutive expression of phyA. Transgenic T. subterraneum grown in agar with P supplied as phytate, took up 1.3- to 3.6-fold more P than controls and had equivalent P uptake to plants supplied with orthophosphate. This unique phenotype was compromised when the plants were grown in soil. None of the five lines showed increased shoot biomass or total P uptake in an unfertilized, low-P soil taken from under permanent pasture. With addition of P, one of the five transgenic lines had consistently greater P nutrition compared with control plants. Despite variable growth and P nutrition responses, P uptake per root length was on average greater for transgenic lines. Exudation of phytase by transgenic T. subterraneum allowed utilization of P from phytate in non-sorbing, sterile laboratory media, but was less effective when plants were grown in soil. Release of extracellular phytase is therefore not the only requirement for the acquisition of P from endogenous soil phytate by plants.
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phytate as a source of phosphorus for the growth of transgenic trifolium subterraneum
Plant nutrition: food security and sustainability of agro-ecosystems through basic and applied research. Fourteenth International Plant Nutrition Coll, 2001Co-Authors: Alan Richardson, P A Hadobas, Richard J SimpsonAbstract:Although phytate is an abundant form of soil organic P, plants generally have only a limited capacity to obtain phosphorus (P) directly from this substrate. Inability of plants to effectively use phytate-P is associated with poor substrate availability in soil environments, and insufficient extracellular root phytase activity. We therefore generated transgenic lines of Trifolium subterraneum L. that expressed the phytase (phyA) gene from Aspergillus niger. Analysis of segregating T1 populations of these plants showed that the presence of an extracellular phytase was effective in improving the P nutrition of the plants when supplied with phytate and grown in agar under sterile conditions.
Alan Richardson - One of the best experts on this subject based on the ideXlab platform.
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expression of a fungal phytase gene in nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils
Plant Biotechnology Journal, 2005Co-Authors: Timothy S George, Richard J Simpson, P A Hadobas, Alan RichardsonAbstract:Transgenic Nicotiana tabacum plants expressing a chimeric phytase gene (ex::phyA) from the soil fungus Aspergillus niger were generated. Three independently transformed lines showed increased extracellular phytase activity compared with a vector control and wild-type plants, both of which had no detectable extracellular phytase. Transgenic N. tabacum plants grown in sterile agar supplied with phosphorus (P) as phytate accumulated 3.7-fold more P than vector control plants. Despite this, the expression of ex::phyA in plants did not lead to an improved accumulation of P from two unamended P-deficient soils. However, when soils were amended with either phytate or phosphate and lime, transgenic plants accumulated up to 52% more P than controls. Positive responses by transgenic plants were, in some instances, coincident with a putative increase in soil phytate. We conclude that the development of plants that exude phytase to the soil may not ensure improved plant P nutrition, as the availability of phytate in the soil also appears to be critical. Nevertheless, if plants that express ex::phyA are combined with soil amendments that promote the availability of phytate, there is the potential to enhance the P nutrition of crop plants and to improve the efficiency of P fertilizer use in agricultural systems.
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characterization of transgenic trifolium subterraneum l which expresses phya and releases extracellular phytase growth and p nutrition in laboratory media and soil
Plant Cell and Environment, 2004Co-Authors: Timothy S George, P A Hadobas, Alan Richardson, Richard J SimpsonAbstract:Transgenic Trifolium subterraneum expressing a phytase gene (phyA) from Aspergillus niger were generated. Five independently transformed lines showed an average 77-fold increase in exuded phytase activity in comparison with null segregant and wild-type controls. Unlike other phosphatases, exuded phytase activity was unaffected by P supply, verifying the constitutive expression of phyA. Transgenic T. subterraneum grown in agar with P supplied as phytate, took up 1.3- to 3.6-fold more P than controls and had equivalent P uptake to plants supplied with orthophosphate. This unique phenotype was compromised when the plants were grown in soil. None of the five lines showed increased shoot biomass or total P uptake in an unfertilized, low-P soil taken from under permanent pasture. With addition of P, one of the five transgenic lines had consistently greater P nutrition compared with control plants. Despite variable growth and P nutrition responses, P uptake per root length was on average greater for transgenic lines. Exudation of phytase by transgenic T. subterraneum allowed utilization of P from phytate in non-sorbing, sterile laboratory media, but was less effective when plants were grown in soil. Release of extracellular phytase is therefore not the only requirement for the acquisition of P from endogenous soil phytate by plants.
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extracellular secretion of aspergillus phytase from arabidopsis roots enables plants to obtain phosphorus from phytate
Plant Journal, 2001Co-Authors: Alan Richardson, P A Hadobas, Julie HayesAbstract:Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild-type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20-fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.
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phytate as a source of phosphorus for the growth of transgenic trifolium subterraneum
Plant nutrition: food security and sustainability of agro-ecosystems through basic and applied research. Fourteenth International Plant Nutrition Coll, 2001Co-Authors: Alan Richardson, P A Hadobas, Richard J SimpsonAbstract:Although phytate is an abundant form of soil organic P, plants generally have only a limited capacity to obtain phosphorus (P) directly from this substrate. Inability of plants to effectively use phytate-P is associated with poor substrate availability in soil environments, and insufficient extracellular root phytase activity. We therefore generated transgenic lines of Trifolium subterraneum L. that expressed the phytase (phyA) gene from Aspergillus niger. Analysis of segregating T1 populations of these plants showed that the presence of an extracellular phytase was effective in improving the P nutrition of the plants when supplied with phytate and grown in agar under sterile conditions.