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Purification and Characterization of Extracellular Phytase from a Marine Yeast Kodamaea ohmeri BG3

Li, Xiaoyu, Chi, Zhenming, Liu, Zhiqiang, Li, Jing, Wang, Xianghong, Hirimuthugoda, Nalini Yasoda
Marine biotechnology 2008 v.10 no.2 pp. 190-197
EDTA (chelating agent), Kodamaea ohmeri, ammonium sulfate, anion exchange, anion exchange chromatography, barium, calcium, cell culture, cobalt, copper, fractionation, gel chromatography, iodoacetic acid, iron, lithium, magnesium, manganese, mercury, molecular weight, pH, phytases, phytic acid, polyacrylamide gel electrophoresis, potassium, silver, sodium, temperature, yeasts, zinc
The extracellular phytase in the supernatant of cell culture of the marine yeast Kodamaea ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex[trade mark sign] G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow Anion-Exchange). According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 98.2 kDa while the molecular mass of the purified enzyme was estimated to be 92.9 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography. The optimal pH and temperature of the purified enzyme were 5.0 and 65°C, respectively. The enzyme was stimulated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Ba²⁺, Mg²⁺ and Co²⁺ (at a concentrations of 5.0 mM), but it was inhibited by Cu²⁺, Hg²⁺, Fe²⁺, Fe³⁺, Ag⁺, and Zn²⁺ (at a concentration of 5.0 mM). The enzyme was also inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid (at a concentration of 1.0 mM), and phenylgloxal hydrate (at a concentration of 5.0 mM), and not inhibited by EDTA and 1,10-phenanthroline (at concentrations of 1.0 mM and 5.0 mM). The K m, V max, and K cat values of the purified enzyme for phytate were 1.45 mM, 0.083 μmol/ml · min, and 0.93 s⁻¹, respectively.