PURIFICATION AND CHARACTERISATION OF AN EXTRACELLULAR PHYTASE FROM ASPERGILLUS NIGER 11T53A9

An extracellular phytase from Aspergillus niger 11T53A9 was purified about 51-fold to apparent homogeneity with a recovery of 20.3% referred to the phytase activity in the crude extract. Purification was achieved by ammonium sulphate precipitation, ion chromataography and gel filtration. The purified enzyme behaved as a monomeric protein with a molecular mass of about 85 kDa and exhibited maximal phytate-degrading activity at pH 5.0. Optimum temperature for the degradation of phytate was 55 C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K-M = 54 mu mol l(-1) and k(cat) = 190 sec(-1) at pH 5.0 and 37 degrees C. The purified enzyme was rather specific for phytate dephosphorylation. It was shown that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P-5, D-Ins(1,2,5,6)P-4, D-Ins(1,2,6)P-3, D-Ins(1,2)P-2 to finally Ins(2)P.