ENZYME IMMOBILIZATION

- restricts the mobility of an enzyme or protein and fixes the enzyme into a state without disturbing its functional
ability
- can reduce the sensitivity of a native enzyme hence increasing the functional efficiency of the enzyme
- “Amino Cyclase” from Aspergillus oryzae in Japan is the first immobilized enzyme

 Methods of Immobilization

A. Adsorption
- Enzyme is adsorbed on the physical outer surface of the support. It can affect the functional ability of enzyme
by blocking its active site.
Carriers used in adsorption can be
(a) Mineral-based support - aluminum oxide, alginate beads
(b) Organic Bimolecular based support – starch, cellulose
(c) Modified ion exchange resin – sepharose

The adsorptive immobilization of enzymes can be done by

1. Static Method - Enzyme is immobilized by allowing it to be in contact with the carrier without
agitation. This is most efficient technique but requires maximum time.
2. Dynamic Method - This process typically involves the admixing of enzyme with the carrier under
constant agitation using mechanical shaker.
3. Reactor loading Method - The carrier is placed into the reactor and enzyme solution is
transferred to the reactor with agitation of the whole content in the reactor. This process is
employed for the commercial production of immobilized enzymes.
4. Electro-deposition Method - In this technique, carrier is placed in the vicinity of an electrode and
the enzymes migrate to carrier in presence of electric current.

B. Covalent Bonding
- The method utilizes chemical groups present on both enzyme and carrier for immobilization.

Example of chemical groups of carriers and enzymes used in bond formation

 Carrier: Carboxyl Group | Enzyme: Phenol ring of tyrosine

Carriers used in covalent bonding can be

(a) Biomolecules - carbohydrates like cellulose
(b) Synthetic molecules – polyacrylamide
(c) Protein carriers – collagen, gelatin
(d) Inorganic molecules – porous glass, silica

Covalent bonding can be done by

1. Diazoation – the reaction occurs between amino group of the carrier and Tyrosil and Histidyl group of
the enzyme.
2. Peptide Bond – occurs in amino and carboxyl groups of enzyme and carrier.
3. Polyfunctional agent – a multi-functional agent like Gluteraldehyde is used to form bond between
amino group of enzyme and carrier.

C. Entrapment
- the enzymes or cells trapped inside the polymer matrix. Entrapment is carried out by mixing the biocatalyst
into a monomer solution, followed by polymerization initiated by a chemical reaction.
 Matrices used in this method are polyacrylamide, collagen, agar, gelatin, alginate and carrageenan.

Methods of entrapment are

1. Inclusion in the Gel – enzyme is trapped inside the gel, which is formed by the polymer.
2. Inclusions in fibers – enzymes are supported on the fibers (skeleton of the matrix in which the
enzyme is trapped) of the supporting material forming the matrix.
3. Microcapsules – enzymes are trapped in the microcapsules. Most common microcapsules are
polyamines and sodium alginate.

D. Cross-linking process / Copolymerization

- This method is based on the formation of covalent bonds between the enzyme molecules, by means of
multifunctional reagents, leading to three dimensional cross linked aggregates.
- The most common reagents used for cross-linking are gluteraldehyde and diazonium salts.

E. Encapsulation
- An enzyme is encapsulated within a capsule made up of semi-permeable membrane like nitrocellulose, nylon
and hemi-cellulosic structures.
- The effectiveness depends on the stability of the enzyme inside the capsule.

 Effect of Mass Transfer Resistance

- Immobilization of an enzyme transforms a homogeneous (soluble) catalyst into a heterogeneous (insoluble)
system.
- Carrier binding techniques introduce external mass transfer effects between the liquid phase and the solid
surface.
- An enzyme immobilized through binding to a carrier bead and placed in a simple flow may be represented by
the following illustration.

- The change in concentration of a reagent A from [A] bulk to [A]surface takes place in a narrow fluid layer next to the
surface of the sphere.
- In all but the simplest cases, we express the mass transfer rate as:

N A  kc Ap ([ A]s  [ A])
where NA = transfer rate: mole/s
kc = convective mass transfer coefficient: m/s
AP = surface area of the particle: m2
[A] = concentration of solute at the surface and in the bulk,
respectively: mole/m3

 Immobilized Enzyme Systems

 Electrostatic and Steric Effects
When enzymes are immobilized in a charged matrix as a result of a change in the microenvironment of the
enzyme, the apparent bulk pH optimum of the immobilized enzyme will shift from that of soluble enzyme.
The charged matrix will repel or attract substrates depending on the type and quantity of surface charge.
For an enzyme immobilized onto a charged support, the shift in the pH-activity profile is given by

Where:
pHi = Internal pH value
pHe = External pH value
Z = charge (valence) on the substrate
NF = 96 500 coulumb/ eq.g (Faraday Constant )
Ψ = Electrostatic Potential
R= gas constant

The activity of an enzyme toward a high-molecular-weight substrate is usually reduced upon

immobilization to a much greater extent than for a low-molecular-weight substrate. This is mainly because
of steric hindrance by the support.
Immobilization also affects the thermal stability of enzymes. Thermal stability often increases upon
immobilization due to the presence of thermal diffusion barriers and the constraints on protein unfolding.