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Ulrich Schwaneberg - One of the best experts on this subject based on the ideXlab platform.
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Directed evolution of an acid Yersinia mollaretii Phytase for broadened activity at neutral pH
Applied Microbiology and Biotechnology, 2018Co-Authors: Georgette Körfer, Catalina Novoa, Janina Kern, Elisabeta Balla, Carolin Grütering, Mehdi D. Davari, Ronny Martinez, Ljubica Vojcic, Ulrich SchwanebergAbstract:Phytases are phosphohydrolases that initiate the sequential hydrolysis of phosphate from phytate, which is the main storage form of phosphorous in numerous plant seeds, especially in cereals and grains. Phytate is indigestible for most monogastric animals, such as poultry, swine, fish, and humans; therefore, microbial Phytases have been widely used in plant (specially soy)-based animal feeding to improve nutrition by enhanced phosphorus, mineral, and trace element absorption, and reducing phosphorus pollution by animal waste. Most Phytases used as animal feed additives have an acid pH optimum (pH 2.5 and 5.5 for Aspergillus and pH 4.5 for E. coli Phytases) and show a sharp decrease in performance at neutral pH, correlating with intestinal digestion. Directed evolution of Phytases has been previously reported to improve enzyme thermostability, pH, or specific activity. In this manuscript, we report a directed evolution campaign of the highly active bacterial Phytase from Yersinia mollaretii (YmPh) towards a broadened pH activity spectrum. Directed evolution identified the key positions T44 and K45 for increased YmPh activity at neutral pH. Both positions are located in the active site loop of the Phytase and have a synergistic effect on activity with a broadened pH spectrum. Kinetic characterization of the improved variants, YmPh-M10 and -M16, showed up to sevenfold increased specific activity and up to 2.2-fold reduced K_half at pH 6.6 under screening conditions compared to Yersinia mollaretii Phytase wild type (YmPhWT).
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Recent Advances in Directed Phytase Evolution and Rational Phytase Engineering
Directed Enzyme Evolution: Advances and Applications, 2017Co-Authors: Amol V. Shivange, Ulrich SchwanebergAbstract:Phytases are hydrolytic enzymes that initiate stepwise removal of phosphate from phytate. Phytate is the major phosphorous storage compound in cereal gains, oilseeds, and legumes and is indigestible by monogastric animals such as poultry and swine. Supplementation of Phytase in animal feed proved to improve animal nutrition and decrease phosphorous pollution. Several Phytases were discovered in the last century, and today a highly competitive market situation emerged the demands for Phytases that are redesigned to excellently match industrial demands. Phytase engineering by directed evolution and rational design has offered a robust approach to tailor-made Phytases with high specific activity, broad thermal and pH profile, and protease resistance. In this chapter, we summarized challenges and successful approaches employed in Phytase engineering. Factors influencing Phytase thermostability, pH stability, pH optima, and protease resistance have been discussed with respect to structural perspective and potential molecular mechanism for improvement. Importance of cooperative substitutions and a way to identify these interactions are discussed. Recent development in screening technology and molecular insights in combining key beneficial substitutions are detailed. In addition, strategies and approaches for rapid and efficient evolution of Phytases and to understand structure function relationships on a molecular level have been proposed.
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directed evolution of a highly active yersinia mollaretii Phytase
Applied Microbiology and Biotechnology, 2012Co-Authors: Amol V. Shivange, Stefan Haefner, Ulrich Schwaneberg, Annegret Serwe, Alexander Dennig, Danilo RoccatanoAbstract:Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed Phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii Phytase (YmPhytase). YmPhytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073 U/mg which is ∼10 times higher than widely used fungal Phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active Phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ∼8,400 clones) a Phytase variant with ∼20% improved thermostability (58°C for 20 min; residual activity wild type ∼34%; variant ∼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993 U/mg).
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 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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distribution of Phytase activity total phosphorus and phytate phosphorus in legume seeds cereals and cereal by products as influenced by harvest year and cultivar
Animal Feed Science and Technology, 2007Co-Authors: Tobias Steiner, Ralf Greiner, R Mosenthin, B Zimmermann, S RothAbstract:Samples of legume seeds, cereals and cereal by-products (n = 113) grown in south-western Germany and originating from different cultivars and harvest years were analyzed for Phytase activity, total phosphorus (P) and phytate P. Phytase activities determined by means of a direct incubation method were lowest in legume seeds and oats (262–496 U/kg dry matter), intermediate in cereals (except oats) (2323–6016 U/kg DM) and highest in cereal by-products (9241–9945 U/kg DM). However, the application of an extraction procedure for the determination of Phytase activities in legume seeds resulted in values below the detection limit of 50 U/kg. On average, about 0.67 of total P in legume seeds, cereals and their by-products is bound to phytate. There was a significant influence (P<0.001) of harvest year (1998–2000) on phytate P contents in wheat. Furthermore, total P and phytate P concentrations differed (P<0.05) between different cultivars of wheat. Moreover, Phytase activities differed (P=0.023) between different cultivars of barley. Total P and phytate P concentrations were highly correlated in legume seeds (r = 0.95) and cereal by-products (r = 0.96) and, to a smaller extent, in cereals (r = 0.66). Milling of cereal grains to bran and flour revealed that Phytase activity, total P and phytate P are highly concentrated (P<0.001) in the outer grain layers of cereals. The influence of preservation of intact legume seeds with propionic acid over a period of 4, 8 or 12 weeks resulted only in a marginal decrease in Phytase activity. Due to high native Phytase activities in cereals (except oats) and their by-products these feedstuffs may contribute substantially to the gastrointestinal hydrolysis of phytate in non-ruminant animals, whereas the contribution of native Phytases originating from legume seeds in terms of improving the availability of plant P seems to be rather limited.
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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
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extracellular Phytase activity of bacillus amyloliquefaciens fzb45 contributes to its plant growth promoting effect
Microbiology, 2002Co-Authors: Elsorra E Idriss, Ralf Greiner, Oliwia Makarewicz, Abdelazim Farouk, Kristin Rosner, Helmut Bochow, Thomas Richter, Rainer BorrissAbstract:Several Bacillus strains belonging to the B. subtilis/amyloliquefaciens group isolated from plant-pathogen-infested soil possess plant-growth-promoting activity [Krebs, B. et al. (1998) R26 J Plant Dis Prot 105, 181–197]. Three out of the four strains investigated were identified as B. amyloliquefaciens and were able to degrade extracellular phytate (myo-inositol hexakisphosphate). The highest extracellular Phytase activity was detected in strain FZB45, and diluted culture filtrates of this strain stimulated growth of maize seedlings under phosphate limitation in the presence of phytate. The amino acid sequence deduced from the Phytase phyA gene cloned from FZB45 displayed a high degree of similarity to known Bacillus Phytases. Weak similarity between FZB45 Phytase and B. subtilis alkaline phosphatase IV pointed to a possible common origin of these two enzymes. The recombinant protein expressed by B. subtilis MU331 displayed 3(1)-Phytase activity yielding D/L-Ins(1,2,4,5,6)P5 as the first product of phytate hydrolysis. A Phytase-negative mutant strain, FZB45/M2, whose phyA gene is disrupted, was generated by replacing the entire wild-type gene on the chromosome of FZB45 with a km::phyA fragment, and culture filtrates obtained from FZB45/M2 did not stimulate plant growth. In addition, the growth of maize seedlings was promoted in the presence of purified Phytase and the absence of culture filtrate. These genetic and biochemical experiments provide strong evidence that Phytase activity of B. amyloliquefaciens FZB45 is important for plant growth stimulation under phosphate limitation.
Eugene V Shakirov - One of the best experts on this subject based on the ideXlab platform.
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Histidine Acid Phytases of Microbial Origin
Microbiology, 2018Co-Authors: Nelly P. Balaban, Aliya D Suleimanova, Eugene V Shakirov, Margarita R. SharipovaAbstract:— This review is focused on analysis of the biological diversity of Phytase-producing microorganisms capable of degrading phytate to inorganic phosphate. General approaches to microbial Phytase classification are discussed, with a particular emphasis on histidine acid Phytases (HAPs), which catalyze specific cleaving of myo -inositol hexakisphosphate. The effect of glycosylation and various effectors on enzyme thermostability and activity of Phytases are described. The data on the biosynthesis of histidine acid Phytases, their substrate specificity, and on the mechanism of myo -inositol hexakisphosphate hydrolysis are considered. A conclusion is made concerning the biotechnological potential of this group of microbial enzymes.
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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.
Byungchul Oh - One of the best experts on this subject based on the ideXlab platform.
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supplementation of alkaline Phytase ds11 in whole wheat bread reduces phytate content and improves mineral solubility
Journal of Food Science, 2011Co-Authors: Yunjong Park, Kihwan Park, Jiwon Park, Byungchul OhAbstract:: In this study, alkaline Phytase was added to whole-wheat bread and the phytate content and mineral profiles were compared to commercially available acidic Phytase. At neutral pH, some phytate (approximately 20%) was degraded by endogenous Phytase in wheat flour, while 40% of phytate was hydrolyzed by alkaline Phytase DS11 and a 35% reduction was observed with acidic Phytase. Most of the enzymatic activity occurred during the proofing stage, and the rate of reaction depended on pH. DS11 Phytase effectively degraded the phytate level within a 30 min treatment at pH 7; however, at least 60 min was needed with acidic Phytase to achieve the same hydrolysis level. Mineral profiles were also dramatically affected by the phytate reduction. The biggest increase was observed in Fe2+ by the Phytase treatment. The Fe2+ content increased 10-fold at pH 7 and 8-fold at pH 5 with alkaline Phytase DS11. Alkaline Phytase DS11 was shown to be effective at phytate reduction in whole-wheat bread preparation. Additionally, phytate degradation enhanced the mineral availability of bread.
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Supplementation of Alkaline Phytase (Ds11) in Whole‐Wheat Bread Reduces Phytate Content and Improves Mineral Solubility
Journal of Food Science, 2011Co-Authors: Yunjong Park, Kihwan Park, Jiwon Park, Byungchul OhAbstract:: In this study, alkaline Phytase was added to whole-wheat bread and the phytate content and mineral profiles were compared to commercially available acidic Phytase. At neutral pH, some phytate (approximately 20%) was degraded by endogenous Phytase in wheat flour, while 40% of phytate was hydrolyzed by alkaline Phytase DS11 and a 35% reduction was observed with acidic Phytase. Most of the enzymatic activity occurred during the proofing stage, and the rate of reaction depended on pH. DS11 Phytase effectively degraded the phytate level within a 30 min treatment at pH 7; however, at least 60 min was needed with acidic Phytase to achieve the same hydrolysis level. Mineral profiles were also dramatically affected by the phytate reduction. The biggest increase was observed in Fe2+ by the Phytase treatment. The Fe2+ content increased 10-fold at pH 7 and 8-fold at pH 5 with alkaline Phytase DS11. Alkaline Phytase DS11 was shown to be effective at phytate reduction in whole-wheat bread preparation. Additionally, phytate degradation enhanced the mineral availability of bread.
Lia R. Valeeva - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Arabidopsis thaliana Plants Expressing Bacterial Phytase
Russian Journal of Plant Physiology, 2019Co-Authors: Lia R. Valeeva, Chuluuntsetseg Nyamsuren, E. V. Shakirov, Margarita R. SharipovaAbstract:Transgenic plants containing genes of bacterial Phytases represent one of the promising ways to solve the problem of phosphorus deficiency in the nutrition of plants and monogastric animals. Histidine acid Phytase PaPhyC from Pantoea agglomerans has a high activity and represents a promising basis for the biotechnology of plants. In this study, the analysis of morphological characteristics, Phytase activity, and phosphorus content in tissues of the earlier obtained, genetically modified Arabidopsis thaliana (L.) Heynh. plants producing extracellular Phytase (PaPhyC) has been carried out. According to the obtained results, modified plants are able to grow on a medium supplemented with phytate as the sole source of phosphorus. Exterior characteristics (rosette diameter and area) of Phytase-expressing plants grown on media containing phytate or inorganic phosphorus do not differ, which confirms that the plants use phytate as the phosphorus source. In the case of plant cultivation on a phytate-containing medium, a high Phytase activity is observed in the cell walls of modified plants. The content of inorganic phosphorus in tissues of modified plants does not change in the case of their cultivation on the medium containing phytate as the sole source of phosphorus.
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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.