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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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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, Ralf Greiner, Astrid Beinhauer, Liia R Valeeva, Inna B Chastukhina, N P Balaban, Eugene V Shakirov, 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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phytase production using citric pulp and other residues of the agroindustry in ssf by fungal isolates
Food Technology and Biotechnology, 2008Co-Authors: Michele Rigon Spier, Ralf Greiner, Jose A Rodriguezleon, Adenise Lorenci Woiciechowski, Ashok Pandey, Vanete Thomaz Soccol, Carlos Ricardo SoccolAbstract:Summary Phytases have important applications in human and animal nutrition because they hydrolyze the phytate present in legumes, cereal grains and oil seeds. This results in an increased availability of minerals, trace elements and amino acids as well as phosphate. Fifty potential phytase-producing fungal strains were isolated from a fertile soil obtained from the northern part of Parana State in Brazil and other alternative sources using a selective media. Thereafter phytase production was evaluated in solid-state fermentation using different residues from the agroindustry supplemented with a nitrogen source at 60 % of moisture after 96 hours at 30 °C. The highest phytase activity (51.53 units per gram of dry substrate, U/g) was achieved with citric pulp and the soil isolate FS3 in solid-state fermentation. Furthermore, treatment of the substrates prior to fermentation in order to reduce microbial contamination was shown to affect phytase production during solid-state fermentation. Heat treatment resulted in an increase of the concentration of inorganic phosphate, a well known repressor of microbial phytase production, and therefore in a reduction of phytase production. UV exposure of the substrate was shown to reduce microbial contamination without affecting phytase production.
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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.
T Satyanarayana - One of the best experts on this subject based on the ideXlab platform.
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Plant Growth Promotion by an Extracellular HAP-Phytase of a Thermophilic Mold Sporotrichum thermophile
Applied Biochemistry and Biotechnology, 2010Co-Authors: Bijender Singh, T SatyanarayanaAbstract:Phytase of the thermophilic mold Sporotrichum thermophile Apinis hydrolyzed and liberated inorganic phosphate from Ca^+2, Mg^+2, and Co^+2 phytates more efficiently than those of Al^3+, Fe^2+, Fe^3+, and Zn^2+. The hydrolysis rate was higher at 60 °C as compared to 26 °C. Among all the organic acids tested, citrate was more effective in enhancing solubilization of insoluble phytate salts by phytase than others. The dry weight and inorganic phosphate contents of the wheat plants were high when supplemented with phytase or fungal spores. The plants provided with 5 mg phytate per plant exhibited enhanced growth and inorganic phosphate. With increase in the dosage of phytase, there was increase in growth and inorganic phosphate of plants, the highest being at 20 U per plant. The compost made employing the combined native microflora of the wheat straw and S . thermophile promoted growth of the plants. The plant-growth-promoting effect was also higher with the compost made using S. thermophile than that from only the native microflora.
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characterization of a hap phytase from a thermophilic mould sporotrichum thermophile
Bioresource Technology, 2009Co-Authors: Ijende Singh, T SatyanarayanaAbstract:Abstract The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ∼456 kDa and p I of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T 1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol −1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg 2+ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate ( V max ) and apparent Michaelis–Menten constant ( K m ) for sodium phytate were 83 nmol mg −1 s −1 and 0.156 mM, respectively. The catalytic turnover number ( K cat ) and catalytic efficiency ( K cat / K m ) of phytase were 37.8 s −1 and 2.4 × 10 5 M −1 s −1 , respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.
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improved phytase production by a thermophilic mould sporotrichum thermophile in submerged fermentation due to statistical optimization
Bioresource Technology, 2008Co-Authors: Ijende Singh, T SatyanarayanaAbstract:Abstract Culture variables affecting phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation were optimized. Soluble starch, peptone, Tween-80 and sodium phytate were identified by Plackett–Burman design as the most significant factors to affect phytase production. The 24 full factorial central composite design of response surface methodology was applied for optimizing the concentrations of the significant variables and to delineate their interactions. Starch, Tween-80, peptone and sodium phytate at 0.4%, 1.0%, 0.3% and 0.3% supported maximum enzyme titres, respectively. An overall 3.73-fold improvement in phytase production was achieved due to optimization. When sodium phytate was substituted with wheat bran (3%), the phytase titre in the former was comparable with that in the latter.
Henry L. Classen - One of the best experts on this subject based on the ideXlab platform.
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in vitro hydrolysis of phytate in canola meal with purified and crude sources of phytase
Animal Feed Science and Technology, 1998Co-Authors: Ryan W. Newkirk, Henry L. ClassenAbstract:Pre-treatment conditions required to hydrolyze phytate in canola meal with crude and purified phytases were investigated. Phytase (10,000, 100,000, 340,000 U/kg Natuphos®) and the same phytase (10,000, 100,000 U/kg) that had been purified by gel filtration were added to canola meal without pH adjustment (pH 5.8, 2:1 H2O: meal). Phytate hydrolysis was incomplete after 30 and 60 min (50°C) indicating that a portion of the phytate was resistant to hydrolysis. Purified and crude phytases (10,000 U/kg) were used to hydrolyze canola meal at pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and 5.8. Phytase was more effective between pH 3 and 5. Crude phytase was significantly more effective (P<0.0001) than purified enzyme, suggesting that non phytase enzymes facilitated the action of phytase. The levels of lower inositol phosphates in response to incubation pH were similar to phytate, suggesting that they too are susceptible to the formation of complexes which prevent hydrolysis. Complete hydrolysis with crude phytase (pH 5.0, 5000 U/kg, 50°C, 2:1 H2O: meal) in 250 g batches was accomplished within 23 h. In conclusion, a portion of the phytate in canola meal is resistant to hydrolysis by phytase. Therefore, meal pretreatment, which includes pH modification, temperature and moisture control, and the addition of phytase and other enzymes, is required for the effective hydrolysis of phytate.
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Phytase activity in the small intestinal brush border membrane of the chicken. Poult. Sci
1998Co-Authors: David D Maenz, Henry L. ClassenAbstract:ABSTRACT The kinetics, mineral dependency, and pH dependency of phytate hydrolysis by preparations of chicken small intestinal brush border membrane vesicles were determined. Substantial phytate hydrolysis occurred over the pH range from 5 to 6.5 with a maximum hydrolysis at pH of 6. Inclusion of 25 mM MgCl2 in the media doubled the rate of phytate hydrolysis. The brush border was shown to have no nonspecific acid phosphatase activity and excess phytate had no effect on alkaline phosphatase activity at pH 11. Under optimal conditions of pH 6 plus 25 mM MgCl2, a kinetic model of a single Michaelis-Menten type of enzymatic activity with a Km of 0.160 ± 0.008 mM and a Vmax of 42.5 ± 1.0 nmol/mg vesicle protein per min plus a small unsaturable component converged to the data (P < 0.05). The specific and total activities of intestinal brush border phytase were highest in the duodenum (P < 0.05) and decreased progressively down the length of the gut. Intestinal brush border vesicles prepared from broiler chicks and mature laying hens had comparable specific phytase activity. However, the total activity of brush border phytase was 35 % higher in the small intestine of laying hens (P < 0.05). Intestinal brush border phytase could contribute to phytate-phosphorus digestibility and may be subject to regulation in response to the dietary phosphorus and vitamin D status of the chicken
H L Classen - One of the best experts on this subject based on the ideXlab platform.
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the effect of minerals and mineral chelators on the formation of phytase resistant and phytase susceptible forms of phytic acid in solution and in a slurry of canola meal
Animal Feed Science and Technology, 1999Co-Authors: David D Maenz, Carmen M Engeleschaan, Rex W Newkirk, H L ClassenAbstract:Abstract Minerals can readily bind to phytic acid and thus have the potential to form mineral–phytate complexes that may be resistant to hydrolysis by phytase activity of animal, plant and microbial origin. In simple solution, at pH 7.0, mineral concentrations from 0.053mM for Zn 2+ up to 4.87mM for Mg 2+ caused a 50% inhibition of phytate-P hydrolysis by microbial phytase. The rank order of mineral potency as inhibitors of phytate hydrolysis was Zn 2+ ⪢Fe 2+ >Mn 2+ >Fe 3+ >Ca 2+ >Mg 2+ at neutral pH. Acidification of the media to pH 4.0 decreased the inhibitory potency of all of the divalent cations tested. The inhibitory potency of Fe 3+ showed a moderate increase with declining pH. Inclusion of 25mM ethylenediamine-tetraacetic acid (EDTA) completely blocked Ca 2+ inhibition of phytate hydrolysis at pH 7. Inorganic P comprised 0.20–0.25 of the total P in a slurry of canola meal. Incubation with microbial phytase increased inorganic P up to 0.50 of total P levels. Supplementation with chelators such as EDTA, citrate and phthalate increased the efficacy of microbial phytase in hydrolyzing phytic acid. Incubation of canola meal with 100mM phthalic acid plus microbial phytase resulted in complete hydrolysis of phytate-P. Competitive chelation by compounds such as EDTA, citric acid or phthalic acid has the potential to decrease enzyme-resistant forms of phytic acid and thereby improve the efficacy of microbial phytase in hydrolyzing phytic acid.
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phytase activity in the small intestinal brush border membrane of the chicken
Poultry Science, 1998Co-Authors: D D Maenz, H L ClassenAbstract:The kinetics, mineral dependency, and pH dependency of phytate hydrolysis by preparations of chicken small intestinal brush border membrane vesicles were determined. Substantial phytate hydrolysis occurred over the pH range from 5 to 6.5 with a maximum hydrolysis at pH of 6. Inclusion of 25 mM MgCl2 in the media doubled the rate of phytate hydrolysis. The brush border was shown to have no nonspecific acid phosphatase activity and excess phytate had no effect on alkaline phosphatase activity at pH 11. Under optimal conditions of pH 6 plus 25 mM MgCl2, a kinetic model of a single Michaelis-Menten type of enzymatic activity with a Km of 0.160 +/- 0.008 mM and a Vmax of 42.5 +/- 1.0 nmol/mg vesicle protein per min plus a small unsaturable component converged to the data (P < 0.05). The specific and total activities of intestinal brush border phytase were highest in the duodenum (P < 0.05) and decreased progressively down the length of the gut. Intestinal brush border vesicles prepared from broiler chicks and mature laying hens had comparable specific phytase activity. However, the total activity of brush border phytase was 35% higher in the small intestine of laying hens (P < 0.05). Intestinal brush border phytase could contribute to phytate-phosphorus digestibility and may be subject to regulation in response to the dietary phosphorus and vitamin D status of the chicken.
Ijende Singh - One of the best experts on this subject based on the ideXlab platform.
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characterization of a hap phytase from a thermophilic mould sporotrichum thermophile
Bioresource Technology, 2009Co-Authors: Ijende Singh, T SatyanarayanaAbstract:Abstract The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ∼456 kDa and p I of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T 1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol −1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg 2+ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate ( V max ) and apparent Michaelis–Menten constant ( K m ) for sodium phytate were 83 nmol mg −1 s −1 and 0.156 mM, respectively. The catalytic turnover number ( K cat ) and catalytic efficiency ( K cat / K m ) of phytase were 37.8 s −1 and 2.4 × 10 5 M −1 s −1 , respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.
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improved phytase production by a thermophilic mould sporotrichum thermophile in submerged fermentation due to statistical optimization
Bioresource Technology, 2008Co-Authors: Ijende Singh, T SatyanarayanaAbstract:Abstract Culture variables affecting phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation were optimized. Soluble starch, peptone, Tween-80 and sodium phytate were identified by Plackett–Burman design as the most significant factors to affect phytase production. The 24 full factorial central composite design of response surface methodology was applied for optimizing the concentrations of the significant variables and to delineate their interactions. Starch, Tween-80, peptone and sodium phytate at 0.4%, 1.0%, 0.3% and 0.3% supported maximum enzyme titres, respectively. An overall 3.73-fold improvement in phytase production was achieved due to optimization. When sodium phytate was substituted with wheat bran (3%), the phytase titre in the former was comparable with that in the latter.
