ENZYME IMMOBILIZATION (Pt.1)

Enzymes are biological catalyst that promotes the transformation of chemical species in living systems. From biotechnological view, they are of immense importance and diverse potential. However, for large extent commercialization of these bioderived catalyst, enzymes  reusable factor becomes mandatory. Failing which they would no longer be economical. Moreover, most enzymes are relatively unstable, there cost of purification is high, it is technically very difficult to recover the active enzyme from the reaction mixture after use and maintenance of their structural stability during biochemical reaction is also very high challenging. Enzymes can catalyzes reactions in different states- they can act as individual molecules in reactions, they can be attach to surfaces or they can work in aggregates with other entities. The attachment or rather "immobilized" state of enzyme has been of interest for industrial purpose.
 The term "immobilized enzymes" refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities and which can be used repeatedly and continuously".

METHODS OF ENZYME IMMOBILIZATION:

1. IRREVERSIBLE :

As the name suggests during this process of Irreversible enzyme immobilization, enzyme when binds to the support cannot be retrieve back after the process is complete. That is to say that there is a complete loss of enzyme activity or the support becomes inactive when the support and enzyme are detached.

• COVALENT BONDING: 

  It is a conventional method for immobilization. This method can be achieved by attachment of the concerned enzymes to the support material through covalent linkage. In most instances, the immobilized procedure consists of at least two steps : (a) activation of the support material and (b) enzyme attachment . The covalent binding is usually formed between the functional group of the support material and the enzyme surface that contains the amino acids residues. The Amino acid residues in the covalent binding are the sulfhydryl groups of cystine, hydroxyl group of serine and threonine. In labs, a wide variety of support have been used for enzyme immobilization. for e.g., charcoal, cellulose, synthetic polymers. One prime advantage of this method is that because of the stable nature of bond formed between enzyme and support material (matrix), the enzyme is not realized into the solution upon use i.e., the enzyme should not be present in the product. However, in order to achieve high levels of bound activity the amino acid residues essential for catalytic activity should not be involve in the covalent linkage to the support matrix. However, because of the covalent nature of the bond, the support material has to be discarded together with the enzyme once the activity decays. The attachment between the enzyme and the support material could either be a direct linkage or through a spacer arm. The potentiality of using a spacer arm is that it provides a greater degree of mobility to the enzyme thus, resulting in higher activity than direct attachment.

• ENTRAPMENT:   

  It is caging of enzymes by covalent or non-covalent bonds within the gels or fibers such that it allows the substrate and products to pass through but retains the enzymes. In this approach, an enzyme is added to the solution of monomers before the gel is formed. Gel formation is very initiative either by altering the temperature or by adding a gel inducing chemical. As a result, the enzyme becomes trap in the gel. This method differs from the covalent coupling methods described above as over here the enzyme is not bound to matrix or membrane but rather is entrapped inside the gel. The advantage of entrapment method is that it is quick, inexpensive or requires mild conditions for reaction process. Moreover, the support matrix protects the enzyme from contamination of microbes, proteins and other enzyme. However, the practical use of this method is limited by restriction of mass transfer through membrane or gels. Therefore, entrapment of enzyme within gel or membrane is a convenient method for use in processes involving low molecular weight substrates and products. Efficient entrapment has been achieved with alginate-gelatin-calcium-hybrid carriers which prevents enzyme leakage and provide increased mechanical stability.

• ENCAPSULATION:

  It provides large surface area to enzyme to contact with the substrate. This method is useful because several enzymes can be immobilized in a single step. Enzymes are immobilized  within micro-capsules. These are prepared from organic polymers. The membrane encloses the enzyme and remains semi-permeable to the substrate and products. This is an easy and economical technique. The stability of a biocatalyst in a solution is an important aspect of this technique. This method is not applicable for high molecular weight substrates. Leakage of enzymes takes place through  micro-capsules. Using this technique. capsules of suitable size can be obtained. The diffusion limitation can be also controlled.

• CROSS-LINKING:

  This  method is based on the formation of covalent bonds between the enzyme molecules by means of multi-functional reagents leading to 3-D cross-linked aggregates. Among the most popular cross-linkers are glutaraldehyde aliphatic diamines. Cross-linking can be both intra- and inter molecular. The major advantage of cross-linking is that very less deabsorption of enzyme takes place as it is strongly bound. This method is best used in conjunction with other methods. However, the major disadvantage of it is that it causes significant chances to active site. thus, resulting in partial loss of enzymes activity,. 

  2. REVERSIBLE :

  Contrary to irreversible enzyme immobilization, during reversible enzyme immobilization, the enzyme under mild conditions can be separated from the support after the process is complete. This is due to the type of interaction involved (non-covalent bonds- hydrophobic interactions, Hydrogen bonding, Vander Waals etc.) in the formation of enzyme-support complex. This method of enzyme immobilization is highly economical because of the advantage presented by it. The support can be regenerated and attached to fresh enzyme for the next cycle of the process. The cost of the support is a prime aspect in the overall process economics.

• ADSORPTION:

  Besides being an easy method to perform with broad applications, this method offers high enzyme-loading. The catalyst went under suitable reaction conditions like temp., pH, incubation time would be absorbed on to the surface of support by simple mixing of the two. Further to this, all the loosely and unbound enzyme could be removed by washing and the stable enzyme support complex can be directly used. Since no reactive forces are involved in the process. Therefore, there are no conformational changes in the enzyme on immobilization. the enzyme can be adsorbed on the matrix by one of the following ways:

(a) NON - SPECIFIC : This is the one of the simplest method for enzyme immob. which is based on                                           ionic binding or physical adsorptions. During ionic binding the enzyme are bound to support matrix by sol-linkages, whereas in the case of physical adsorption it is the non-covalent bonds i.e., H-bonds, Vander Waal forces through which the enzyme is bound to the matrix. The nature of forces involved in no-specific adsorption results in an immob. process that can be reversed by changing the conditions that influence the strength of interaction. Based on the charges of the matrix and protein, strong bonds are formed between the two. However, the enzyme is not distorted and it usually reserves the catalytic activity of enzyme. Such methods are therefore economically attractive but may differ from problems such as enzyme leakage from the matrix when the interactions are relatively weak.

(b) IONIC BINDING: The basic principle of ionic binding is the protein-ligand interactions which                                           involve the salt linkages. Although this method is reversible and simple but in general, it is difficult to find conditions under which the enzyme remains both strongly bound and fully active. The nature of this type of non-covalent immob. could be reversed by changing the polarity, temp. or ionic strengths. However, problems may arise from the use of a highly charged support, when the substrate or product themselves are charged.

(c) HYDROPHOBIC INTERACTIONS:  The bases of this method is not the chemical bonds                                                                                     between the enzyme and support but its an entropically governed interaction that defines this method of immob. The foundation of this method depends on experimental variable like salt conc. , pH, temp. ionic strength. The degree of interaction depends on the chemical nature (specifically the hydrophobicity) of support and the enzymes . The extent of substitution and the size of hydrophobic ligand governs the strength of interaction between the support and enzyme.

(d) AFFINITY BINDING: It is the immob. of enzyme on to the matrix through specific interactions.                                               This method exploits the specificity of the enzyme towards its support (matrix) under different physiological conditions. The remarkable selectivity of the interaction between the matrix and enzyme is a major benefit of the method. Two methods are being followed in affinity immob. either the matrix is pre-coupled to an affinity ligand for target enzyme or the enzyme is conjugated to an entity that develops affinity towards the matrix.

• CHELATION OR METAL BINDING:

  In this method, mainly Zirconium or titanium salts are used and often this method is known as 'metal-linked immob.' The metal hydroxyl or its salt is  precipitated on the support material by either neutralization or heating. The metal salts or hydroxides have the potential to bind with the nucleophilic groups present on the surface of the support material because of the stearic factors, it is impossible for the matrix to occupy all co-ordination positions of the matrix. Therefore, some of the positions remain free to co-ordinate with groups from the enzyme. From practical and operational point of view, this method is simple and specific activities of immob. enzymes by this method have been reported to be relatively high. The major advantage with this method is that the carrier and the enzyme can be separated and hence regenerated by altering the pH. Thus making it a reversible process.