**1. Introduction**

Even today, the widespread use of magnetic particles in areas such as biotechnology, engineering, material sciences, biomedicine, and microbiology, among others, still is disclosed in the literature. So, a large part of the scientific community including biomedical, biologists, and pharmacists, in addition to chemists and physicists, is looking for novel applications for magnetic bio-derivatives obtained from biomolecules immobilized on iron oxide particles. The use of magnetic particles is further than matrices for biomolecules immobilization such particles can also be used as a potential contrast agent in magnetic resonance imaging (MRI), drug delivery, magnetic hyperthermia, photothermal therapy, and food treatment in order to better the organoleptic properties [1].

The great importance of developing attractive and promising magnetic bioderivatives as well as bringing to light new applications is evident. Our research group has proposed a considerable number of magnetic materials from inorganic (e.g., diatomaceous earth) or organic (e.g., azocasein) compounds and iron oxides as support to enzyme immobilization or protein purification. Recently, magnetic

#### *Applied Surface Science*

diatomaceous earth coated with polyaniline (mDE@PANI) exhibited good features as a matrix for covalent immobilization of three industrial enzymes (invertase, trypsin, and β-galactosidase) [2]. This magnetic composite was also promising for the treatment of boldo tea after immobilization of the enzyme tannase [3]. Alves et al. [4] reported a novel magnetic composite from magnetite and azocasein to trypsin purification of fish Nile tilapia (crude extract). The obtainment of antioxidant peptides for use in food products was possible by immobilization of protease on magnetic support [5]. These are some of our research involving magnetic materials and their applications that we will address in more detail later.

#### **1.1 Historical background: enzyme immobilization on magnetic particles**

In 1916, the first scientific report on immobilization of enzymes was announced. This finding involved the invertase, a hydrolytic enzyme, which preserved their catalytic behavior after being absorbed on charcoal or aluminum hydroxide surface [6]. Robinson et al. [7] reported the first work of the use of magnetic particles as support to enzyme immobilization in 1973. The authors purposed two magnetic materials from iron oxide (magnetite) and another compound as a matrix to immobilize the enzymes: α-chymotrypsin and β-galactosidase for applications in bioreactors. A year later, Van Leemputten and Horisberger [8] immobilized trypsin and invertase on functionalized magnetite. Since then, magnetic separation has become an increasingly popular tool for the process of separating biological molecules and cells.

According to IUPAC gold book [9], an immobilized enzyme is defined as "a soluble enzyme bound to an insoluble organic or inorganic matrix, or encapsulated within a membrane in order to increase its stability and make possible its repeated or continued use." An efficient and robust immobilized derivative must preserve good retention of the catalytic activity, possess greater thermal and operational stability, be reused without considerable loss of activity, allow the easy separation of products and enzyme, and be resistant to microbial attack. In addition, a derivative immobilized on magnetic particles has advantages such as (i) easy and fast separation of the reaction medium by application of an external magnetic field, (ii) enzyme which is not stressed since conventional methods of separation (e.g., centrifugation and filtration) can be avoided, and (iii) large loading of biomolecules onto small particles (nanoparticles (NPs)) as a consequence of their high surface area.

Since the last decade, several scientific works about immobilization of enzymes have been published. In the first month of 2019, over 100 articles with the keyword "enzyme immobilization" have been reported in the PubMed database.

#### **1.2 Immobilization strategies: Choosing the better approach**

The choice of the immobilization method is as important as the nature of biomolecule (e.g., biochemical properties) and the experimental conditions chosen to obtain an immobilized derivative with desired features. So special attention should be given to immobilization approaches since the applicability of the immobilized derivative depends on this. The characteristics of the support are also relevant; however, it will be discussed later.

Overall, the methods of immobilization are categorized as irreversible and reversible since those interactions between enzyme and support are from weak physical adsorption to strong covalent bonds. Irreversible immobilization is understood as the attachment of the biocatalyst to the support with retention of the biological activity. However, the detachment of the biocatalyst will lead to the loss of its activity. Covalent bond, entrapment or microencapsulation, and cross-linking belong to this category. Already the adsorption and the affinity methodologies are

**29**

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

associated with the reversible category. Additionally, the immobilization methods can be classified into chemical and physical according to support binding. **Figure 1** displays the most used immobilization methods together with some advantages and

*Scheme with major strategies used for enzyme immobilization. Advantages and disadvantages are also highlighted.*

Currently combinations of two or more immobilization methods can be employed to obtain derivative immobilized with features desired. Thus, the limitations from one type of immobilization could be avoided. Briefly, the main features

• **Covalent bond**. This method is based on the formation of covalent bonds between the biocatalyst and the support. For this, the presence of active chemical groups on surface of both components (i.e., biomolecule and support) is necessary. It is important to mention that these functional groups in the biocatalyst are not responsible for their catalytic activity. Moreover, the use of an activating agent, e.g., glutaraldehyde, which will lead the covalent attachment is required. Covalent bond is one of the most immobilization technique used.

• **Entrapment**. In this approach, the biocatalyst is restricted within a polymeric network. The substrate and products can reach the biocatalyst since their molecular weight is low. So, this method is suitable when mass transfer limitations through polymeric network are not a problem. Porous gels, fibers, and microencapsulation are some strategies to entrapping the biocatalyst. The immobilization by entrapment could be by essentially physical forces or include

• **Cross-linking**. An arrangement of insoluble aggregates with high molecular weight is formed by a simple process involving the biocatalyst and bi- or multifunctional reagents or ligands. Due to this method which did not use a support and involve covalent bonds, conformation changes of the biocatalyst are possible leading to the loss of their activity. Glutaraldehyde, a bifunctional agent, is the most generally used for this immobilization technique. Cross-linked enzyme aggregates (CLEAs) have emerged as attractive alternative to produce physical aggregates with preservation of biocatalyst structure and hence their catalytic activity.

• **Adsorption**. This simple, fast, and inexpensive method leads to the formation of noncovalent interactions. Electrostatic adsorption is the linkage approach more used. Unfortunately, the interaction between the biocatalyst and the support may be modulated by some operational parameters such as ionic strength, pH, and temperature. Therefore, this approach is more appropriate

of the immobilization methods will be mentioned as follows.

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

disadvantages.

**Figure 1.**

covalent bonding.

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

**Figure 1.**

*Applied Surface Science*

diatomaceous earth coated with polyaniline (mDE@PANI) exhibited good features as a matrix for covalent immobilization of three industrial enzymes (invertase, trypsin, and β-galactosidase) [2]. This magnetic composite was also promising for the treatment of boldo tea after immobilization of the enzyme tannase [3]. Alves et al. [4] reported a novel magnetic composite from magnetite and azocasein to trypsin purification of fish Nile tilapia (crude extract). The obtainment of antioxidant peptides for use in food products was possible by immobilization of protease on magnetic support [5]. These are some of our research involving magnetic materi-

als and their applications that we will address in more detail later.

(nanoparticles (NPs)) as a consequence of their high surface area.

**1.2 Immobilization strategies: Choosing the better approach**

however, it will be discussed later.

"enzyme immobilization" have been reported in the PubMed database.

Since the last decade, several scientific works about immobilization of enzymes have been published. In the first month of 2019, over 100 articles with the keyword

The choice of the immobilization method is as important as the nature of biomolecule (e.g., biochemical properties) and the experimental conditions chosen to obtain an immobilized derivative with desired features. So special attention should be given to immobilization approaches since the applicability of the immobilized derivative depends on this. The characteristics of the support are also relevant;

Overall, the methods of immobilization are categorized as irreversible and reversible since those interactions between enzyme and support are from weak physical adsorption to strong covalent bonds. Irreversible immobilization is

understood as the attachment of the biocatalyst to the support with retention of the biological activity. However, the detachment of the biocatalyst will lead to the loss of its activity. Covalent bond, entrapment or microencapsulation, and cross-linking belong to this category. Already the adsorption and the affinity methodologies are

**1.1 Historical background: enzyme immobilization on magnetic particles**

In 1916, the first scientific report on immobilization of enzymes was announced. This finding involved the invertase, a hydrolytic enzyme, which preserved their catalytic behavior after being absorbed on charcoal or aluminum hydroxide surface [6]. Robinson et al. [7] reported the first work of the use of magnetic particles as support to enzyme immobilization in 1973. The authors purposed two magnetic materials from iron oxide (magnetite) and another compound as a matrix to immobilize the enzymes: α-chymotrypsin and β-galactosidase for applications in bioreactors. A year later, Van Leemputten and Horisberger [8] immobilized trypsin and invertase on functionalized magnetite. Since then, magnetic separation has become an increasingly popular tool for the process of separating biological molecules and cells. According to IUPAC gold book [9], an immobilized enzyme is defined as "a soluble enzyme bound to an insoluble organic or inorganic matrix, or encapsulated within a membrane in order to increase its stability and make possible its repeated or continued use." An efficient and robust immobilized derivative must preserve good retention of the catalytic activity, possess greater thermal and operational stability, be reused without considerable loss of activity, allow the easy separation of products and enzyme, and be resistant to microbial attack. In addition, a derivative immobilized on magnetic particles has advantages such as (i) easy and fast separation of the reaction medium by application of an external magnetic field, (ii) enzyme which is not stressed since conventional methods of separation (e.g., centrifugation and filtration) can be avoided, and (iii) large loading of biomolecules onto small particles

**28**

*Scheme with major strategies used for enzyme immobilization. Advantages and disadvantages are also highlighted.*

associated with the reversible category. Additionally, the immobilization methods can be classified into chemical and physical according to support binding. **Figure 1** displays the most used immobilization methods together with some advantages and disadvantages.

Currently combinations of two or more immobilization methods can be employed to obtain derivative immobilized with features desired. Thus, the limitations from one type of immobilization could be avoided. Briefly, the main features of the immobilization methods will be mentioned as follows.


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

• **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 antibodies or lectins can be used.
