Isolation and characterisation of phytase from dormant Corylus avellana seeds.
Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)(2)SO(4) precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS-PAGE it was found to be a monomeric protein with molecular mass 72+/-2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 microM) and highest specificity constant (V(max)/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 microM and Ki=205 microM, respectively).
D010832:6-Phytase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods; D003208:Concanavalin A / Q000737:chemistry; D031211:Corylus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008670:Metals / Q000737:chemistry / Q000494:pharmacology; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D010710:Phosphates; D003208:Concanavalin A; D010832:6-Phytase