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CHE1009-BIOCHEMICAL ENGINEERING
MODULE-II
LECTURE – 6

Dr.A.Babu Ponnusami
Associate Professor
SCHEME
 Immobilized Enzyme Systems

Enzyme Immobilization:

- Easy separation from reaction mixture, providing the ability to

control reaction times and minimize the enzymes lost in the
product.

- Re-use of enzymes for many reaction cycles, lowering the total

production cost of enzyme mediated reactions.

- Ability of enzymes to provide pure products.

- Possible provision of a better environment for enzyme activity

- Diffusional limitation
Immobilization Methods
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 Selecting an Immobilization Technique

 It is well recognized that no one method can be regarded as the

universal method for all applications or all enzymes. Consider,
 Widely different chemical characteristics of enzymes
 Different properties of substrates and products
 Range of potential processes employed
 Immobilization by Entrapment
Entrapment Immobilization is based on the
localization of an enzyme within the lattice of a polymer
matrix or membrane.
- retain enzyme
- allow the penetration of substrate.

It can be classified into matrix Entrapment and micro

encapsulation
Matrix Entrapment

Matrix Gel entrapment places the enzyme within the interstitial

spaces of crosslinked, water-insoluble polymer gels.
Example: Polyacrylamide gels
 The solubility of alginate and k-Carrageenan varies with the
cation, allowing these soluble polymers to be crosslinked upon the
addition of CaCl2 and KCl, respectively.
Variations of pore size result in enzyme leakage, even after
washing. The effect of initiator used in polyacrylamide gels can be
problematic.
Advantages:

 Enzymes are not chemically modified

 Enzymes properties are not altered.

Disadvantages:

 Deactivation of enzyme is possible while gel formation

 Enzyme Leakage possible depending on the pore size.

 Diffusional limitation may pose reduced accessibility for

the substrate.
Acrylamide+N-N’-methylenedisacrylamide+Enzyme---Polymer
matrix of cross linked acrylamide

Potasium persulfate- Initiator

Dimethyl Amino Propio Nitrate(DMAPN)-Accelerator
 Immobilization by Entrapment in microcapsule

Microencapsulation : Enclosing the enzymes within spherical,

semi-permeable membranes of 1-300 μm diameter.
Ex: Urethane prepolymers, when mixed with an aqueous enzyme

solution crosslink via urea bonds to generate membranes of varying

hydrophilicity.
Alternatively, photo-crosslinkable resins can be gelled by UV-

irradiation.
Membrane have pores permitting small substrate and product

molecules to enter and leave the capsule.

Advantage of Encapsulation:

 Enzymes are much closure contact with the substrate in the

surroundings.
Disadvantage of Encapsulation:

 High molecular weight substrates have limited diffusivity, and

cannot be treated enzymes.
Entrapment
- Matrix Entrapment - Membrane Entrapment
(microencapsulation)
• Matrix Materials used in Entrapment :

• Organics: polysaccharides, proteins, carbon, vinyl and allyl

polymers, and polyamides.
• Example: Ca-alginate, agar, collagen.

• Immobilization procedures:
Enzyme + polymer solution → polymerization
→ extrusion/shape the particles

• Inorganics: activated carbon, porous ceramic.

• Shapes: particle, membrane, fiber

Challenges in Entrapment Method

- enzyme leakage into solution

- diffusional limitation

- reduced enzyme activity and stability

- lack of control micro-environmental conditions.

It could be improved by modifying matrix or membrane.

Immobilization by Carrier Binding or Surface Immobilization

Attachment of an enzyme to an insoluble carrier creates an active

surface catalyst. Modes of surface attachment classify carrier
methods into physical adsorption, ionic binding and covalent
binding.
Physical Adsorption: Enzymes can be bound to carriers by
physical interaction such as hydrogen bonding and/or van der
Waal’s forces.
 The enzyme structure is unmodified
 Carriers include chitosan, acrylamide polymers and silica-
alumina
 Binding strength is usually weak and affected by temperature
and the concentration of reactants.
Ionic Binding: Stronger enzyme-carrier binding is obtained with
solid supports containing ion-exchange residues.
 cellulose, glass-fibre paper, polystyrene sulfonate

 pH and ionic strength effects can be significant

 Covalent attachment
Covalent attachment of soluble enzymes to an insoluble
support is the most common immobilization technique.
 Amino acid residues not involved in the active site can be
used to fix the enzyme to a solid carrier
Advantages:
1. Minimal enzyme leaching from the support results in stable
productivity
2. Surface placement permits enzyme contact with large
substrates
Disadvantages:
1. Partial modification of residues that constitute the active
site decreases activity
2. Immobilization conditions can be difficult to optimize
(often done in the presence of a competitive inhibitor)
 Covalent Attachment Techniques
Cyanogen bromide activates supports with vicinal hydroxyl groups (polysaccharides, glass beads) to
yield reactive imidocarbonate derivatives:

Diazonium derivatives of supports having aromatic amino groups are activated for enzyme
immobilization:

Under the action of condensing agents (Woodward’s reagent K), carboxyl or amino groups of
supports and amino acid residues can be condensed to yield peptide linkages.
Other methods include diazo coupling, alkylation, etc.
 Most Convenient Residues for Covalent Binding

• Amino acid residues with polar and reactive functional groups are
best for covalent binding, given that they are most often found on
the surface of the enzyme.

• The data shown in next slide is the most convenient residues for
binding in descending order.

• The average percent composition of proteins (reactive residues

only) is shown, along with the number of potential binding
reactions sites.
 Abundance(%)Reactions

+ Lysine (Lys)
 7.0 27
CH2 4 NH3
 3.4 31
CH2 SH Cysteine (Cys)
 3.4 16
CH2 OH Tyrosine (Tyr)

 2.2 13
HN N
Histidine (His)
CH2
O  4.8 4
CH2 C O Aspartic Acid (Asp)

O  4.8 4
CH2 CH2 C O Glutamic Acid (Glu)
 3.8 6
CH2 3 NH C NH2 Arginine (Arg)
+NH2
CH2  1.2 7
Tryptophan (Trp)
N
H
 Immobilization methods
a) adsorption
b) covalent binding
c) entrapment
d) encapsulation
 Immobilization by Crosslinking

Bi- or multi-functional compounds serve as reagents for

intermolecular crosslinking of enzymes,

Creating insoluble aggregates that are effective heterogeneous

catalysts.

Reagents commonly have two identical functional groups which

react with specific amino acid residues.

Common reagents include glutaraldehyde, carbodimide and

diisocyanates,

Involvement of the active site in crosslinking can lead to great

reductions in activity, and the gelatinous nature of the product can
complicate processing.
Cross-linking is to cross link enzyme molecules with each other
using agents such as glutaraldehyde.

Features: similar to covalent binding.

Several methods are combined.

 Properties of support material
 The form, shape, density, porosity, pore size distribution, operational
stability and particle size distribution of the supporting matrix will
influence the result
 The ideal support is cheap, inert, physically strong and stable
 Ideally, it should:
 Increase the enzyme specificity
 Shift the pH optimum to the desired value for the process
 Discourage microbial growth and non-specific adsorption
 Some matrices may possess other properties which are useful for
particular purposes such as
 Ferromagnetism (e.g. magnetic iron oxide, enabling transfer of
the biocatalyst by means of magnetic fields)
 A catalytic surface (e.g. manganese dioxide, which catalytically
removes the inactivating hydrogen peroxide produced by most
oxidases)