Purification and characterization of three phytases from germinated lupine seeds (Lupinus albus var. amiga).
Three phytases were purified about 14200-fold (LP11), 16000-fold (LP12), and 13100-fold (LP2) from germinated 4-day-old lupine seedlings to apparent homogeneity with recoveries of 13% (LP11), 8% (LP12), and 9% (LP2) referred to the phytase activity in the crude extract. They behave as monomeric proteins of a molecular mass of about 57 kDa (LP11 and LP12) and 64 kDa (LP2), respectively. The purified proteins belong to the acid phytases. They exhibit a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 80 microM (LP11), 300 microM (LP12), and 130 microM (LP2) and k(cat) = 523 s(-1) (LP11), 589 s(-1) (LP12), and 533 s(-1) (LP2) at pH 5.0 and 35 degrees C. The phytases from lupine seeds exhibit a broad affinity for various phosphorylated compounds and hydrolyze phytate in a stepwise manner.
Greiner
Ralf
R
Federal Research Centre for Nutrition, Centre for Molecular Biology, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany.
D010832:6-Phytase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D018525:Germination; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D031297:Lupinus / Q000201:enzymology*; D008970:Molecular Weight; D010833:Phytic Acid / Q000378:metabolism; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D004791:Enzyme Inhibitors; D010833:Phytic Acid; D010832:6-Phytase