Immobilization of Purified Phytase Enzyme from Tirmit (Lactarius volemus) on Coated Chitosan with Iron Nanoparticles and Investigation of Its Usability in Cereal Industry

摘要

In this study, phytase enzyme was purified and characterized from Tirmit ( Lactarius volemus ) mushroom and purified phytase enzyme was immobilized on the surface of modified chitosan with nano-Fe~(3)O~(4)nanoparticles. Then, free and immobilized enzyme applications had been carried out in cereal crops. Phytase enzyme was purified 120.2-fold and characterized with 11.6 % yield in range of 40–80 % from Tirmit (L. volemus) by using, ammonium sulfate precipitation, DEAE-Sephadex ion exchange chromatography and Sephacryl S200 gel filtration chromatography techniques. The purification fold was calculated by determining the activity and amount of protein for each step. Then, purified phytase enzyme was immobilized on modified chitosan support material with Fe~(3)O~(4)nanoparticles. For the immobilized and free enzyme, optimum pH was 5.5 and optimum temperature was 50 °C. First, the effects of some metal ions, such as CuCl~(2), HgCl~(2), FeCl~(2), MgCl~(2), ZnCl~(2)and CaCl~(2), were investigated on the bound and free purified phytase enzyme. Second, molecular weight of the enzyme was determined by SDS-PAGE electrophoresis method. Third, substrate specificity was determined for the purified phytase enzyme using some natural polymers, such as locust bean gum, gelatin, xylene, cellulose, phytate and starch. Finally, investigated whether the immobilized and free enzyme could be hydrolyzed the phytic acid in green lentils, red lentils, peas, pinto beans, beans, brass, corn, dried corn, oat, rye, wheat, broad bean, chickpea and peanuts or not.