Immobilization of Enzyme

Definition: Immobilization simply refers to the enticement of an enzyme to a solid support. Enzyme immobilization defines as a process, which encloses the enzyme molecules to an absolute phase from a bulk phase. The bulk phase consists of substrates, effectors and inhibitors.

Enzyme molecule impounds in or on some suitable matrix having definite porosity. The carrier or matrix allows the exchange of medium (containing substrate and product) to which an enzyme molecule confines.

Immobilization restricts the movement of enzymes. It is a very popular method which is now used in many fields like Industrial, Medical, Bioresearch, Food science etc.

Content: Immobilization of Enzyme

  1. History
  2. Components of Immobilization
  3. Methods of immobilization
  4. Advantages of immobilization
  5. Disadvantages of Immobilization
  6. Applications of Immobilized enzymes

History

History of enzyme immobilization includes three steps in the development of immobilized enzyme.

First step: During 1815, immobilized enzymes were used empirically in industrial processes such as the production of acetic acid and wastewater treatment.

Second step: During the 1960s, a single enzyme immobilization system was introduced. Then this system used in the production of L-amino acids, Isomerization of glucose etc.

Third step: During 1970-1995, multiple enzyme immobilization including co-factor regeneration and cell immobilization was introduced.

Components of Immobilization

Major components of an enzyme immobilization include An enzyme, a support matrix and mode of attachment of a catalyst to the carrier.
components of immobilization

Enzyme

It defines as a biomolecule that accelerates many biochemical reactions. It acts as biocatalyst that mediates the conversion of substrate into a product but never used up.

Support matrix

It is a material that uses for the imprisonment of an enzyme.

Ideal properties of support matrix:

  • Mechanical strength.
  • Biocompatible
  • Stable
  • Inertness
  • Regenerability
  • Ease of derivatization (transformation of the substrate into a product)
  • Expansion of enzyme specificity
  • Reduction in product inhibition
  • Reduction of microbial contamination
  • Cost-effective

Classification of a matrix

On the basis of chemical composition, it is of two types: Organic and Inorganic carrier matrix.
types of matrix

Organic matrix: It subdivides into Natural and synthetic polymers.

Natural polymers: It shows favourable compatibility with proteins.

E.g. : Polysaccharides (Cellulose, dextran, agar, agarose, chitin, alginate etc.), Proteins (Collagen, albumin) and Carbon

Synthetic polymers: It shows high chemical and mechanical stability. Polystyrene, polyacrylate, polyacrylamide, polyamides etc. are examples of synthetic polymers.

Inorganic matrix: It subdivides into Natural and Processed minerals.

Natural minerals: E.g.Bentonite, celite, centolite, silica, charcoal etc.

Processed materials: E.g.Porous glass, metals and metal oxides.

Physical characteristics of the matrices:

  • Mean particle size
  • Hydrophilic character
  • Mechanical strength

Mean particle size: It is the parameter which decides the porosity of a carrier. Porous matrix prefers over non-porous matrix because it increases the surface area by which the loading capacity of an enzyme also increases. This property of the matrix determines the total surface area and affect the binding ability of a catalyst. A porous material should have optimized pore distribution to optimize the flow of particles.

Hydrophilic character: It determines the level of activity of an immobilized enzyme.

Mechanical strength: It defines the binding of an enzyme that is inversely related to the ease with which it can inverse.

Methods of immobilization

On the basis of binding property, it classifies into physical and chemical processes.
methods of immobilization

Physical methods of immobilization: It consists of the following types

Adsorption

It is the oldest and simplest method. In this type, enzyme adheres to the surface of the water-insoluble carrier matrix. The binding is nonspecific like electrostatic or hydrophobic affinity binding to a particular ligand. Binding between enzymes to the carrier matrix is usually firm, but weakened by many factors:

  • Addition of substrate
  • pH or ionic strength

Bonding is non-permanent and accomplished by weak bonds mainly: hydrogen bonds and Vander Waal forces.
adsorption type

The matrix used: The matrix particle size must be small (500Å-1mm D).

Examples: Carrier used in this, can be of different types:

  • Mineral support (E.g. Aluminium oxide, clay)
  • Organic support (E.g. Starch)
  • Modified sepharose and ion exchange resins

Methods of immobilization by adsorption:

Static method: It is an efficient but time-consuming method. It involves immobilization of enzyme and carrier molecule without agitation.
Dynamic process: It involves the mixing of an enzyme with the carrier under constant agitation.
Reactor loading: It involves transferring of both enzyme and carrier in the reactor with the agitation of the whole content. Use for the commercial production of the immobilized enzyme.
Electro-deposition: In this, first a carrier places proximal to the electrode in an enzyme bath. Then an electric current is passed.  This result in movement of the enzyme towards the carrier. At last, the enzyme gets deposited on the surface.

Advantages:

  • No pore diffusion limitation
  • Simple method
  • No reagents are required
  • Limited loss of enzyme activity
  • Economical
  • Less disruption to an enzyme.
  • Requires minimum activation steps
  • Can be recycled, regenerated and reused.
  • The high loading efficiency of an enzyme.

Disadvantages:

  • Low surface area for binding
  • Desorption of an enzyme from the carrier
  • Yield is low

Entrapment

In this method, enzyme traps inside a porous polymer or gel matrix. This method is called lattice entrapment. The bonding between an enzyme and matrix can be covalent or non-covalent.

The matrix used: It is water soluble, nature varies with different enzymes.

Examples: Following carrier used in this are:

Polyacrylamide gels, cellulose triacetate, agar, gelatine, alginate etc.

Methods of enzyme entrapment: It consists of the following ways

Inclusion in gels: Involves entrapment of enzyme inside the gel matrix.
Inclusion in fibres: Entrapment of enzyme inside the fibre matrix.
Inclusion in microcapsules: Involves entrapment inside a microcapsule.

entrapment type

Advantages:

  • Enzyme loading capacity is high
  • Fast method
  • Less or no structural distortion
  • Easy to practice

Disadvantages:

  • Diffusion of substrate and product create difficulties
  • Leakage of low molecular weight enzymes.
  • Chances of microbial contamination
  • Enzyme inactivation
  • Loss of enzyme activity
  • Limited industrial use

Encapsulation

It is the membrane confinement method. Enzyme confines within the semipermeable membrane of a capsule in an aqueous solution.

This process allows the exchange of medium (substrate & product), but not an enzyme. The effectiveness of this method depends on the stability of an enzyme.

Matrix used: The capsule made of a semi-permeable membrane can be: polymeric, lipoid, non-ionic etc. in nature.

Examples of semipermeable membrane: nitrocellulose, nylon etc.

Methods of encapsulation: It includes the following means:

Encapsulation in reaction vessel: It involves partitioning of a chamber by a semipermeable membrane. One chamber contains enzymes whereas other contains substrate and product.
Encapsulation by hollow fibre membrane: Involves entrapment of enzyme inside a semipermeable matrix (cellulose, triacetate etc.). In this, enzyme traps inside the empty space of the matrix.
Microencapsulation: By chemical polymerization, enzyme molecules enclose inside a microcapsule by the use of 1-6-diaminohexane.
Encapsulation by liposomes: In this, the enzyme binds to the concentric lipoidal membrane of the liposome by the use of phospholipid.

Advantages:

  • There is no enzyme leakage
  • Does not affect the enzyme activity
  • Simple method
  • The high loading efficiency of an enzyme

Disadvantages:

  • Pore size limitation
  • Not so cost effective

Covalent Binding

It is a widely used method. In this system, enzyme molecule binds to the carrier by a covalent bond. The binding strength is powerful. A complex form through this bonding is stable. There is no enzyme loss during the process.

Covalent binding occurs between the active part, the, i.e. functional group of an enzyme and carrier molecule. The functional group takes part in binding are -NH2, -NH3, -COO, -OH, -SH, -O, -S etc. The order of reactivity of these functional group to the carrier depends upon their charged status:

-S > -SH > -O > -NH2 > -COO > -OH >> -NH3+

Examples of a polymeric carrier used in this process are:

  • Carboxylic acid and related groups of polyglutamic acid
  • Amide group of a polypeptide
  • Amino and related groups of polysaccharides

Some most commonly used polymers are the polysaccharide (celluloses, agarose, sepharose etc.), polyvinyl alcohol, silica and porous glasses.

Methods used for covalent binding:

Diazotation: This process involves bonding between the amino group of the matrix and tyrosyl or histidyl group of an enzyme on reaction with NaNO2 and HCl.

DIAZOTATION

By peptide bond: It involves bonding between amino or carboxyl group of the matrix to the carboxyl or amino group of an enzyme. During this, a matrix is chemically treated to bind with the active functional group.

PEPTIDE BOND

Cyanogen bromide activation: It involves binding of glycol groups of a matrix with an enzyme by the activation of CNBr.

CNBR ACTIVATION

By polyfunctional reagents: This process involves bonding between the amino group of the matrix and amino group of an enzyme. Example: Glutaraldehyde (Bi-functional reagent).

POLY FUNCTIONAL AGENT

Advantages:

  • Strong binding strength
  • Leakage of an enzyme from a carrier is absent
  • Simple and widely used method
  • Not affected by pH or ionic strength.

Disadvantages:

  • Denaturation of the enzyme during immobilization
  • Only a small amount of enzyme can be immobilized
  • Not so cost effective

Cross Linking

It is also called “Copolymerization“. In this, immobilized enzymes covalently link to the various groups of an enzyme via polyfunctional reagents. It does not require a support matrix. Cross-linking leads to the formation of 3D crosslinked aggregates.
Commonly used polyfunctional agents are Glutaraldehyde, diazonium salts etc.

Advantages:

  • Low or no enzyme leakage
  • Higher enzyme stability
  • Simple and cheap method
  • Wide applicability in commercial production

Disadvantages:

  • Cause enzyme inactivation
  • Polyfunctional reagents cause denaturation
  • Not so cost effective

pictorial representation

Advantages of Immobilization

  • Can be reused
  • Decrease the labour input
  • Can be used continuously
  • Increase enzyme and substrate ratio
  • Requires minimum activation time

Disadvantages of Immobilization

  • Industrial use is limited
  • Enzymes  can lose their catalytic property
  • Some proteins lose their stability
  • Enzymes inactivate by heat generation
  • Expensive to carry out

Applications of Immobilized enzymes

Industrial use: E.g. Production of antibiotics, amino acids etc.
Biomedical use: E.g. For the treatment, diagnosis and drug delivery.
In the food industry: E.g. Production of jams, jellies, syrups etc.
Sewage treatment: In the treatment of sewage and industrial effluent for wastewater management.
In the detergent industry: E.g. immobilization of lipases to digest lipid present in stains or dirt.
Textile industry: E.g. Scouring, bio-polishing etc.

Thus immobilization process is widely used in all the fields where the enzyme is immobilized to the certain phase where an enzyme can be reused and stabilize to carry out many reactions

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