The document discusses various methods of immobilizing enzymes and whole cells, including physical and chemical methods. Physical methods include adsorption, entrapment, and microencapsulation, which attach the enzyme without covalent bonds. Chemical methods include covalent binding and cross-linking, which use covalent bonds to strongly attach the enzyme. The properties of immobilized enzymes depend on the method used, with covalent binding providing the strongest attachment but potentially reducing enzyme activity. Immobilization provides benefits such as reusability and stability under process conditions.

2. “The permanent attachment or fixation of
enzymes, Bacteria or other cells (plant or
animal) to a hard substrate or within a
stable matrix via any of a number of
physical and/or chemical means”

3. “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.”

4.  Enzyme
 Matrix
 Mode of attachment
 They are soluble in water. So it is very difficult to
separate the enzyme for reuse in a batch process.
 Enzymes can be immobilized on the surface or
inside of an insoluble matrix either by chemical or
physical methods.

5.  They can also be immobilized in their soluble
forms by retaining them with a
semipermeable membrane.
 The enzymes can be attached to the support
by interactions ranging from reversible
physical adsorption and ionic linkages to
stable covalent bonds.
 As a consequence of enzyme immobilization,
some properties such as catalytic activity or
thermal stability become altered.

6. “Cell immobilization is a technique
to fix whole cells either on a
suitable matrix or on
any support with
retention of its function”.

7.  This method is implemented when the
enzymes are difficult or expensive to extract.
E.g intracellular enzymes
 If a series of enzymes are required in the
reaction; whole cell immobilization may be
used for convenience
 This is only done on a commercial basis when
the need for the product is more justified.
 Cells are absorbed onto support materials

8. PhysicalChemical
Cross
Linking
IonicCovalent
Carrier
Binding
Entrapment
Micro
Encapsulation
Adsorption
Ionic Network
Formation
Precipitation
Performed
Structures
Polymerization

9.  It is intermolecular cross-linking of enzymes by
means of multifunctional
reagents(glutaraldehyde, etc)
 Performed by the formation of intermolecular
cross-linkages between the enzyme molecules by
reagents.

10.  Oldest Method
 Binding of enzymes to water-insoluble
carriers (polysaccharide derivatives)
 Care is required in the selection of
carriers as well as in binding
techniques

11.  Carried out under severe conditions.
 If we need high activity of immobilized enzymes
than immobilization of an enzyme by covalent
binding is carried out under well controlled
conditions.
 The binding forces between the enzyme and
carrier are strong, and the enzyme cannot easily be
lost from carriers even in the presence of
substrates or salts at high concentrations.
 Covalent methods for immobilization are
employed when there is a strict requirement for
the absence of the enzyme in the product.

12.  Carried out under mild
conditions
 Weak Binding forces relative
to others methods
 Leakage of the enzyme from
the carrier may occur after
changes in the ionic strength,
pH of the substrate, or
product solution

13. In physical methods, there is physical
immobilization of enzymes or cell. A physical
connection is established between the support and
the enzyme to immobilize it partly or completely.
Physical methods is divided into following
categories:
 Adsorption
 Microencapsulation
 Entrapment

14.  Simplest way to immobilize enzymes.
 Enzymes can be adsorbed physically on
a surface-active adsorbent by contacting
an aqueous solution of enzyme with an
adsorbent.
 Commonly employed adsorbents are
alumina, ammonium-exchange resins,
calcium carbonate, carbon, cation
exchange resins, celluloses, clays,
collagen, colloid-ion.

15.  Enzymes can be immobilized within
semipermeable membrane microcapsules.
 This can be done by the interfacial
polymerization technique.
 Organic solvent containing one component
of copolymer with surfactant is agitated in
a vessel and aqueous enzyme solution is
introduced.
 The polymer membrane is formed at the
liquid-liquid interface while the aqueous
phase is dispersed as small droplets.

16. “Entrapping is the incorporation
of enzymes into the lattice
of a semipermeable gel,
or enclosing of enzymes in a
semipermeable polymer
membrane, such as
collagen, gelatin, cellulose, triacetate,
polyacrylamide”.

17.  It is the most widely used method, especially in the form of
alginate beads.
 Alginate is a polysaccharide that forms a stable gel in the
presence of cations, with calcium the most frequently used.
Beads of Alginate-containing cells, are formed by dipping a
cell suspension-sodium alginate solution mixture into a
stirred calcium chloride solution.
 Κ-Carrageenan can also be used in similar manner instead
of alginate, using either calcium or potassium.
 Advantage of this method is that the gel can be reversible
by adding EDTA.

18.  Preparations of agar and agarose can be used to
trap plant cells by precipitation.
 The polysaccharides form gel when a heated
aqueous solution is cooled. The gel can be
dispersed into particles in the warm liquid state
by mixing in a hydrophobic phase, e. g. olive oil.
 When particles of the desired size are obtained,
the entire mixture is cooled and this results in
solidification

19.  Gel entrapment by polymerization is
most commonly carried out using
polyacrylamide.
 The toxicity of the initiator and cross-
linking agents used in the
polymerization has caused a loss of cell
viability.

20.  Entrapment in preformed structures involves
some form of open network through which
nutrient medium may pass, but which entraps
plant cells or cell aggregates.
 Such structures can be facilitated by using
cotton fiber, fiberglass mats, reticulate
polyurethane foam, and in a cloth
nonwoven polyester short fibers.

21. Preparation & Characterization Of Immobilized Enzymes
Sr.
#
Charac-
teristics
Physical
Adsorp-
tion
Ionic
Binding
Covalent
Binding
Cross-
Linking
Method
Entrapping
Method
1. Preparation Easy Easy Difficult Difficult Difficult
2. Enzyme
Activity
Low High High Moderate High
3. Substrate
Specificity
Unchange
able
Unchange-
able
Unchange-
able
Unchange-
able
Unchange-
able
4. Binding
Force
Weak Moderate Strong Strong Strong
5. Regeneratio
n
Possible Possible Impossible Impossible Impossible
6. General
Applicability
Low Moderate Moderate Low High
7. Cost Low Low High Moderate Low

22. Cell Matrix
Interaction
Light
Mass
Transfer
Aeration

23.  When using reticulated polyurethane foam, in order
for any immobilized cells to function well, the
volumetric fraction of the foam has to be sufficient
enough for all the cells and the reticulated pores of
the foam is large enough to contain the cells.
 The importance of initial interaction of cells with the
surface of the polyurethane particles, intrusion of
cells into the foam, simultaneous growth,
coalescence of cells in the foam and retention of cells
in the foam must occur.

24.  The transfer of compounds through
immobilized cell matrix is usually assumed to
be by molecular diffusion, as in gel
entrapment.
 The microstructure of immobilization matrix
may bring other types into action such as
capillary and active transports.
 These types of transport mechanism are true
for reticulated foam, membrane and fiber mats
matrices.

25.  The resistance towards mass transfer that
results in a decrease in the transport of
nutrients, can be an advantage in creating
a stress factor for undesired secondary
metabolite synthesis.
 Mass transfer restriction within the
biomass can be minimized by surface
immobilization in a layer form where
there is maximum contact between the
surface of the immobilized cells and the
liquid phase.

26. Relationship between metabolism and
dissolved oxygen concentration is
complex, a conclusion cannot be reached
about the effect of reduced availability of
oxygen in immobilized plant cell system
on secondary metabolite production and
growth.

27.  Metabolism can be affected by periodic
exposure to light, and the quality and intensity
of the light are significant.
 Only the outer cell layers of the cultures in the
immobilized matrix may receive some light.
 This may be advantageous in the case where
some precursors are form in light and some in
dark condition.
 The supply of light to the interior of the
immobilized cell matrix may be possible by the
use of optical fibers.

28. The characteristics of the matrix are of paramount
importance in determining the performance of the mobilized
enzyme system. Ideal support properties include :
 Physical resistance to compression
 Hydrophilicity
 Inertness toward enzymes ease of derivation
 Biocompatibility
 Resistance to microbial attack
 Availability at low cost
 High stability against physical, chemical, and microbial
degradation Pore parameters
 Particle size determine the total surface area

30.  Nonporous supports show few diffusional
limitations and have a low loading capacity.
 Porous supports are generally preferred because the
high surface area allows for a higher enzyme loading
and the immobilized enzyme receives greater
protection from the environment.
 Porous supports should have a controlled pore
distribution in order to optimize capacity and flow
properties.

31. The hydrophilic character is one of the most
important factors determining the level of
activity of an immobilized enzyme.
An excellent matrix that has been extensively
used is agarose.
In addition to its high porosity, which leads to
a high capacity for proteins, some other
advantages of using agarose as a matrix are
hydrophilic character, ease of derivation,
absence of charged groups and commercial
availability

32.  Immobilized enzymes are widely used
for variety of applications.
 The immobilized enzymes are also
widely used in food industry,
pharmaceutical industry, bioremediation,
detergent industry, textile industry, etc.
 Several hundreds of enzymes are
immobilized and used for various large
scale industries.

33.  In food industry, the purified enzymes are used
but during the purification the enzymes will
denature.
 Hence the immobilization technique makes the
enzyme stable.
 The immobilized enzymes are used for the
production of syrups.
 Immobilized beta-galactosidase used for lactose
hydrolysis in whey for the production of baker’s
yeast.

34.  The detergent industry also employs enzymes for
removal of stains.
 The enzymes used in detergent industry are
protease which is used to remove the stains of
blood, egg, grass and human sweat.
 Amylase used to remove the starch based stains
like potatoes, gravies, chocolate.
 Lipase used to remove the stains of oil and fats
and also used to remove the stains in cuffs and
collars.

35.  Cellulase is used for cotton based fabrics in order
to improve softening, color brightening and to
remove soil stains.
 Nowadays Biotech cleaning agents are widely
used in the detergent industries. When compared
to synthetic detergents the bio based detergents
have good cleaning property.

36.  The increasing consumption of fresh water and
water bodies are mixed up with polluted industrial
waste water and the waste water treatments are
necessary at present.
 The sources of dye effluents are textile industry,
paper industry, leather industry and the effluents
are rich in dye colorants.
 Nowadays enzymes are used to degrade the dye
stuffs.
 The enzymes used in the wastewater treatments are

37.  The Horse radish Peroxidases are entrapped in
calcium alginate beads, this method is still in lab
scale research.
 The immobilized laccase enzyme has the ability
to degrade dyes anthracinoid dye, Lancet blue
and Ponceau red.
 Adsorption method is widely used because of its
easy regeneration.
 During the covalent method of immobilization
the conformational change in the enzyme occurs
which will affect the activity of the enzyme.

38.  The enzymes derived from microbial origin are of
great interest in textile industry. The enzymes
such as cellulase, amylase, laccase, pectinase and
these are used for various textile applications such
as scouring, bio polishing, designing, denim
finishing, treating wools etc.
 The processing of fabrics with enzymes requires
high temperatures and increased pH, the free
enzymes does not able to withstand the extreme
conditions.

39.  Hence, enzyme immobilization for this process
able to withstand at extreme and able to maintains
its activity for more than 5-6 cycles.

40.  Enzymes are used for diagnosis and treatment of
diseases in the medical field.
 The inborn metabolic deficiency can be overcome by
replacing the encapsulated enzymes (i.e enzymes
encapsulated by erythrocytes) instead of waste
metabolites, the RBC acts as a carrier for the
exogenous enzyme drugs
 The enzyme encapsulation through the
electroporation is an easiest way of immobilization in
the biomedical field and it is a reversible process for