**1.3 Why covalent immobilization?**

There is not a universal method of immobilization and support for the enzymes and their uses in biotechnology. Several factors could drive the choice of a particular method, e.g., physicochemical properties of support or different features of substrates and products. As shown in **Figure 1**, all methods present advantages and disadvantages. However, why should the covalent bond be chosen? Covalent bonding is the most used strategy of the irreversible category. The removal of the covalent immobilized enzyme from the support without affecting its catalytic activity as an attempt to recover either the biomolecule or the support is meaningless due to hard process usually involved to disrupt the covalent bond. Moreover, better thermal and operational stability, as well as major resistance to pH, temperature, and solvent variations, are some of the well-known benefits of the immobilization approach. A good reason to choose this method is when a system with high stable protein coverage is recommended. Obtainment of the product of high purity, i.e., no contaminants including the enzyme, is another excuse to employ the covalent bond for immobilization.

### **2. About the particles used as support**

In order to prepare an immobilized derivative, at least the biomolecule, the support, and the method of immobilization are required. Choosing the support is an essential step in the immobilization process since the characteristics of the material can influence in the performance of the biocatalyst. Even today there is no general rule for selecting the ideal support to attach the biomolecule. However, materials with characteristics such as chemical inertia, hydrophilic character, low cost, mechanical resistance, and resistance to microbial attack are widely used. Often in the literature, terms as matrix and carrier are found as synonyms of support.

Magnetic particles as a carrier to biomolecule immobilization are desirable materials due to easy separation of the biocatalyst from the reaction medium by application of an external magnetic field. Among the particles with magnetic property, the iron oxides, in particular, magnetite (Fe3O4), are the materials with notable uses in the immobilization process. Maghemite (γ-Fe2O3) and hematite (α-Fe2O3) are iron oxides derived from magnetite and can be found together with it.

#### **2.1 Iron oxides: Why use it as a support of biomolecules?**

Until now several magnetic particles obtained from pure iron oxide or a mixture of phases and composites materials have been presented as attractive support to enzyme immobilization or protein purification. To understand why these particles are so attractive, it might be useful to review the characteristics of the magnetite. The iron oxide is inorganic in nature, which presents a ratio of ferric/ferrous ions equal to 2:1, in stoichiometric magnetite. The ions positively charged (ferric,

**31**

*Magnetic Bio-Derivatives: Preparation and Their Uses in Biotechnology*

denoted as Fe3+, and ferrous, expressed as Fe2+) are distributed in two different sites with a crystal inverse spinel structure, formed by a cubic network face centered (fcc) of oxygen anions. Tetrahedral sites are filled by Fe3+ ions, while the octahedral

. Maghemite is also a ferrimagnetic mate-

), while the hematite is weakly ferromagnetic (*M*s minor

). It is important to mention that there is a correlation between the

magnetic properties of the material with their size. So, when the magnetite presents nanometer size (below 15 nm), the superparamagnetic behavior is observed [12]. In addition to physicochemical and magnetic properties of the magnetite, using these particles as a carrier of the biomolecules could avoid laborious procedures

At present, many synthesis methods to produce iron oxide particles have been developed. Evaluating the preparation method together with the features desires of the material (i.e., size, shape, size distribution, and surface chemistry) is very important once the magnetic property, as well as applications of material synthesized, depends on it. Moreover, the degree of structural defects (i.e., the presence of impurities) and distribution of the defects are also related to the synthesis methodology [13]. In general, the main preparation methods of the iron oxide particles can be classified as chemical, physical, and biological. Among these methodologies, the chemical route is the most used due to high yield along with low-cost production. Coprecipitation, hydrothermal, microemulsion, sonochemical, thermal decomposition, sol-gel synthesis, and electrochemical decomposition are some of the chemical methods [14]. A complete description about the most common preparation methods of the iron oxide particles together with their benefits and drawbacks can be

The "coprecipitation method" presents interesting characteristics that make it the chosen one among the chemical route category. For instance, this technique is simple and inexpensive and can be used for large-scale production. Briefly, iron oxide particles are produced by addition of a precipitating agent (e.g., ammonium hydroxide) to the solution containing a mixture of ferrous and ferric salts (e.g., FeCl2.4H2O and FeCl3.6H2O) at room temperature or high temperature. A molar ratio equal to 2:1 for Fe3+/Fe2+ ions is generally used. The obtainment of a black precipitate (Fe3O4) is a good indication that the synthesis was successful. The size, shape, composition and magnetic property of the particles can be modified depending on the iron precursors, iron concentrations, ferric/ferrous ratio, precipitating agent, pH, temperature, ionic strength, stirring velocity, surfactants addition, and

Processes of the nucleation and the growth of crystals are involved in this technique. Both processes should take place separately, i.e., first the nucleation and then the growth. According to Wu et al. [10], pH is an important parameter to address toward the nucleation (solution pH minor than 11) and the growth (solution pH major than 11) of crystals. Even though the coprecipitation method is widely used to produce iron oxide particles with high saturation magnetization, some limitations like broad and large size distribution, poor crystallinity,

According to the magnetic response, iron oxides can be classified as ferromagnetic, antiferromagnetic, paramagnetic, and ferrimagnetic. At room temperature, bulk magnetite is a ferrimagnetic material due to the combination of ferromagnetic and antiferromagnetic comportments that happens below the Curie temperature (850 K) [11]. This iron oxide presents a saturation magnetization

*DOI: http://dx.doi.org/10.5772/intechopen.85748*

sites are occupied by Fe3+ and Fe2+ ions [10].

(*M*s) value between 92 and 100 emu g<sup>−</sup><sup>1</sup>

such as decantation, centrifugation, or filtration.

rial (*M*s = 60–80 emu g<sup>−</sup><sup>1</sup>

**2.2 Synthesis of iron oxides**

found in the literature [1, 12, 14].

working under inert atmosphere [10, 15].

than 1 emu g<sup>−</sup><sup>1</sup>

*Magnetic Bio-Derivatives: Preparation and Their Uses in Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.85748*

denoted as Fe3+, and ferrous, expressed as Fe2+) are distributed in two different sites with a crystal inverse spinel structure, formed by a cubic network face centered (fcc) of oxygen anions. Tetrahedral sites are filled by Fe3+ ions, while the octahedral sites are occupied by Fe3+ and Fe2+ ions [10].

According to the magnetic response, iron oxides can be classified as ferromagnetic, antiferromagnetic, paramagnetic, and ferrimagnetic. At room temperature, bulk magnetite is a ferrimagnetic material due to the combination of ferromagnetic and antiferromagnetic comportments that happens below the Curie temperature (850 K) [11]. This iron oxide presents a saturation magnetization (*M*s) value between 92 and 100 emu g<sup>−</sup><sup>1</sup> . Maghemite is also a ferrimagnetic material (*M*s = 60–80 emu g<sup>−</sup><sup>1</sup> ), while the hematite is weakly ferromagnetic (*M*s minor than 1 emu g<sup>−</sup><sup>1</sup> ). It is important to mention that there is a correlation between the magnetic properties of the material with their size. So, when the magnetite presents nanometer size (below 15 nm), the superparamagnetic behavior is observed [12].

In addition to physicochemical and magnetic properties of the magnetite, using these particles as a carrier of the biomolecules could avoid laborious procedures such as decantation, centrifugation, or filtration.

#### **2.2 Synthesis of iron oxides**

*Applied Surface Science*

environments.

antibodies or lectins can be used.

**2. About the particles used as support**

**1.3 Why covalent immobilization?**

when the physical adsorption of the biocatalyst is carried out in hydrophobic

• **Affinity**. The immobilization by affinity interaction requires that the biocatalyst as well as the support present specific chemical groups on their surfaces, that is, the presence of complementary species, e.g., streptavidin-biotin interaction. As a consequence of the specific interaction, notable selectivity is a major benefit of this method. However, this procedure is expensive since

There is not a universal method of immobilization and support for the enzymes and their uses in biotechnology. Several factors could drive the choice of a particular method, e.g., physicochemical properties of support or different features of substrates and products. As shown in **Figure 1**, all methods present advantages and disadvantages. However, why should the covalent bond be chosen? Covalent bonding is the most used strategy of the irreversible category. The removal of the covalent immobilized enzyme from the support without affecting its catalytic activity as an attempt to recover either the biomolecule or the support is meaningless due to hard process usually involved to disrupt the covalent bond. Moreover, better thermal and operational stability, as well as major resistance to pH, temperature, and solvent variations, are some of the well-known benefits of the immobilization approach. A good reason to choose this method is when a system with high stable protein coverage is recommended. Obtainment of the product of high purity, i.e., no contaminants including the

enzyme, is another excuse to employ the covalent bond for immobilization.

In order to prepare an immobilized derivative, at least the biomolecule, the support, and the method of immobilization are required. Choosing the support is an essential step in the immobilization process since the characteristics of the material can influence in the performance of the biocatalyst. Even today there is no general rule for selecting the ideal support to attach the biomolecule. However, materials with characteristics such as chemical inertia, hydrophilic character, low cost, mechanical resistance, and resistance to microbial attack are widely used. Often in the literature, terms as matrix and carrier are found as synonyms of support.

Magnetic particles as a carrier to biomolecule immobilization are desirable materials due to easy separation of the biocatalyst from the reaction medium by application of an external magnetic field. Among the particles with magnetic property, the iron oxides, in particular, magnetite (Fe3O4), are the materials with notable uses in the immobilization process. Maghemite (γ-Fe2O3) and hematite (α-Fe2O3) are iron

Until now several magnetic particles obtained from pure iron oxide or a mixture of phases and composites materials have been presented as attractive support to enzyme immobilization or protein purification. To understand why these particles are so attractive, it might be useful to review the characteristics of the magnetite. The iron oxide is inorganic in nature, which presents a ratio of ferric/ferrous ions equal to 2:1, in stoichiometric magnetite. The ions positively charged (ferric,

oxides derived from magnetite and can be found together with it.

**2.1 Iron oxides: Why use it as a support of biomolecules?**

**30**

At present, many synthesis methods to produce iron oxide particles have been developed. Evaluating the preparation method together with the features desires of the material (i.e., size, shape, size distribution, and surface chemistry) is very important once the magnetic property, as well as applications of material synthesized, depends on it. Moreover, the degree of structural defects (i.e., the presence of impurities) and distribution of the defects are also related to the synthesis methodology [13]. In general, the main preparation methods of the iron oxide particles can be classified as chemical, physical, and biological. Among these methodologies, the chemical route is the most used due to high yield along with low-cost production. Coprecipitation, hydrothermal, microemulsion, sonochemical, thermal decomposition, sol-gel synthesis, and electrochemical decomposition are some of the chemical methods [14]. A complete description about the most common preparation methods of the iron oxide particles together with their benefits and drawbacks can be found in the literature [1, 12, 14].

The "coprecipitation method" presents interesting characteristics that make it the chosen one among the chemical route category. For instance, this technique is simple and inexpensive and can be used for large-scale production. Briefly, iron oxide particles are produced by addition of a precipitating agent (e.g., ammonium hydroxide) to the solution containing a mixture of ferrous and ferric salts (e.g., FeCl2.4H2O and FeCl3.6H2O) at room temperature or high temperature. A molar ratio equal to 2:1 for Fe3+/Fe2+ ions is generally used. The obtainment of a black precipitate (Fe3O4) is a good indication that the synthesis was successful. The size, shape, composition and magnetic property of the particles can be modified depending on the iron precursors, iron concentrations, ferric/ferrous ratio, precipitating agent, pH, temperature, ionic strength, stirring velocity, surfactants addition, and working under inert atmosphere [10, 15].

Processes of the nucleation and the growth of crystals are involved in this technique. Both processes should take place separately, i.e., first the nucleation and then the growth. According to Wu et al. [10], pH is an important parameter to address toward the nucleation (solution pH minor than 11) and the growth (solution pH major than 11) of crystals. Even though the coprecipitation method is widely used to produce iron oxide particles with high saturation magnetization, some limitations like broad and large size distribution, poor crystallinity,

aggregation, and tendency to oxidize can be cited. For instance, the formation of impurities (e.g., maghemite) depends on the initial and final solution pH as well as the reaction temperature [16]. Moreover, impurities are also observed in very small particles (minor than 20 nm) because of the high surface area/volume ratio, which allows a great number of surface atoms. This happening could lead to the formation of maghemite, for example, as a consequence of the oxidation of Fe2+ to Fe3+ ions. The maghemite can also be present when the magnetic sample is stored for an extensive period (6 months) as well as exposure to high temperatures (superior to 180°C) [16]. Since maghemite is also a ferrimagnetic material but with a slightly minor saturation magnetization than magnetite, the presence of maghemite in the magnetic sample may not be considered a disadvantage. However, the presence of hematite is an unwanted impurity since this oxide is weakly ferromagnetic and exhibits low saturation magnetization.

Our research group has used the coprecipitation method for the obtainment of magnetic particles to be used as a matrix for biomolecules immobilization [17, 18]. Among these materials, some magnetic composites were also synthesized. For this, materials (without magnetic property) can be added to the FeCl3.6H2O/FeCl2.4H2O mixture before the magnetization aiming different purposes: magnetic particle composite synthesis.

Maciel et al. [19] synthesized a magnetic levan; that is, under a polymer of fructose, the iron oxide particles were formed. The magnetic levan particles were treated with sodium periodate (NaIO4) and employed as a support for trypsin covalent immobilization.

Rêgo et al. [20] have reported a gum magnetic from *Parkia pendula* seeds as a matrix for concanavalin A (Con A) covalent immobilization. For this to be possible, seed gum was included in the solution containing the Fe3+ and Fe2+ ions. Afterward, it was functionalized with NaIO4 allowing the covalent immobilization of the lectin Con A.

Mercês et al. [21] described the process to convert Dacron to magnetic Dacron-hydrazide (mDAC). Heparin (HEP) was activated by carbodiimide and N-hydroxysuccinimide and covalently linked to mDAC (mDAC-HEP). Human antithrombin was then purified by affinity chromatography using the mDAC-HEP.

Alves et al. [4] prepared a magnetic composite from azocasein and iron oxide particles (mAzo). The presence of azocasein, azo-dye-insoluble casein derivative, on the surface of the magnetic particles allowed trypsin to be purified by affinity binding. The enzyme forms complex with the modified substrate but does not hydrolyze it. Washing the enzyme-azocasein-magnetic particles removes unspecific proteins of the mixture. Afterward, the complex is disrupted by increasing the ionic strength, and the enzyme is collected in the supernatant.

Due to the inorganic nature of the iron oxides, the magnetic particles do not have chemical groups to enable biomolecules to bind covalently. Therefore, additional procedures should be performed for this purpose (functionalization). Two approaches can be carried out: (1) coating them with polymers containing these chemical groups and (2) adding materials encompassing these chemical arms during or after the magnetic particle synthesis (composites). In this context, Cabrera et al. [22] used the 3-aminopropyltriethoxysilane (APTES) as a silane agent to available amine groups on the surface of the magnetic diatomaceous earth (mDE). The treatment with APTES was carried out after the synthesis of the mDE particles. The composite material (mDE-APTES) showed efficiency as a matrix to immobilize invertase. Furthermore, Cabrera et al. [2] have also reported a simple, effective, and inexpensive synthesis methodology to obtain a magnetic composite made from mDE particles coated with polyaniline (mDE@PANI). The coating with PANI was carried out after preparation of the mDE particles. Three industrial enzymes (invertase, β-galactosidase, and trypsin) were successfully immobilized using glutaraldehyde as

**33**

**Figure 2.**

*Magnetic Bio-Derivatives: Preparation and Their Uses in Biotechnology*

a chemical arm on the mDE@PANI particles. All magnetic bio-derivatives displayed superior performance (related to catalytic activity and stability) compared to the free enzyme. Thus, the mDE@PANI particles showed potential as a matrix to immobilize other biomolecules. Tannase was also covalently immobilized onto the mDE@ PANI particles [3]. This enzyme catalyzes the hydrolysis of tannins present in several beverages. **Figure 2** displays the preparation of functionalized magnetic particles for

*Approaches to prepare functionalized magnetic particles. (A) Functionalized by coating process and* 

Before proceeding with the immobilization process, essential aspects such as material and surface science, biomolecule chemistry, and reaction engineering should be evaluated. A satisfactory immobilization will be achieved when all these aspects are properly integrated. However, this is not very easy due to the multidisciplinary nature of problems along with the main effects occurring in different length

It is well known that an immobilized derivative presents, in the majority of times, minor retention of their catalytic activity when compared to its free counterpart. This happens for two reasons: (i) conformational changes of the biomolecule due to covalent bond with the support and (ii) problems arising of the catalytic reaction occur in a heterogeneous environment [24]. So, analyzing the behavior of biomolecule immobilized could help in choosing the most suitable particle size for

In the last times, the "nanoimmobilization" is widely used for several researchers to indicate nanostructures as support for the immobilization of biomolecules. The employment of NPs presents various benefits due to unique physical properties resulting from their nanometer size (below 100 nm). Higher surface area, significant biomolecule loading, superior mass transfer resistance, and minor diffusion

biomolecule immobilization or protein purification.

**2.3 Micro or nano iron oxide particles?**

*(B) functionalized by the addition of chemical groups.*

scales from nanometer to millimeter [23].

the material used as support.

*DOI: http://dx.doi.org/10.5772/intechopen.85748*

*Magnetic Bio-Derivatives: Preparation and Their Uses in Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.85748*

**Figure 2.**

*Applied Surface Science*

composite synthesis.

covalent immobilization.

exhibits low saturation magnetization.

aggregation, and tendency to oxidize can be cited. For instance, the formation of impurities (e.g., maghemite) depends on the initial and final solution pH as well as the reaction temperature [16]. Moreover, impurities are also observed in very small particles (minor than 20 nm) because of the high surface area/volume ratio, which allows a great number of surface atoms. This happening could lead to the formation of maghemite, for example, as a consequence of the oxidation of Fe2+ to Fe3+ ions. The maghemite can also be present when the magnetic sample is stored for an extensive period (6 months) as well as exposure to high temperatures (superior to 180°C) [16]. Since maghemite is also a ferrimagnetic material but with a slightly minor saturation magnetization than magnetite, the presence of maghemite in the magnetic sample may not be considered a disadvantage. However, the presence of hematite is an unwanted impurity since this oxide is weakly ferromagnetic and

Our research group has used the coprecipitation method for the obtainment of magnetic particles to be used as a matrix for biomolecules immobilization [17, 18]. Among these materials, some magnetic composites were also synthesized. For this, materials (without magnetic property) can be added to the FeCl3.6H2O/FeCl2.4H2O mixture before the magnetization aiming different purposes: magnetic particle

Maciel et al. [19] synthesized a magnetic levan; that is, under a polymer of fructose, the iron oxide particles were formed. The magnetic levan particles were treated with sodium periodate (NaIO4) and employed as a support for trypsin

for concanavalin A (Con A) covalent immobilization. For this to be possible, seed gum was included in the solution containing the Fe3+ and Fe2+ ions. Afterward, it was functionalized with NaIO4 allowing the covalent immobilization of the lectin Con A. Mercês et al. [21] described the process to convert Dacron to magnetic Dacron-hydrazide (mDAC). Heparin (HEP) was activated by carbodiimide and N-hydroxysuccinimide and covalently linked to mDAC (mDAC-HEP). Human antithrombin was then purified by affinity chromatography using the mDAC-HEP. Alves et al. [4] prepared a magnetic composite from azocasein and iron oxide particles (mAzo). The presence of azocasein, azo-dye-insoluble casein derivative, on the surface of the magnetic particles allowed trypsin to be purified by affinity binding. The enzyme forms complex with the modified substrate but does not hydrolyze it. Washing the enzyme-azocasein-magnetic particles removes unspecific proteins of the mixture. Afterward, the complex is disrupted by increasing the ionic

Due to the inorganic nature of the iron oxides, the magnetic particles do not have chemical groups to enable biomolecules to bind covalently. Therefore, additional procedures should be performed for this purpose (functionalization). Two approaches can be carried out: (1) coating them with polymers containing these chemical groups and (2) adding materials encompassing these chemical arms during or after the magnetic particle synthesis (composites). In this context, Cabrera et al. [22] used the 3-aminopropyltriethoxysilane (APTES) as a silane agent to available amine groups on the surface of the magnetic diatomaceous earth (mDE). The treatment with APTES was carried out after the synthesis of the mDE particles. The composite material (mDE-APTES) showed efficiency as a matrix to immobilize invertase. Furthermore, Cabrera et al. [2] have also reported a simple, effective, and inexpensive synthesis methodology to obtain a magnetic composite made from mDE particles coated with polyaniline (mDE@PANI). The coating with PANI was carried out after preparation of the mDE particles. Three industrial enzymes (invertase, β-galactosidase, and trypsin) were successfully immobilized using glutaraldehyde as

strength, and the enzyme is collected in the supernatant.

Rêgo et al. [20] have reported a gum magnetic from *Parkia pendula* seeds as a matrix

**32**

*Approaches to prepare functionalized magnetic particles. (A) Functionalized by coating process and (B) functionalized by the addition of chemical groups.*

a chemical arm on the mDE@PANI particles. All magnetic bio-derivatives displayed superior performance (related to catalytic activity and stability) compared to the free enzyme. Thus, the mDE@PANI particles showed potential as a matrix to immobilize other biomolecules. Tannase was also covalently immobilized onto the mDE@ PANI particles [3]. This enzyme catalyzes the hydrolysis of tannins present in several beverages. **Figure 2** displays the preparation of functionalized magnetic particles for biomolecule immobilization or protein purification.

#### **2.3 Micro or nano iron oxide particles?**

Before proceeding with the immobilization process, essential aspects such as material and surface science, biomolecule chemistry, and reaction engineering should be evaluated. A satisfactory immobilization will be achieved when all these aspects are properly integrated. However, this is not very easy due to the multidisciplinary nature of problems along with the main effects occurring in different length scales from nanometer to millimeter [23].

It is well known that an immobilized derivative presents, in the majority of times, minor retention of their catalytic activity when compared to its free counterpart. This happens for two reasons: (i) conformational changes of the biomolecule due to covalent bond with the support and (ii) problems arising of the catalytic reaction occur in a heterogeneous environment [24]. So, analyzing the behavior of biomolecule immobilized could help in choosing the most suitable particle size for the material used as support.

In the last times, the "nanoimmobilization" is widely used for several researchers to indicate nanostructures as support for the immobilization of biomolecules. The employment of NPs presents various benefits due to unique physical properties resulting from their nanometer size (below 100 nm). Higher surface area, significant biomolecule loading, superior mass transfer resistance, and minor diffusion

problems are some of the advantages of the use of nanoparticles as a matrix. Also better stability and performance of biocatalyst immobilized along with a low protein unfolding were also reported [25, 26]. Some disadvantages for the NPs (e.g., large-scale application and price of material preparation) have been presented [26]. On the other hand, magnetic nanoparticles (MNPs) have been used as a support of several biological molecules as well as present superparamagnetism (i.e., magnetic response is only observed after application of magnetic field) and can be recovered by the use of a magnet [27]. So, the magnetic property as a plus feature to the nanoparticles would make them more attractive not only for potential uses in biotechnology [14] but also for a lot of biomedical applications including magnetic hyperthermia [28], drug delivery [29], contrast agent in magnetic resonance imaging (MRI) [30], and cellular therapy [31].

As above mentioned (Section 2.2), some factors influence the size (micro or nano) of iron oxide particles. For instance, Maciel et al. [17] assessed the effect of the temperature and the nature of the precipitating agent (strong base) to produce iron oxide nanoparticles. The authors employed sodium hydroxide (NaOH) as the precipitating agent and carried out the synthesis at low temperature (50°C). Small magnetic nanoparticles with a diameter near to 15 nm were obtained and used as a matrix to immobilize trypsin. The magnetic bio-derivative displayed about 90% retention of specific activity after five reuses. Despite Cabrera et al. [2] have reported mDE@PANI nanoparticles (~12 nm) as a promising matrix to immobilize trypsin, the immobilized derivative (mDE@PANI-TRYP) retained 75 and 60% of its initial activity after five and nine cycles of reusability, respectively. The decrease of catalytic activity could be attributed to the loss of the magnetic bio-derivative (mDE@PANI-TRYP) during the washing process after each reusability cycle. After coating with polyaniline, the mDE@PANI showed better stability in suspension. Thus, working with very small particles and good colloidal stability can lead to loss of the immobilized derivative.

Therefore, the choice of micro or nanoparticles as support will depend on several factors including colloidal stability of the particles, operating conditions, and application of the immobilized derivative, among others.
