title: Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance.
abstract: An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The K M for sodium phytate hydrolysis was 30.9 mM, while the k cat and k cat / K M were 1.46 ×10 (5) s (-1) and 4.7 × 10 (6) s (-1) .M (-1) , respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg (2+) , Cd (2+) , K (+) and Ca (2+) , and it was drastically inhibited by F (-) . The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t 1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.
authors.affiliation: Instituto de Ciências Agrárias, Universidade Federal de Viçosa, Rio Paranaíba, MG, Brasil. ; Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brasil.
authors.forename: Paulo S
authors.initials: PS
authors.lastname: Monteiro
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brasil.
authors.forename: Valéria M
authors.initials: VM
authors.lastname: Guimarães
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brasil.
authors.forename: Ricardo R
authors.initials: RR
authors.lastname: de Melo
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brasil.
authors.forename: Sebastião T
authors.initials: ST
authors.lastname: de Rezende
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism; D055503:Protein Multimerization; D059748:Proteolysis; D013379:Substrate Specificity; D013696:Temperature; D014462:Ultrafiltration
chemical_list: D004791:Enzyme Inhibitors; D010833:Phytic Acid; D010832:6-Phytase; D010447:Peptide Hydrolases
keywords: dephosphorylation; phosphatase; phytic acid