Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization

Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.     

Purification and characterization of acid phosphatase from cotyledons of germinating soybean seeds

Soybean acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was completely separated from phytase (EC 3.1.3.8) isolated from cotyledons of germinating seeds and purified to homogeneity. A four-step purification regimen consisting of ammonium sulfate fractionation, and ion-exchange, affinity, and chromatofocusing gel chromatographies was employed to achieve a homogeneous preparation. Acid phosphatase activity appeared as a major band of the three forms of acid phosphatase identified on native gels. The purified enzyme had a molecular weight of 53,000 when electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular weight of 53,000 from its mobility in a Fracto-gel TSK HW-50F gel permeation column. The molar extinction coefficient of the enzyme at 278 nm was estimated to be 4.2 X 10(4) M-1 cm-1. The isoelectric point of the protein, as revealed by chromatofocusing, was about 6.7. The optimal pH for activity, like other plant acid phosphatases, was 5.0. While the enzyme failed to accommodate phytate as a substrate, the enzyme did exhibit a broad substrate selectivity. The affinity of the enzyme for p-nitrophenyl phosphate was high (Km = 70 microM), and activity was competitively inhibited by orthophosphate (Ki = 280 microM). The estimated catalytic turnover number (Kcat) of the enzyme for p-nitrophenyl phosphate was about 430 per second. Although the purified enzyme was stable at 0 degrees C and exhibited maximum catalytic activity at 60 degrees C, thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 68 degrees C for 10 min.     

Purification, N-terminal amino acid sequence and characterization of pH 2.5 optimum acid phosphatase (E.C. 3.1.3.2) from Aspergillus ficuum

An acid phosphatase from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using three ion exchange chromatographic steps. SDS-PAGE of the purified enzyme gave a single stained band at approximately 68-KDa. The mobility of the native enzyme in gel filtration chromatography, however, indicated that the molecular mass to be about 130-KDa implying the active form to be a dimer. On the basis of a molecular mass of 68-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 3.4 x 10(5) M-1 cm-1. The isoelectric point of the enzyme, as judged by chromatofocusing, was about 4.0. The purified enzyme is highly stable at 0 degree C. Thermal inactivation studies have indicated that the enzyme is unstable at 70 degrees C. The enzyme, however, exhibited a broad temperature optima with a maximum catalytic activity at 63 degrees C. The Km of the enzyme for p-nitrophenylphosphate is about 270 microM with an estimated turnover number of 2550 per sec. The enzyme is a glycoprotein as evidenced by the positive PAS staining; the sugar composition suggests the presence of N-linked high mannose-oligosaccharides. A partial N-terminal amino acid sequence up to the twenty-third residue was obtained. The enzyme was inhibited competitively by inorganic orthophosphate (Ki = 185 microM) and non-competitively by phosphomycin (Ki = 600 microM).     

Phytase produced on citric byproducts: purification and characterization

Citric pulp is an agro-industrial residue from the citrus processing industry with low inorganic phosphorus content applied in animal feed. A new bioprocess was developed to produce and purify a new phytase generated on citric pulp fermentation by Aspergillus niger FS3. The phytase was purified by cationic-exchange, anionic-exchange chromatography and chromatofocusing steps. From SDS–PAGE analysis, the molecular weight of the purified phytase was calculated to be 108 kDa. The phytase had an optimum pH of 5.0–5.5 and an optimum temperature of 60°C. The phytase displayed high affinity for phytate, and the K _m was 0.52 mM. The purified phytase was sufficiently able to withstand pelleting temperatures, retaining sufficiently high phytate-degrading activity.     

Purification and characterization of a methyltransferase from Aspergillus parasiticus SRRC 163 involved in aflatoxin biosynthetic pathway

A five step scheme has been developed for the purification of a methyltransferase (MT) from mycelia of 3-day old Aspergillus parasiticus (SRRC 163), which catalyzes one step in the aflatoxin biosynthetic pathway. The S-adenosylmethionine (SAM) requiring MT activity is essential for the conversion of sterigmatocystin (ST) to O-methylsterigmatocystin (OMST) prior to being converted to aflatoxin B1. The purification of the MT was carried out from cell-free extracts by CDR (Cell Debris Remover, a cellulosic weak anion exchanger, Whatman) treatment, QMA ACELL, Hydroxylapatite-Ultrogel, PBE 94 chromatofocusing and FractoGel TSK HW-50F filtration chromatography. The purified enzyme was only about 0.1% of the total extractable proteins. The pI of the protein was about 5.0 as judged by chromatofocusing. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 160-KDa. SDS-polyacrylamide gel electrophoresis revealed two protein subunit bands of molecular masses approximately 110-KDa and 58-KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.87 X 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5). The reaction catalyzed by the MT was optimum at pH 7.5 and between 25-35 degrees C. The Km of the enzyme for ST and SAM was determined to be 1.8 microM and 42 microM, respectively with an estimated turnover number of the enzyme for ST of 2.2 X 10(-2) per sec.     

Purification and Characterization of a Bacterial Phytase Whose Properties Make it Exceptionally Useful as a Feed Supplement

A periplasmatic phytase from a bacterium isolated from Malaysian waste water was purified about 173-fold to apparent homogeneity with a recovery of 10% referred to the phytase activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 65°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K _M = 0.15 mmol/l and k _cat = 1164 s⁻¹ at pH 4.5 and 37°C. The purified enzyme was shown to be highly specific. Among the phosphorylated compounds tested, phytate was the only one which was significantly hydrolysed. Some properties such as considerable activity below pH 3.0, thermal stability and resistance to pepsin make the enzyme attractive for an application as a feed supplement.     

Purification and Biochemical Characterization of Phytase Enzyme from <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> (<Emphasis Type="Italic">MH121153</Emphasis>)

Phytase (myo-inositol hexaphosphate phosphohydrolase) belongs to phosphatases. It catalyzes the hydrolysis of phytate to less-phosphorylated inorganic phosphates and phytate. Phytase is used primarily for the feeding of simple hermit animals in order to increase the usability of amino acids, minerals, phosphorus and energy. In the present study, phytase isolation from the Lactobacillus coryniformis strain, isolated from Lor cheese sources, phytase purification and characterization were studied. The phytase was purified in simple three steps. The enzyme was obtained with 2.60% recovery and a specific activity of 202.25 (EU/mg protein). The molecular mass of the enzyme was determined to be 43.25 kDa with the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The optimum temperature and pH for the enzyme were found as 60 °C and 5.0 and respectively. To defined the substrate specificity of the phytase, the hydrolysis of several phosphorylated compounds by the purified enzyme was studied and sodium phytate showed high specificity. Furthermore, the effects of Ca²⁺, Ag⁺, Mg²⁺, Cu²⁺, Co²⁺, Pb²⁺, Zn²⁺ and Ni²⁺ metal ions on the enzyme were studied.     

Characterization,Gene Cloning,and Sequencing of a Fungal Phytase,PhyA, From Penicillium oxalicum PJ3

A phytase from Penicillium oxalicum PJ3, PhyA, was purified near to homogeneity with 427-fold increase in specific phytase activity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis of the purified enzyme indicated an estimated molecular mass of 65 kD. The optimal pH and temperature of the purified enzyme were pH 4.5 and 55°C, respectively. The enzyme activity was strongly inhibited by Ca²⁺, Cu²⁺, Zn²⁺, and phenylmethylsulfonyl fluoride (PMSF). The K_m value for sodium phytate was 0.545 mM with a V_max of 600 U/mg of protein. The phyA gene was cloned, and it contains an open reading frame of 1,383 with a single intron (118 bp), and encodes a protein of 461 amino acids.     

Cloning, Expression, and Enzyme Characterization of an Acid Heat-Stable Phytase from Aspergillus fumigatus WY-2

A novel thermostable phytase gene was cloned from Aspergillus fumigatus WY-2. It was 1459 bp in size and encoded a polypeptide of 465 amino acids. The gene was expressed in Pichia pastoris GS115 as an extracellular enzyme. The expressed enzyme was purified to homogeneity and biochemically characterized. The purified enzyme had a specific activity of 51 U/mg with an approximate molecular mass of 88 kDa. The optimum pH and temperature for activity were pH 5.5 and 55°C, respectively. After incubation at 90°C for 15 min, it still remained at 43.7% of the initial activity. The enzyme showed higher affinity for sodium phytate than other phosphate conjugates, and the K_m and K_cat for sodium phytate were 114 μM and 102 s⁻¹, respectively. Incubated with pepsin at 37°C for 2 h at the ratio (pepsin/phytase, wt/wt) of 0.1, it still retained 90.1% residual activity. These exceptional properties give the newly cloned enzyme good potential in animal feed applications.     

Purification and characterization of phytase from cotyledons of germinating soybean seeds

Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.     

A novel extracellular low‐temperature active phytase from Bacillus aryabhattai RS1 with potential application in plant growth

Bacillus aryabhattai RS1 isolated from rhizosphere produced an extracellular, low temperature active phytase. The cultural conditions for enzyme production were optimized to obtain 35 U mL^?1 of activity. Purified phytase had specific activity and molecular weight of 72.97 U mg^?1 and ～40 kDa, respectively. The enzyme was optimally active at pH 6.5 and 40°C and was highly specific to phytate. It exhibited higher catalytic activity at low temperature, retaining over 40% activity at 10°C. Phytase was more thermostable in presence of Ca²⁺ ion and retained 100% residual activity on preincubation at 20–50°C for 30 min. Partial phytase encoding gene, phy_B (816 bp) was cloned and sequenced. The encoded amino acid sequence (272 aa) contained two conserved motifs, DA[A/T/E]DDPA[I/L/V]W and NN[V/I]D[I/L/V]R[Y/D/Q] of β‐propellar phytase and had lower sequence homology with other Bacillus phytases, indicating its novelty. Phytase and the bacterial inoculum were effective in improving germination and growth of chickpea seedlings under phosphate limiting condition. Moreover, the potential applications of the enzyme with relatively high activity at lower temperatures (20–30°C) could also be extended to aquaculture and food processing. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:633–641, 2017     

Purification and biochemical properties of an extracellular acid phytase produced by the Saccharomyces cerevisiae CY strain

An extracellular acid phytase was purified to homogeneity from the culture supernatant of the Saccharomyces cerevisiae CY strain by ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 630 kDa by gel filtration. Removing the sugar chain by endoglycosidase H digestion revealed that the molecular mass of the protein decreased to 446 kDa by gel filtration and gave a band of 55 kDa by SDS-PAGE. The purified enzyme was most active at pH 3.6 and 40 °C and was fairly stable from pH 2.5 to 5.0. The phytase displayed broad substrate specificity and had a K_m value of 0.66 mM (sodium phytate, pH 3.6, 40 °C). The phytase activity was completely inhibited by Fe³⁺ and Hg²⁺, and strongly inhibited (maximum of 91%) by Ba²⁺, Co²⁺, Cu⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Sn²⁺ at 5 mM concentrations.     

弗氏柠檬酸杆菌植酸酶的分离纯化及其酶学性质研究

从弗氏柠檬酸杆菌(Citrobacter freundii)中分离纯化了一种植酸酶并进行了酶学性质研究,其反应最适pH为4.0~4.5,最适温度为40℃,在37℃下以植酸钠为底物的Km值为0.85nmol/L,Vmax为0.53IU/(mg.min),具有较好的抗胰蛋白酶的能力。酶蛋白的分子量大小约为45kDa,成熟酶蛋白N端序列为QCAPEGYQLQQVLMM。     

Purification and characterization of a phytase from Pseudomonas syringae MOK1

A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40 degrees C. The Michaelis constant (Km) and maximum reaction rate (Vmax) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu2+, Cd2+, Mn2+, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal.     

A Thermostable phytase from <Emphasis Type="Italic">Neosartorya spinosa</Emphasis> BCC 41923 and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K _m and V _max for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe²⁺, Fe³⁺, and Al³⁺. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).     

Purification and Some Properties of ATP: Nucleotide Pyrophospho-transferase of Streptomyces adephospholyticus

ATP: nucleotide pyrophosphotransferase was purified from culture filtrate of Streptomyces adephospholyticus A–4668 about 13,000 fold by the method including ammonium sulfate fractionation, Amberlite IRC–50 treatment and column chromatography with DEAE-cellulose, DEAE-Sephadex A–25, SP-Sephadex C–25 and Sephadex G–75. The purified enzyme was homogenous on disk gel electrophoresis and ultracentrifugation and the specific activity was 915 units per mg protein, The molecular weight was determined as 28,000 by gel filtration on Sephadex G–75. The enzyme was found to be stable in the pH range of 5.5 to 10.5. More than 80% of the activity was remained after heating at 60°C for 30 min. The enzyme exhibited maximum activity at 50°C.     

Purification and characterization of extracellular phytase from Aspergillus niger ATCC 9142

Extracellular phytase produced by Aspergillus niger ATCC 9142 was purified to homogeneity by employing an initial ultrafiltration step, followed by chromatography using ion exchange, gel filtration and chromatofocusing steps. The purified enzyme was an 84 kDa, monomeric protein. It possessed a temperature optimum of 65 degrees C, and a pH optimum of 5.0. Km and Vmax values of 100 microM and 7 nmol/s, respectively, were recorded and these values fall well within the range of those previously reported for microbial phytases. Substrate specificity studies indicated that, while the enzyme could hydrolyse a range of non-phytate-based phosphorylated substrates, its preferred substrate was phytate. Phytase activity was moderately stimulated in the presence of Mg2+, Mn2+, Cu2+, Cd2+, Hg2+, Zn2+ and F- ions. Activity was not significantly affected by Fe2- or Fe3- and was moderately inhibited by Ca2+. The enzyme displayed higher thermostability at 80 degrees C than did two commercial phytase products. Initial characterisation of the purified enzyme suggested that it could be a potential candidate for use as an animal feed supplement.     

Identification of two calcineurin alpha isoforms in bovine brain by two different monoclonal antibodies.

Calcineurin (calcium- and calmodulin-stimulated phosphatase) alpha subunit purified from bovine brain was found to be composed of two polypeptides, 61 KDa (alpha 1) and 59 KDa (alpha 2). The two peptides were separated and extracted from polyacrylamide gel. The immuno-peptide mapping of the purified peptides by partial proteolysis showed that the 59-KDa polypeptide was not a degradative product of the 61-KDa polypeptide. The interaction of the enzyme with two monoclonal antibodies, Vj6 and Vd3, raised against bovine brain calcineurin revealed that the 61-KDa polypeptide was recognized by both Vj6 and Vd3, whereas the 59-KDa one was recognized only by Vj6. These results indicate that there are at least two isoforms of calcineurin alpha subunits in bovine brain.     

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Schistosoma mansoni. A potential target for chemotherapy

Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities are essential for the supply of guanine nucleotides in Schistosoma mansoni schistosomules. In crude extracts of adult S. mansoni, these two activities co-elute in size exclusion, ion exchange, and chromatofocusing chromatography and exhibit similar stabilities to heat treatment, suggesting that they are associated in one enzyme protein hypoxanthine-guanine phosphoribosyltransferase. This enzyme has been purified by a combination of heat treatment at 85 degrees C and chromatofocusing chromatography with elution at an apparent pI of 5.27 +/- 0.15. Pore gradient electrophoresis of the native enzyme followed by subsequent activity staining demonstrate an enzyme molecular weight of 105,000. The activity staining pattern remains the same whether hypoxanthine or guanine is used as the substrate, further supporting the existence of a single protein, hypoxanthine-guanine phosphoribosyltransferase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein results in a single protein band with a subunit molecular weight estimate of 64,000, suggesting that the native enzyme is a dimer. Preliminary kinetic studies showed that the purified hypoxanthine-guanine phosphoribosyltransferase reacted with guanine at a rate twice as fast as it did with hypoxanthine, but it did not act on xanthine at all. A full-length mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase cDNA clone pHPT5 and a plasmid pSV2-gpt containing the xanthine-guanine phosphoribosyltransferase gene for Escherichia coli were utilized as probes on Southern blots of S. mansoni DNA digests, and no significant hybridization was found under relatively relaxed conditions. Polyclonal antibodies made against human erythrocyte hypoxanthine-guanine phosphoribosyltransferase and E. coli xanthine-guanine phosphoribosyltransferase were tested in enzyme-linked immunosorbent assays of S. mansoni protein extracts, and no detectable cross-reacting protein was found. S. mansoni hypoxanthine-guanine phosphoribosyltransferase thus may bear rather limited homology to mammalian hypoxanthine-guanine phosphoribosyltransferase or bacterial xanthine-guanine phosphoribosyltransferase and could be an attractive target for antischistosomal chemotherapeutic drug design.     

Purification and Properties of Extracellular Acid Phosphatase from Zizania latifolia-pavasite,stilago esculenta

An extracellular acid phosphatase from Ustilago esculenta was purified to homogeneity on the basis of polyacrylamide gel electrophoresis. It was a glycoprotein with an isoelectric point of 4.7. The molecular weight of the enzyme was estimated to be about 343,000 by gel filtration on Sephadex G-200, whereas on SDS-polyacrylamide gel electrophoresis, the enzyme gave a single protein band with a molecular weight of 116,000. This result suggests that the enzyme consists of three identical subunits. The enzyme showed an optimum activity at pH 4.5, retained 90% of its activity for 10 min at 55°C and had a Km value of 0.25 mm for p-nitrophenylphosphate. No definite substrate specificity of the enzyme was observed.