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)SO4 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 SIDS-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 K-m (162 muM) and highest specificity constant (V-max/K-m) 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 (K-i = 407 muM and K-i = 205 muM, respectively).