α-amylase was immobilized covalently on iron oxide magnetic nanoparticles. The synthesis of magnetic nanoparticles was done by the coprecipitation conventional method. The chemical composition and particle size of the synthesized particles was confirmed via X-ray diffraction. Tyrosine, Lucien and chitosan and glutaraldehyde were investigated to make a covalent binding between the iron oxide magnetic core and the immobilized enzyme. Immobilization using chitosan and glutaraldehyde show the best result. Finally the immobilization efficiency was tested by determination of protein concentration in a solution before and after mixing with the magnetic nanoparticles.
In the last two decades, new terms with the prefix `nano’ have rushed into the scientific vocabulary; nanoparticle, nanostructure, nanotechnology, nanomaterial, nanocluster, nanochemistry, nanocolloids, nanoreactor and so on. Nanoparticles, are defined as particulate dispersions with a size in the range of 10-100nm (Gubin et al, 2005). Magnetic nanoparticles have gained a remarkable interest in the last years both for basic research and applied studies. The use of magnetic nanostructures has been proven in biochemistry, biomedicine, and waste treatment among other fields. This broad range of applications is based on the fact that magnetic particles have very large magnetic moments, which allow them to be transported and driven by external magnetic fields. The magnetic nanostructures have also a great potential in biotechnological processes taking into account that they can be utilized as a carrier for enzymes during different biocatalytic transformations (Dussán et al, 2007).
Different types of biomolecules such as proteins, enzymes, antibodies, and anticancer agents can be immobilized on these nanoparticles. Magnetic supports for immobilization purpose are either prepared by incorporating magnetic particles during the synthesis of the supporting polymer or magnetic particles itself be coated with common support materials such as dextran or agarose. Recently, a new method for the direct binding of proteins on magnetic nanoparticles via carbodiimide activation was proposed (Ren et al, 2011).
Immobilization is one of the efficient methods to improve enzyme stability. There are various methods for immobilization of enzymes on many different types of supports. It can be a chemical method in which ionic or covalent bond formations occur between the enzyme and the carrier, or it can be a physical method, such as adsorption or entrapment of the enzyme in or on a solid support material. Magnetic nanoparticles as immobilization materials have advantage based on its property and size that make it desirable for using it in various applications (Mateo et al, 2007). Iron oxide nanoparticles, Fe3O4, are one of the widely used types of magnetic nanoparticles and have great potential for applications in biology and medicine due to their strong magnetic properties and low toxicity (Jalal et al, 2011)
Review of literature
I) Magnetic nanoparticles:
The historical development of nanoparticles starting with Paul Ehrlich and then first attempts by Ursula Scheffel and colleagues and the extensive work by the group of Professor Peter Speiser at the ETH Zürich in the late 1960s and early 1970s (Jörg Kreuter 2007). They are solid particles with a size from 10 to 100nm which can be manipulated using magnetic field. Such particles commonly consist of magnetic elements such as iron, nickel and cobalt. They have been used in catalysis, biomedicine, magnetic resonance imaging, magnetic particle imaging, data storage , environmental remediation and optical filters (Gubin et al, 2005).
Magnetic nanoparticles as immobilization materials have the following advantages: simple and inexpensive production, can be released in controlled manner, stable magnetic properties of complexed nanoparticles and easy isolation steps in short time. Among these materials, Fe3O4 magnetic nanoparticles are the most commonly studied. Fe3O4 magnetic nanoparticles have good biocompatibility, strong superparamagnetism, low toxicity, and an easy preparation process, and their use in biosensors has already shown attractive prospects (Sheng-Fu Wang and Yu-Mei Tan, 2007).
II) Magnetic core material:
There are many magnetic materials available with a wide range of magnetic properties. such as cobalt, chromium and iron oxide-based materials such as magnetite and maghemite. The suitable magnetic materials depend on applications the MNP will apply in (Dobson et al, 2007). Magnetite Fe3O4:
Magnetite is a common mineral which exhibits ferro (ferri) magnetic properties. The structure of magnetite belongs to the spinel group, which has a formula of AB2O4. Its ferromagnetic structures arise from alternating lattices of Fe(II) and Fe(III). This gives it a very strong magnetization compared to naturally occurring antiferromagnetic compounds such as the ferrihydrite core of the ferritin protein (McBain et al, 2008). III) Synthesis of iron Magnetic nanoparticles:
There were many synthesis methods for magnetic nanoparticles one of these is Co-precipitation. This method may be the most promising one because of its simplicity and productivity (zhao et al., 2008). It is widely used for biomedical applications because of ease of implementation and need for less hazardous materials and procedures. Co-precipitation is specifically the precipitation of an unbound “antigen along with an antigen-antibody complex” in terms of medicine (Indira and Lakshmi, 2010).The reaction principle is simply as:
Fe2+ + 2Fe3+ + 8OH– ⇔ Fe (OH)2 + 2Fe(OH)3 → Fe3O4 + 4H2O (Guo et al., 2009).
Other method used for synthesis like: Thermolysis of metal-containing compounds, synthesis of magnetic nanoparticles at a gas-liquid interface, synthesis in reverse micelles and sol-gel method (Gubin et al, 2005).
IV) Characterization of MNP:
There is no unique method for determination of the nanoparticle composition and dimensions; as a rule, a set of methods including X-ray diffraction, Transmission electron microscope and Extended X-ray Absorption Fine Structure (EXAFS) Spectroscopy are used (Gubin et al, 2005).
X-Ray diffraction analysis of nanomaterial seldom produces diffraction patterns with a set of narrow reflections adequate for identification of the composition of the particles they contain. Some X-ray diffraction patterns exhibit only two or three broadened peaks of the whole set of reflections typical of the given phase (Moroz 2011).
In the case of larger particles (provided that high-quality X-ray diffraction patterns can be obtained), it is often possible not only to determine the phase composition but also to estimate, based on the reflection width, the size of coherent X-ray scattering areas, corresponding to the average crystallite (nanoparticle) size. This is usually done by the Scherer formula (Gubin et al, 2005).
The nanoparticle dimensions are determined most often using Transmission electron microscope, which directly shows the presence of nanoparticles in the material under examination and their arrangement relative to one another. The phase composition of nanoparticles can be derived from electron diffraction patterns recorded for the same sample during the investigation. Note that in some cases, TEM investigations of dynamic processes are also possible. For example, the development of dislocations and disclinations in the nanocrystalline during the mechanochemical treatment has been observed (Woehrle et al, 2000).
More comprehensive information is provided by high resolution transmission electron microscopy, which allows one to study the structure of both the core and the shell of a nanoparticle with atomic resolution, and in some cases, even to determine their stoichiometric composition (Woehrle et al, 2000).
The structures of non-crystalline samples are often studied by EXAFS spectroscopy. An important advantage of these methods is its selectivity, because it provides the radial distribution (RDA) curve for the atoms of the local environment of the chosen chemical element in the sample. The interatomic distances (R) and coordination numbers (N) obtained by EXAFS are then compared with the known values for the particular phase (Gubin et al, 2005).
Other methods are used more rarely to study the nanoparticle structures. Integrated research makes it possible to determine rather reliably the structures of simple nanoparticles; however, determination of the structures of nanoparticles composed of a core and a shell of different compositions are often faced with difficulties (Gubin et al, 2005).
V) Stabilization of Magnetic Nanoparticles:
Although there have been many significant developments in the synthesis of magnetic nanoparticles, maintaining the stability of these particles for a long time without agglomeration or precipitation is an important issue. Stability is a crucial requirement for almost any application of magnetic nanoparticles. Especially pure metals, such as Fe, Co, and Ni and their metal alloys, are very sensitive to air. Thus, the main difficulty for the use of pure metals or alloys arises from their instability towards oxidation in air, and the susceptibility towards oxidation becomes higher the smaller the particles are (Lu et al, 2007). Therefore, it is necessary to develop efficient strategies to improve the chemical stability of magnetic nanoparticles:
Surface Passivation by Mild Oxidation:
A very simple approach to protect the magnetic particles is to induce a controlled oxidation of a pure metal core, a technique long known for the passivation of air-sensitive supported catalysts. This oxidation can be achieved by various methods (Peng et al, 1999).
For example, Peng et al. developed a method for oxidizing gas-phase nanoparticles by using a plasma-gas-condensation-type cluster deposition apparatus. Demonstrated that very good control over the chemical state of the cobalt nanoparticles was achieved by their exposure to an oxygen plasma. The control of the oxide layer has a tremendous impact on exchange-biased systems, where a well-defined thickness of the ferromagnetic core and the anti-ferromagnetic shell are desirable. Moreover, a direct correlation of the structure and magnetism in the small particles can be determined. developed a mild oxidation method, using synthetic air to smoothly oxidize the as-synthesized cobalt nanoparticles to form a stable outer layer which can stabilize the nanoparticles against further oxidation (Peng et al, 1999).
Other methods: Matrix-Dispersed Magnetic Nanoparticles, Carbon Coating, Silica Coating , Precious-Metal Coating and Surfactant and Polymer Coating
Typical strategies for immobilizing catalysis enzyme onto MNPs rely on surface grafting via low molecular weight linkers or polymers containing amino or epoxy functional groups to which enzyme are reacted via covalent conjugation methods (Ren et al, 2011).
Due to their high specific surface area and easy separation from the reaction medium by the use of a magnetic field, they have been employed in enzymatic catalysis applications ex amylase EC 3.2.1 (Ren et al, 2011).
The maximum reported loading capacity of amylase is approximately 81.97 mg/g (Aktaş et al, 2011). One drawback of existing immobilization technologies is that the activity of enzyme decreases significantly upon immobilization due possibly to changes in enzyme secondary structure, or limited access of substrate to the active site of the surface bound enzyme (Lei et al, 2009). Thus, despite numerous reported approaches for immobilization of catalysis enzyme on magnetic nanoparticles, there is still the need for simple, cost-effective and high loading capacity methods.
Aim of work
Is to Synthesis of iron magnetic nanoparticle (MNP) then immobilize amylase on MNP and test the efficiency of immobilization method then study the activity of immobilized amylase.