Preparation and Properties of Purified Phytase from Oakbug Milkcap (Lactarius Quietus) Immobilised on Coated Chitosan with Iron Nano Particles and Investigation of Its Usability in Food Industry

Hicran Onem¹ and Hayrunnisa Nadaroglu^{1, 2,}

¹Ataturk University, Faculty of Engineering, Department of Nano-Science and Nano-Engineering, Erzurum, TURKEY

²Ataturk University, Erzurum Vocational Training School, Department of Food Technology, Erzurum, TURKEY

Cite this paper:
Hicran Onem and Hayrunnisa Nadaroglu. Preparation and Properties of Purified Phytase from Oakbug Milkcap (Lactarius Quietus) Immobilised on Coated Chitosan with Iron Nano Particles and Investigation of Its Usability in Food Industry. Journal of Food and Nutrition Research. 2014; 2(12):938-945. doi: 10.12691/jfnr-2-12-13

Abstract

In this study, phytase enzyme was purified and characterized from Oakbug Milkcap mushrooms (Lactarius quietus) and the purified phytase enzyme was immobilized on the surface of modified chitosan with nano-Fe₃O₄ nanoparticles. The phytase was purified from Oakbug Milkcap mushrooms using ammonium sulphate precipitation in the range of 40-80%. and DEAE-sephadex ion-exchange chromatography. The purification fold was calculated by determining the activity and amount of protein for each step. The purified phytase enzyme was then immobilized on modified chitosan support material with Fe₃O₄ nanoparticles. The optimum pHs for the immobilized and free enzyme were 6.0 and the optimum temperatures were 60°C. The effects of some metal ions such as CuCl₂, Hg₂Cl₂, FeCl₂, MgCl₂, ZnCl₂, CaCl₂ were investigated on both the bound and free purified phytase. The molecular weight of the enzyme was determined using the SDS-PAGE electrophoresis method. Finally, we investigated whether or not the immobilized and free enzyme could hydrolyzed the phytic acid in green lentils, red lentils, peas, pinto beans, beans, brass, corn, dried corn, oat, rye, wheat, broad bean, chickpeas and peanuts. It was concluded that the immobilized phytase was quite resistant to temperature, pH and metal ions and it could be safely used in the hydrolysis of phytic acid in the legumes in the food industry.

Keywords:
Oakbug Milkcap (Lactarius quietus) mushroom phytase phytic acid immobilization

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References:

[1]	Kumar, V., Sinha, A. K., Makkar, H. P. S., Becker, K. 2010. Dietary roles of phytate and phytase in human nutrition: a review. Food Chem. 120 (4), 945-959.
[2]	Zhang, G. Q., Dong, X. F., Wang, Z. H., Zhang, Q., Wang, H. X., Tong, J. M. 2010. Purification, characterization, and cloning of a novel phytase with low pH optimum and strong proteolysis resistance from Aspergillus ficuum NTG-23, Bioresource Technology, 101 (11), 4125-4131.
[3]	Greiner, R., Konietzny U. 2006. Phytase for food application. Food Technol. Biotechnol. 442, 125-140.
[4]	Nayini, N. R., Markakis, P. 1984. The phytase of yeast. Lebensm.-Wiss. Technology, 17, 24-26.
[5]	Weremko, D., Fandrejewski, H., Zebrowska, T., Han, K., Kim, J. H., Cho, W. T. 1997. Bioavailability of phosphorus in feeds of plant origin for pigs -Review. Asian-Austral. J. Anim. Sci. 10, 551-566.
[6]	Lassen, S. F., Breinholt, J., Ostergaard, P. R., Brugger, R., Bischoff, A., Wyss, M., Fuglsang, C. C. 2001. Appl. Env. Microbiol. 67, 4701-4707.
[7]	Collopy, P. D., Royse, D. J. 2004. Characterization of phytase activity fromcultivated ediblemushrooms and t.heir production substrates. J. Agr. Food Chem. 52, 7518-7524.
[8]	Nadaroglu, H., Tasgin E. 2013. Purification and characterisation of laccase from Lactarius volemus and its application of removal phenolic compounds from some fruit juices. Int. J. Food Agr. Env. 11 (3&4), 109-114.
[9]	Sarıbuga, E., Dikbas, N., Nadaroglu, H., Senol M., 2014. Purification, characterization of phytase enzyme from Lactobacillus acidophilus bacteria and determination of it's some kinetic properties. J. Pure Appl. Microbio. 8, 91-96.
[10]	Sarıbuga, E., Nadaroglu, H., Dikbas, N., Senol, M., Cetin, B., 2014. Purification, characterization of phytase enzyme from Lactobacillus plantarum bacteria and determination of it's some kinetic properties. Afr. J. Biotechnol. 13 (23), 2373-2378.
[11]	Laemmli, D. K. 1970. Cleavage of structural proteins during in assembly of the heat of bacteriophage T4. Nature, London, 227, 680-685.
[12]	Chiu, S-H., Chung, T-W., Giridhar, R., Wu, W-T., 2004. Immobilization of β-cyclodextrin in chitosan beads for separation of cholesterol from egg yolk. Food Res. Int. 37, 217-231.
[13]	Iqbal T.H., Lewisi K.O., Cooper B.T. 1994. Phytase activity in the human and rat small intestine. Gut, 35, 1233-1236.
[14]	Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantites of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7, 248-254.
[15]	Konietzny, U., Greiner, R. 2004. Bacterial phytase: Potential application, in vivo function and regulation of its synthesis. Brazilian J. Microbiol. 35, 11-18.
[16]	Lei, X. G., Porres, J. M., 2003. Phytase enzymology, applications, and biotechnology. Biotech. Lett. 25, 1787-1794.
[17]	Nuge, T., Hashim, Y. Z. H-Y. H., Farouk, A-E. A., Salleh H. M. 2014, Cloning and expression of a novel phytase gene phyMS from Mycobacterium smegmatis, Adv. Enz Res. 2, 27-38.
[18]	Ostanin, K., Van Etten, R. L. 1993. Asp304 of Escherichia coli acid phosphatase is involved in leaving group protonation. Turkish J. Biol. Chem. 268, 20778-20784.
[19]	Ogawa, K., Hirano, S., Miyanishi, T., Yui, T., Watanabe, T. 1984. A new polymorph of chitosan, Macromol. 17, 973-975.
[20]	Cao, C., Xiao, L., Chen, C., Shi, X., Cao, Q., Gao, L. 2014. In situ preparation of magnetic Fe3O4/chitosan nanoparticles via a novel reduction–precipitation method and their application in adsorption of reactive azo dye, Powder Tech. 260, 90-97.
[21]	Guo, M., Diao, P., Cai, S. 2005. Hydrothermal growth of well aligned ZnO nanorodarrays: dependence of morphology and alignment ordering upon preparing conditions. J. Solid State Chem. 1786, 1864-1873.
[22]	Li, G., Jiang, Y., Huang, K., Ding, P., Chen, J. 2008. Preparation and properties of magnetic Fe3O4-chitosan nanoparticles, J. Alloy. Compd. 466, 451-456.
[23]	Shang, C. Y., Li, W. X., Zhang, R. F., 2014. Immobilized Candida antarctica lipase B on ZnO nanowires/macroporous silica composites for catalyzing chiral resolution of R, S-2-octanol. Enzyme Microb Tech. In press.
[24]	Konietzny, U., Greiner, R. 2003. Phytic acid: Nutritional Impact. In: Encyclopedia of Food Science and Nutrition, B. Caballero, L. Trugo, P. Finglas Eds., Elsevier, London, UK, 4555-4563.
[25]	Lopez, H. W., Vallery, F., Levrat-Verny, M. A., Coudray, C., Deminge, C., Emesy, C. 2000. Dietary phytic acid and wheat bran enhance mucosal phytase activity in rat small intestine. J. Nutr. 130, 2020-2025.
[26]	Ferguson, E. L., Gibson, R. S., Thomson, L. U., Ounpuu, S. 1989. Dietary calcium, phytate, and zinc intakes and the calcium, phytate, and zinc molar ratios of the diets of a selected group of East African children. Am. J. Clin. Nutr. 50, 1450-1456.