title: Purification and characterization of phytase from Klebsiella pneumoniae 9-3B.
abstract: Phytase, an enzyme that catalyzes the hydrolysis of phytate, was purified from Klebsiella pneumoniae 9-3B. The isolate was preferentially selected in a medium which contains phytate as a sole carbon and phosphate source. Phytic acid was utilized for growth and consequently stimulated phytase production. Phytase production was detected throughout growth and the highest phytase production was observed at the onset of stationary phase. The purification scheme including ion exchange chromatography and gel filtration resulted in a 240 and 2077 fold purification of the enzyme with 2% and 15% recovery of the total activity for liberation of inorganic phosphate and inositol, respectively. The purified phytase was a monomeric protein with an estimated molecular weight of 45kDa based on size exclusion chromatography and SDS-PAGE analyses. The phytase has an optimum pH of 4.0 and optimum temperature of 50°C. The phytase activity was slightly stimulated by Ca(2+) and EDTA and inhibited by Zn(2+) and Fe(2+). The phytase exhibited broad substrate specificity and the K(m) value for phytate was 0.04mM. The enzyme completely hydrolyzed myo-inositol hexakisphosphate (phytate) to myo-inositol and inorganic phosphate. The properties of the enzyme prove that it is a good candidate for the hydrolysis of phytate for industrial applications.
authors.affiliation: Laboratory of Applied Microbiology, Graduate Faculty of Agriculture, Hokkaido University, N9 W9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan.
authors.forename: Lotis
authors.initials: L
authors.lastname: Escobin-Mopera
authors.forename: Midori
authors.initials: M
authors.lastname: Ohtani
authors.forename: Sachie
authors.initials: S
authors.lastname: Sekiguchi
authors.forename: Teruo
authors.initials: T
authors.lastname: Sone
authors.forename: Ayumi
authors.initials: A
authors.lastname: Abe
authors.forename: Michiko
authors.initials: M
authors.lastname: Tanaka
authors.forename: Vithaya
authors.initials: V
authors.lastname: Meevootisom
authors.forename: Kozo
authors.initials: K
authors.lastname: Asano
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D010710:Phosphates; D010833:Phytic Acid; D004492:Edetic Acid; D010832:6-Phytase