ENZYME

IMMOBILIZATION
• Enzymes have extensive applications in food industries such as
baking, dairy products, starch conversion and beverage
processing (fruit, vegetable juices, beer and wine).
• In textile industries, they have found a special place due to their
effect on end products.
• In industries such as paper and pulp making and detergents, the
use of enzymes has become a necessary processing strategy.
• Some of the major class of industries such as health care &
pharmaceuticals and chemical manufacturing have been
increased due to the catalytic nature of enzymes.
• Another major application of enzymes is in waste management
especially for solid wastes treatment and waste water purification.
• Past few years have marked the significance of enzymes in
production of biofuels such as biodiesel, bioethanol, biohydrogen
and biogas from biomass conversion.
• However, all these desirable characteristics of enzymes
and their widespread industrial applications are often
obstructed by their lack of long-term operational
stability, shelf life and by their recovery & reusability.
• Enzyme immobilization is one of the strategies to
overcome these problems.
• Immobilization is defined as the physical confinement
of the viable biocatalysts, including enzymes and whole
cells, to a certain defined region of space known as the
carrier or support material to limit its free movement
while preservation of their viability and catalytic
functions.
• The first discovery of this process was by Nelson and Griffin
1961, when they find that invertase is immobilized
(adsorbed) on charcoal and able to hydrolyze sucrose.

• After this publication, several researches were published

about immobilization of several proteins by covalent
bonding with saving its biological activity.

• It can be referred to this process as immobilization of

biocatalysts (enzymes or cells).

• These facts suggest that energy saving, resources saving,

low pollution process and low waste products could be
achieved by using immobilization of the biocatalysis agent.

• Furthermore, the recovery of the product from the reaction

mixture is a problem when the biocatalysts are used in a free
form.
• The main objective of enzyme immobilization is to
maximize the advantages of enzyme catalysis, which is
possible by using a support with low synthesis cost and
high binding capacity.
• Currently commercial application of immobilized
enzyme have been enhanced as they are highly
efficient.
• Further, its resistance to various environmental changes
such as pH or temperature has been increased during
immobilization of enzyme on solid support.
• Compared to their free forms, immobilized enzymes are
generally more stable and easier to handle.
• In addition, the reaction products are not contaminated
with the enzyme which is useful in the food and
pharmaceutical industries.
• Moreover, in the case of proteases, the rate of the
autolysis process can be dramatically reduced upon
immobilization only, if a multipoint or multisubunit
immobilization is achieved, or if a favourable enzyme
environment is obtained.
• Additionally, immobilization also improves many
properties of enzymes such as performance in organic
solvents, pH tolerance, selectivity, heat stability or the
functional stability.
• Increasing the structural rigidity of the protein and
stabilization of multimeric enzymes prevents
dissociation-related inactivation.
• The attached enzyme is again ready for the
subsequent reactions without the need for repeated,
time consuming, and costly extraction and
purification procedures.
• These alterations result from structural changes
introduced into the enzyme molecule by the applied
immobilization procedure and from the creation of a
microenvironment in which the enzyme works,
different from the bulk solution.
• The major components of an immobilized enzyme
system are:
1. The enzyme,
2. The support,
3. The mode of attachment of the enzyme to the
matrix.
• The term solid-phase, solid support, support, carrier,
and matrix are used synonymously.
• The materials used as Carrier matrices are usually
inert polymers or inorganic materials.
• The characteristics of the matrix are of paramount
importance in determining the performance of the
immobilized enzyme system.
• Properties for an Ideal support are:
1. Physical resistance to compression,
2. Hydrophilicity,
3. Inertness towards enzymes,
4. Ease of derivatization,
5. Bio-compatibility,
6. Resistance to microbial attack,
7. Availability at low cost
• The physical characteristics of the matrices (such as
mean particle diameter, swelling behavior, mechanical
strength, compression behavior) will be of major
importance for the performance of the immobilized
systems and determine the type of reactor used under
technical conditions (i.e., stirred tank, fluidized, fixed
beds).

• In particular, pore parameters and particle size

determine the total surface area and thus critically affect
the capacity for binding of enzymes.

• Therefore, porous supports are in general preferred

because the high surface area allows a higher enzyme
loading and the immobilized enzyme is more protected
from the environment.
 TYPES
OF
IMMOBILIZATION
 a. adsorption
b.covalent
binding
c. entrapment
d.membrane
confinement

Immobilised enzyme systems.

(a) enzyme non-covalently adsorbed to an insoluble particle;
(b) enzyme covalently attached to an insoluble particle;
(c) enzyme entrapped within an insoluble particle by a cross-
linked polymer;
(d) enzyme confined within a semipermeable membrane.
 ADSORPTION
• Adsorption of enzymes onto insoluble supports is a very simple
method of wide applicability and capable of high enzyme loading
(about one gram per gram of matrix).
• Simply mixing the enzyme with a suitable adsorbent, under
appropriate conditions of pH and ionic strength, followed, after a
sufficient incubation period, by washing off loosely bound and
unbound enzyme will produce the immobilised enzyme in a
directly usable form.
• The driving force causing this binding is usually due to a
combination of hydrophobic effects and the formation of several
salt links per enzyme molecule.
• The particular choice of adsorbent depends principally upon
minimising leakage of the enzyme during use.
• Although the physical links between the enzyme molecules
and the support are often very strong, they may be reduced by
many factors including the introduction of the substrate.
• Care must be taken that the binding forces are not weakened
during use by inappropriate changes in pH or ionic strength.
• Examples of suitable adsorbents are ion-exchange matrices,
porous carbon, clays, hydrous metal oxides, glasses and
polymeric aromatic resins.
• Ion-exchange matrices, although more expensive than these
other supports, may be used economically due to the ease with
which they may be regenerated when their bound enzyme has
come to the end of its active life; a process which may simply
involve washing off the used enzyme with concentrated salt
solutions and re-suspending the ion exchanger in a solution of
active enzyme.
 Schematic diagram showing the effect of soluble enzyme concentration on the
activity of enzyme immobilised by adsorption to a suitable matrix. The amount
adsorbed depends on the incubation time, pH, ionic strength, surface area, porosity,
and the physical characteristics of both the enzyme and the support.
 COVALENT BONDING METHODS
• One of the most common used techniques for enzyme
immobilization. This is performed through the amino acid
residue which is not included in the active site.
• For example: by using the ε- amino group of lysine, β-carboxyl
group of aspartic, hydroxyl group of serine or threonine,
mercapto group of systeine, imidazole group of histidine.
• Each of these groups can reacts with a suitable function group
of the carrier such as diazonium salt, acid-azide, isocyanate,
activated halide alkyle or aldehyde.
• Commonly used matrices :-
agarose, cellulose, poly acrylamides etc.
• Immobilisation of enzymes by their covalent coupling to insoluble
matrices is an extensively researched technique.
• Only small amounts of enzymes may be immobilised by this method
(about 0.02 gram per gram of matrix) although in exceptional cases as
much as 0.3 gram per gram of matrix has been reported.
• The strength of binding is very strong, however, and very little leakage
of enzyme from the support occurs.
• The relative usefulness of various groups, found in enzymes, for
covalent link formation depends upon their availability and reactivity
(nucleophilicity), in addition to the stability of the covalent link, once
formed.
• Lysine residues are found to be the most generally useful groups for
covalent bonding of enzymes to insoluble supports due to their
widespread surface exposure and high reactivity, especially in slightly
alkaline solutions.
• They also appear to be only very rarely involved in the active sites of
enzymes.
 Activation by CNBr
• The most commonly used method for immobilising enzymes on the
research scale (i.e., using less than a gram of enzyme) involves
Sepharose, activated by cyanogen bromide.
• This is a simple, mild and often successful method of wide
applicability.
• Sepharose is a commercially available beaded polymer which is
highly hydrophilic and generally inert to microbiological attack.
• Chemically it is an agarose (poly-{b-1,3-D-galactose-a-1,4-(3,6-
anhydro)-L-galactose}) gel.
• The hydroxyl groups of this polysaccharide combine with cyanogen
bromide to give the reactive cyclic imido-carbonate. This reacts with
primary amino groups (i.e. mainly lysine residues) on the enzyme
under mildly basic conditions (pH 9 - 11.5,).
 Activation by Ethyl chloroformate
• The high toxicity of cyanogen bromide has led to the commercial, if
rather expensive, production of ready-activated Sepharose and the
investigation of alternative methods, often involving chloroformates,
to produce similar intermediates.
• Carbodiimides are very useful bifunctional reagents as they allow the
coupling of amines to carboxylic acids.
• Careful control of the reaction conditions and choice of carbodiimide
allow a great degree of selectivity in this reaction.
 Immobilization of enzyme using Glutaraldehyde
• Glutaraldehyde is another bifunctional reagent which may be used to
cross-link enzymes or link them to supports.
• It is particularly useful for producing immobilised enzyme
membranes, for use in biosensors, by cross-linking the enzyme plus a
non-catalytic diluent protein within a porous sheet (e.g., lens tissue
paper or nylon net fabric).
• The glutaraldehyde bind with two molecules of enzyme (amino group
of the enzyme with the carbonyl group of the glutaraldehyde).
• Several molecules of glutaraldehyde can bind together to form
oligoglutaraldehyde.
• This polymer can bind with several enzyme molecules (cross-linking)
to form the insoluble immobilized enzyme.
• There are numerous other methods available for the covalent
attachment of enzymes (e.g., the attachment of tyrosine groups
through diazo-linkages, and lysine groups through amide
formation with acyl chlorides or anhydrides).
• It is clearly important that the immobilised enzyme retains as
much catalytic activity as possible after reaction.
• This can, in part, be ensured by reducing the amount of
enzyme bound in non-catalytic conformations.
• Immobilisation of the enzyme in the presence of saturating
concentrations of substrate, product or a competitive inhibitor
ensures that the active site remains unreacted during the
covalent coupling and reduces the occurrence of binding in
unproductive conformations.
• The activity of the immobilised enzyme is then simply restored
by washing the immobilised enzyme to remove these
molecules.
 The effect of covalent coupling on the
expressed activity of an immobilised
enzyme.

(a) Immobilised enzyme (E) with its

active site unchanged and ready
to accept the substrate molecule
(S), as shown in (b).

(c) Enzyme bound in a non-productive

mode due to the inaccessibility of
the active site.

(d) Distortion of the active site

produces an inactive immobilised
enzyme.

Both (c) and (d) may be reduced by use of 'spacer' groups between the enzyme
and support, effectively displacing the enzyme away from the steric influence
of the surface.
 ADVANTAGES AND DISADVANTAGES OF
THE COVALENT BINDING METHOD

Advantages:
1- The enzyme doesn't leak from the carrier.
2- The enzyme can be easily interacts with the substrate because
it is in the surface of the carrier.
3- The enzyme stability is often increased due to its strong
binding with the carrier.

Disadvantages:
1- The yield activity of the enzyme is low due to using of toxic
materials during immobilization.
2- The optimal immobilization conditions are difficult to be find.
3- Renewable of the carrier and recovery of the enzyme is
impossible.
 ENTRAPMENT
• The entrapment method of immobilization is based on the
localization of an enzyme within the lattice of a polymer matrix
or membrane.
• The entrapment process may be a purely physical caging or
involve covalent binding.
• Amounts in excess of 1 g of enzyme per gram of gel or fibre may
be entrapped.
• It is done in such a way as to retain protein while allowing
penetration of substrate.
• Entrapment of enzymes within gels or fibres is a convenient
method for use in processes involving low molecular weight
substrates and products.
• However, the difficulty which large molecules have in
approaching the catalytic sites of entrapped enzymes
precludes the use of entrapped enzymes with high
molecular weight substrates.
• Enzymes may be entrapped in cellulose acetate fibres
by, for example, making up an emulsion of the enzyme
plus cellulose acetate in methylene chloride, followed by
extrusion through a spinneret into a solution of an
aqueous precipitant.
• Entrapment is the method of choice for the
immobilisation of microbial, animal and plant cells,
where calcium alginate is widely used.
• It can be classified into: lattice, microcapsule,
membrane, and reversed micelle types.
1- Lattice-Type
• Entrapment involves
entrapping enzymes within the
spaces of a cross-linked water-
insoluble polymer.
• Some synthetic polymers such
as polyarylamide, calcium
alginate, polyvinylalcohol and
natural polymers such as starch
and agar have been used to
immobilize enzymes using this
technique.
• a) Polyacrylamide: This polymer which is used in
electrophoresis analysis. The disadvantage of this
method is the toxicity pf acryl amide.

• b) Alginate gel method: Alginate is extracted from

seaweeds, such as giant kelp (Macrocystis pyrifera).
The chemical constituents of alginate are random
sequences of chains of β-D-mannuronic and α-L-
guluronic acids attached with 1→4 linkages. Alginates
are insoluble in water, but absorb water readily.
Alginate molecules are cross-linked by calcium ions to
form the in-soluble polymer.
•Procedure: A suspension of mixture of
enzyme solution and alginate is passed
through a narrow tube (1mm diameter)
into a solution of calcium chloride. The
insoluble polymer containing the enzyme
is formed in a form of small beads.

- Because of this, calcium alginate beads

can be formed in extremely mild
conditions, which ensure that enzyme
activity yields of over 80% can be routinely
achieved. The advantage of this method is
that the enzymes or microbial cells can be
easily recovered by dissolving the gel in a
sodium solution.
2- Microcapsule-Type:
• This type of entrapment involves enclosing the enzymes
within semi permeable polymer membranes.

• The preparation of enzyme micro capsules requires

extremely well-controlled conditions and the procedures
for micro capsulation of enzymes can be classified as:
• Interfacial Polymerization Method:

In this procedure, enzymes are enclosed in semi

permeable membranes of polymers as follows:

1- An aqueous mixture of the enzyme and hydrophilic

monomer are emulsified in a water-immiscible
organic solvent.

2- Then the same hydrophilic monomer solution is

added to the organic solvent by stirring.
Polymerization of the monomers then occurs at the
interface between the aqueous and organic solvent
phases in the emulsion.

3- The result is that the enzyme in the aqueous phase

is enclosed in a membrane of polymer.
• Liquid Drying:

1- A polymer is dissolved in a water-immiscible organic solvent

which has a boiling point lower than that of water.

2- An aqueous solution of enzyme is dispersed in the organic

phase to form an emulsion.

3- This emulsion containing aqueous micro droplets is then

dispersed in an aqueous phase containing protective colloidal
substances such as gelatin, and surfactants, and a secondary
emulsion is prepared.

4- The organic solvent is then removed by warming in vacuum. A

polymer membrane is thus produced to give enzyme micro
capsules.
 MEMBRANE CONFINEMENT
• The enzyme molecule here are confined to semi-permiable
membrane. Thus in a aqueous phase beg of membrane is free
to move with out allowing enzyme to come out.
• Membrane confinement of enzymes may be achieved by a
number of quite different methods, all of which depend for
their utility on the semipermeable nature of the membrane.
• This must confine the enzyme while allowing free passage for
the reaction products and, in most configurations, the
substrates.
• The simplest of these methods is achieved by placing the
enzyme on one side of the semipermeable membrane while
the reactant and product stream is present on the other side.
• Hollow fibre membrane units are available commercially with
large surface areas relative to their contained volumes (> 20
m2 L-1) and permeable only to substances of molecular weight
substantially less than the enzymes.
• Although costly, these are very easy to use for a wide variety
of enzymes (including regenerating coenzyme systems)
without the additional research and development costs
associated with other immobilisation methods.
• Enzymes, encapsulated within small membrane-bound
droplets or liposomes, may also be used within such reactors.

• Enzyme membranes can be prepared by attaching enzymes

to membrane-type carriers.
• As an example of the former, the enzyme is dissolved in an
aqueous solution of 1,6-diaminohexane.
• This is then dispersed in a solution of hexanedioic acid in the
immiscible solvent, chloroform.
• The resultant reaction forms a thin polymeric (Nylon-6,6) shell
around the aqueous droplets which traps the enzyme.
• Liposomes are concentric spheres of lipid membranes,
surrounding the soluble enzyme.
• They are formed by the addition of phospholipid to enzyme
solutions.
• The micro-capsules and liposomes are washed free of non-
confined enzyme and transferred back to aqueous solution
before use.
 Generalised Comparison Of Different
Enzyme Immobilisation Techniques
Adsorption Covalent Membrane
Characteristics Entrapment
binding confinement

Preparation Simple Difficult Difficult Simple

Cost Low High Moderate High

Binding force Variable Strong Weak Strong

Enzyme leakage Yes No Yes No

Applicability Wide Selective Wide Very wide

Running Problems High Low High High

Matrix effects Yes Yes Yes No

Large diffusional
No No Yes Yes
barriers

Microbial protection No No Yes Yes

 ADVANTAGES

• Enzymes are costly, Immobilization permits their

repeated use.
• The product can be easily separated without any
additional cost.
• Immobilized enzymes can be used in nonaqueous
system.
• Continuous production system can be used.
• Thermo stability of some enzyme can be increased
by immobilization.
• Enzyme can be used at much higher concentration
then free enzyme.
 DISADVANTAGES

• Additional cost.
• It sometime affects stability and activity of enzyme.
• This approach can not be used when one of the
substrates is insoluble.
• Some immobilization methods restricts the diffusion
of substrate.
 CLASSIFICATION OF SUPPORT
Organic Inorganic
Natural polymers Natural minerals
 Polysaccharides: cellulose,
 Bentonite, silica
dextrans, agar, agarose, chitin,
alginate Processed materials
 Proteins: collagen, albumin  Glass (non-porous and
 Carbon controlled pore), metals,
Synthetic polymers controlled pore metal oxides
 Polystyrene
 Other polymers: polyacrylate,
polymethacrylates,
polyacrylamide, polyamides,
vinyl and allyl-polymers
• Natural organic carriers have many functional groups
which stabilize biocatalysts.
• This class of carriers includes: alginate, κ-carrageenan,
chitosan, sawdust, straw, charcoal, plant fibres, corncob,
bagasse, rice, husks of sunflower seeds, diatomite and
mycelium.
• These supports are hydrophilic, biodegradable,
biocompatible, and inexpensive because they are mostly
waste from the food industry.
• However, the possibility of their application in
bioremediation processes is limited because of low
resistance to biodegradation, sensitivity to organic
solvents, and stability in a narrow pH range.
• Synthetic organic carriers have numerous functional
groups with diversified characters.
• This class includes polypropylene, polyvinyl chloride,
polystyrene, polyurethane foam, polyacrylonitrite and
polyvinyl alcohol.
• Their advantage is the possibility to regulate their structure
at the macromolecular level, the selection of the proper
molecular weight, the spatial structure and the manner and
arrangement order of each active functional group in the
chain.
• Furthermore, synthetic supports can be formed into
various shapes (tubes, membranes, coatings, carriers of
various shapes from spherical to oval), and they are easily
available and relatively inexpensive.
• Moreover, during synthesis, the porosity, pore diameter,
polarity and hydrophobicity of the carrier may be controlled.
• Porous supports should have a controlled pore distribution in
order to optimize capacity and flow properties.
• Inorganic carriers (natural and synthetic) have a high
chemical, physical and biological resistance.
• They are represented by magnetite, volcanic rocks,
vermiculite, porous glass, silica-based materials, ceramics and
nanoparticles.
• A significant disadvantage of these carriers is the presence of
a small number of functional groups, which prevents sufficient
bonding of the biocatalyst.
• For that reason they are used in the formation of hybrid
carriers, combining natural polymers and synthetic
nanoparticles.
 MATRICES FOR ENZYME
IMMOBILIZATION
Surface-Bound Enzymes
The physical and chemical properties of the matrices
used for enzyme immobilization are very important as
they are major governing factors of chemical,
biochemical, mechanical and kinetic properties of
immobilized enzymes. The matrix can be biopolymer,
synthetic organic polymer, hydrogels, smart polymer
or inorganic solid.
 BIOPOLYMERS
• They are water-insoluble polysaccharides (e.g., cellulose, starch,
agarose, chitosan, and proteins such as gelatin and albumin).
• The first industrial application of a biopolymer came in 1960,
using immobilized aminoacylase from Aspergillus oryzae onto
DEAE-Sepahdex (modified cellulose with diethylaminoethyl) by
ionic adsorption for production of amino acids.
• The process was performed in a fixed-bed reactor under
continuous operation.
• Since then, several other commercial enzymes were immobilized
onto DEAE-Sephadex.
• In case of immobilized enzyme onto DEAE-Sephadex, a retention
of 70 % enzyme activity was observed with respect to the soluble
enzyme.
• Agarose is an excellent matrix which has been extensively
used.
• In addition to its high porosity which leads to a high
capacity for proteins, some other advantages of using
agarose are hydrophilic character, absence of charged
groups (which prevents nonspecific adsorption of substrate
and products), and commercial availability.
• However, an important limitation of agarose and other
porous supports is the high cost.
• An approach to avoid this problem is the use of reversible
methods of immobilization that allow matrix regeneration
and reuse.
 SYNTHETIC ORGANIC POLYMERS
• The various synthetic organic polymers are being used
for enzyme immobilization are; Eupergit-C (acrylic
resins), Sepa beads FP-EP, Amberlite XAD-7 (porous
acrylic resins) etc.
• Eupergit-C has surface diameter of 170 mm and pore
diameter of 25 nm.
• It is prepared by using compounds: N,N′-methylene-bi-
(methacrylamide), methacrylamide, allyl glycidyl ether
and glycidyl methacrylate.
• It is hydrophilic in nature with chemical and mechanical
stability over a pH range of 0–14.
• It has a high density of oxirane (Ethylene oxide) moieties
on its surface, which is responsible for multi-point
enzyme binding via covalent bonds at either neutral or
alkaline pH thus giving a long term operational stability
in wide pH range.
• After enzyme attachment, the remaining epoxy groups of
oxirane are blocked by mercaptoethanol, ethanolamine
or glycine to prevent non-specific interactions.
• Eupergit-C has been used for several industrial enzymes.
• Major success has been achieved in case of Penicillin
amidase with a reusability of more than 800 washes.
• However, due to diffusional limitations by Eupergit-C,
kinetic properties of immobilized enzymes have been
greatly interpolated.
• Sepa beads consists of functionalized
polymethacrylate-based resin with oxirane groups,
similar to Eupergit C.
• Amberlite XAD-7 is another synthetic matrix used to
immobilize enzymes by simple adsorption, for
example lipases from Humicola lanuginosa, Candida
antarctica and Rhizomucor miehei.
• Sepa-beads are best immobilizing matrix with
respect to Eupergit-C and Amberlite XAD-7 due to
excellent operational stability, reusability and
kinetic properties of immobilized enzymes.
 INORGANIC SOLIDS
• They include alumina, silica, zeolites and mesoporous
silicas.
• They are known to be the cheapest matrix being used for
the immobilization of several industrial or nonindustrial
enzymes.
• Immobilized enzymes which are specifically used in
organic solvents have been very helpful for various
industrial processes, most importantly in packed bed
reactors based on packed granules.
• The immobilized enzyme is stable for several months under
dried conditions and stability.
• Silica has been found to be an excellent matrix for enzyme
immobilization due to their small size (dia. 2–40 nm) and high
surface area ranging from 300–1500 m2 g–1 with very high
stability at elevated temperatures.
• Its surface can be easily functionalized with any moiety, due to
which it can attach any enzyme.
• Immobilized enzymes are either present on the surface or
inside silica based on immobilization protocols.
• Epoxide hydrolase from Aspergillus niger has been
immobilized onto silica by covalent linkage leading to an
immobilized enzyme with retention of 90 % of enzyme activity
with respect to soluble enzyme.
• Immobilized enzymes have a shelf life of thousand times
greater than that of soluble enzymes.
• Inorganic solids have been used in the generation of
protein-coated microcrystals (PCMCs).
• They are prepared by mixing enzyme solutions with a
concentrated salt solution (potassium sulfate) followed by
its drop-wise addition to water-miscible solvent (isopropyl
alcohol) generating micron-sized crystals containing
enzyme on their surface.
• PCMCs have very high stability in the absence of any
solvent for a long period with applicability for wide range
of solvents.
• This method of immobilization causes the least damage to
the three-dimensional structure of the enzyme.
 SMART POLYMER
• The most studied example of smart polymer is thermostable
biocompatible polymer [poly-N-isopropylacrylamide
(polyNIPAM)].
• PolyNIPAM exhibits the unique property of existing in two states;
the solution state when the temperature is lower than 32 °C, and
the polymer state when the temperature is 32 °C.
• Enzyme immobilization is done by mixing the enzyme solution
with solution state of polyNIPAM, i.e. <32 °C followed by an
increase in temperature, which precipitates immobilized enzyme
and subsequently filtration to get immobilized enzyme.
• This method of immobilization causes the least damage to
enzyme conformation.
• The enzyme is attached by covalent linkage with polyNIPAM
involving its vinyl groups with NH2 groups present on enzyme
surface.
• Penicillin G amidase has been successfully immobilized onto
polyNIPAM leading to an immobilized enzyme with a higher
stability and activity almost similar to soluble enzyme.
• Recently, a thermostable polymer has been made using 2-(2-
methoxyethoxy) ethyl methacrylate and oligo(ethylene glycol)
methacrylate (OEGMA).
• The formed polymer resembles poly(ethylene glycol) in terms of
low toxicity and it is anti-immunogenic.
• It is similar to polyNIPAM in terms of existing in solution and
polymer states as a function of temperature.
• Solution state temperature can vary from 26–90 °C depending on
the amount of OEGMA used.
 Entrapment Carriers
• Enzymes can be immobilized by enclosing them inside the
matrices.
• Sol-gel is a metal alkoxides that has been used for the
entrapment of several enzymes.
• Enzyme immobilization into silica sol-gel is prepared by
hydrolytic polymerization of tetraethoxysilane followed by
drying.
• The immobilization method involves drying, which is the
determining factor in the morphology of sol-gel.
• For example, drying by evaporation produces xerogels with
nano-cages and pores while drying in the presence of CO2
produces aerogel with a delicate structure with phenomenal
porosity.
• Silica aerogel is known to be the lightest solid on earth with a
density of 0.001.
• Furthermore, the addition of Celite to sol-gel has led to
increased thermal stability of immobilized lipase.
• When higher alkyl groups are used for sol-gel preparation in
the presence of additives (e.g., isopropyl alcohol, crown ethers,
surfactants and KCl), an increase in Vmax by a factor of 10 has
been observed.
• The addition of silica quartz fiber has improved the mechanical
properties of immobilized enzyme.
• Sol-gel immobilized lipases have been used for the synthesis
of biodiesel by esterifying oil from sunflower seed.
• However, they cannot be used at high substrate concentrations
due to diffusion constraints.
• Experiments are still ongoing that aim to improve the activity
of immobilized enzymes via entrapment by various additives.