Isolation of phytase producer aspergillus niger, purification, characterization and optimization of the enzyme

In this study, the phytase enzyme from Aspergillus niger FM-7 isolated from decayed plants, water, soil and manure was produced, characterized and purified. Phytase production of this strain was determined by production of clear zone around the colonies on the sodium phytate containing medium, after 72 hours incubation at 30οC. The enzyme isolated from Aspergillus niger FM-7 strain was selected in this study, because of its superior activity. SDS-PAGE analysis of phytase enzyme produced from Aspergillus niger FM-7 strain gave two protein bands corresponding to molecular weights of 93 kDa and 70 kDa in M1 medium and 100 kDa and 83 kDa in M1+% 0.1 Na-phytate medium, respectively. The optimum activity of the enzyme produced in M1 and M1+%0.1 Na-phytate medium was emerged at 70οC, pH 3.8 and 70οC, pH 4.6, respectively, on the otherhand, the optimum activity of commercial phytase enzymes, A and B, produced from Aspergillus niger was emerged at 70 οC, pH 3.4. For 60 minutes at 90οC, the enzymes produced in M1 and M1+%0.1 Na-phytate medium, and the commercial A and B maintained their activity at a rate of 35%, 35%, 1% and 41%, respectively. After dialysis and gel-filtration chromatography, the specific activity of the enzyme produced in M1 medium was found as 941,17 U/mg and a 6-fold purification was achieved when compared with the initial purification. The Km and Vmax values for Na-phytate substrate were 7,7 mM and 5137 U/mg proteins, respectively. Full enzyme activities were significantly stimulated with MgCl2, NiCl2, MgSO4, CuSO4, MnSO4 and acetic acid; however, significantly inhibited with EDTA and SDS. Due to its characteristic properties, we propose that the phytase enzyme, produced from Aspergillus niger FM-7 may be conveniently used in feed additive