1Department of Biochemistry, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
2Falavarjan Branch, Islamic Azad University, Isfahan, Iran
Objective(s): Enzyme immobilization via nanoparticles is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto silica-coated magnetite nanoparticles to improve enzyme properties in the presence of cadmium chloride as an inhibitor. Materials and Methods: The process consists of the following steps: (1) preparing magnetic iron oxide nanoparticles using the co-precipitation method, (2) coating NP with silica (SiO2) by sol–gel reaction, (3) characterizations of NPs were examined by FT-IR, XRD, AGFM and TEM. (4) Immobilization of LPO on the magnetite NPs, (5) Study kinetic and stability of both free and immobilized LPO in the presence of various concentrations of cadmium chloride. Results: The size of the Fe3O4 and silica-coated magnetite nanoparticles were about 9 nm and 12 nm, respectively. The results showed that the highest immobilization yield, nearly 90 %, was attained at 240 to 300 µg of LPO at 15h. It was found that the concentration of cadmium chloride directly affects the LPO activity and changes the kinetic parameters of it. Also, the results showed that immobilized LPO has better tolerance than the free LPO, so that after immobilization, Vmax of immobilized LPO was increased and Km of immobilized LPO was decreased. Conclusion: The results demonstrating that the effect of immobilized lactoperoxidase on silica-coated magnetite nanoparticles increases the stability of the LPO in the presence of cadmium chloride as inhibitor. Michaelis–Menten parameters (Km and Vmax) also revealed the considerable improvement of immobilized.
1. Wu L, Wu S, Xu Z, Qiu Y, Li S, Xu H. Modified nanoporous titanium dioxide as a novel carrier for enzyme immobilization. Biosens & Bioelec. 2010; 59-66.
2. Mehta J, Bhardwaj N, Bhardwaj SK, Kim K-H, Deep A. Recent advances in enzyme immobilization techniques: Metal-organic frameworks as novel substrates. Coordin Chem Reviews. 2016; 322: 30-40.
3. Prado Barragán LA, Buenrostro-Figueroa JJ, Aguilar González CN, Marañon I. Chapter 10 - Production, Stabilization, and Uses of Enzymes From Fruit and Vegetable Byproducts A2 - Poltronieri, Palmiro. In: D’Urso OF, editor. Bio of Agricultural Waste & By-Products: Elsevier; 2016; 271-286.
4. Ward K, Xi J, Stuckey DC. Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Surface and enzyme effects. Colloids and Surfaces B: Biointerfaces. 2015; 136: 424-430.
5. Clark DP, Pazdernik NJ. Chapter 11 - Protein Engineering. Biotechnology (Second Edition). Academic Cell. 2016; 365-392.
6. Albers WM, Vikholm I, Viitala T, Peltonen J. Chapter 1 – interfacial and materials aspects of the immobilization of biomelecules onto solid surfaces A2 - Nalwa, Hari Singh. Handbook of Surfaces & Interfaces of Maters. 2001; 1-31.
7. Wu X-c, Zhang Y, Wu C-y, Wu H-x. Preparation and characterization of magnetic Fe3O4/CRGO nanocomposites for enzyme immobilization. Transactions of Nonferrous Met Soc of China. 2012; 162-168.
8. Liu M-q, Dai X-j, Guan R-f, Xu X. Immobilization of Aspergillus niger xylanase A on Fe3O4-coated chitosan magnetic nanoparticles for xylooligosaccharide preparation. Catal Commun. 2014; 55: 6-10.
9. Long J, Li X, Wu Z, Xu E, Xu X, Jin Z, Immobilization of pullulanase onto activated magnetic chitosan/Fe3O4 nanoparticles prepared by in situ mineralization and effect of surface functional groups on the stability. Colloids & Surfaces. 2015; 69-77.
10. Qiu J, Peng H, Liang R. Ferrocene-modified Fe3O4@SiO2 magnetic nanoparticles as building blocks for construction of reagentless enzyme-based biosensors. Elect Communs. 2007; 2734-2738.
11. Seenuvasan M, Malar CG, Preethi S, Balaji N, Iyyappan J, Kumar MA, Fabrication, characterization and application of pectin degrading Fe3O4–SiO2 nanobiocatalyst. Materials Sci & Engine: C. 2013; 2273-2279.
12. Saravanakumar T, Palvannan T, Kim D-H, Park S-M. Optimized immobilization of peracetic acid producing recombinant acetyl xylan esterase on chitosan coated-Fe3O4 magnetic nanoparticles. Process Biochem. 2014; 1920-8.
13. Minibayeva F, Beckett RP, Kranner I. Roles of apoplastic peroxidases in plant response to wounding. Phytochem. 2015; 112: 122-129.
14. Velde Fvd, Rantwijk Fv, Sheldon RA. Improving the catalytic performance of peroxidases in organic synthesis. Trends in Biotech. 2001; 19(2): 73-80.
15. Pan M, Shen S, Chen L, Dai B, Xu L, Yun J, et al. Separation of lactoperoxidase from bovine whey milk by cation exchange composite cryogel embedded macroporous cellulose beads. Separation & Purification Tech. 2015; 147: 132-138.
16. Hamid M, Khalil ur R. Potential applications of peroxidases. Food chem. 2009; 115(4): 1177-1186.
17. Dumitraºcu L, Stãnciuc N, Stanciu S, Râpeanu G. Thermal inactivation of lactoperoxidase in goat, sheep and bovine milk – A comparative kinetic and thermodynamic study. Food Engine. 2012; 113(1): 47-52.
18. Atasever A, Ozdemir H, Gulcin I, Irfan Kufrevioglu O. One-step purification of lactoperoxidase from bovine milk by affinity chromatography. Food Chem. 2013; 136(2): 864-870.
19. Campbell RE, Kang EJ, Bastian E, Drake MA. The use of lactoperoxidase for the bleaching of fluid whey. Dairy Sci. 2012; 95(6): 2882-2890.
20. Cissé M, Polidori J, Montet D, Loiseau G, Ducamp-Collin MN. Preservation of mango quality by using functional chitosan-lactoperoxidase systems coatings. Postharvest Bio & Tech. 2015; 101: 10-4.
21. Pogorilyi RP, Melnyk IV, Zub YL, Seisenbaeva GA, Kessler VG. Enzyme immobilization on a nanoadsorbent for
improved stability against heavy metal poisoning. Colloid & Surfaces. 2016; 144: 135-142.
22. Keyhani J, Keyhani E, Einollahi N, Minai-Tehrani D, Zarchipour S. Heterogeneous inhibition of horseradish peroxidase activity by cadmium. BBA J . 2003; 1621 (2): 140-148.
23. Khosroshahi ME, Ghazanfari L. Preparation and characterization of silica-coated iron-oxide bionanoparticles under N2 gas. Low-dimensional Sys & Nano. 2010; 42(6): 1824-189.
24. Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977; 83(2): 346-356.
25. Bahamin N. Shareghi B. The Effect of Cadmium Sulfate on the Thermal Stability and Kinetics of Peroxidase at Different Temperatures. Experimental Animal Bio. 2013; 7(16).