title: Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization.
abstract: 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.
mesh_terms: D010832:6-Phytase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D000118:Acetylglucosaminidase; D000911:Antibodies, Monoclonal; D001230:Aspergillus / Q000201:enzymology*; D006358:Hot Temperature; D007526:Isoelectric Point; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight
chemical_list: D000911:Antibodies, Monoclonal; D010832:6-Phytase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase