Immobilization of plant cells

1. IMMOBILIZATION OF
PLANT CELLS
Aachal D. Jain
Msc. II
Paper 1

2. INTRODUCTION
 Plant cell culture has been for sometime
considered as an alternative method for the
production of flavors, colors and pharmaceuticals
to their extraction from plants.
 One of the major limiting factors in the
development of a commercial production system
using plant cell culture has been the production
cost of phytopharmaceuticals.
 Immobilization of plant cells would be one
method of increasing productivity and hence
reducing the costs.

3.  Immobilization is the newest culture technology of
plant cell, and considered as to be the most
“natural”.
 It has been defined as a technique, which
confines the cells to a defined region in a
space while retaining their catalytic activity
and prevents its entry into the mobile phase,
which carries the substrate and product.
 Immobilization of plant cells, protoplast or
embroyos is achieved by binding these materials
onto or within a solid support.

4. NEED FOR IMMOBILIZATION
 Protection from degradation and deactivation.
 Retention of enzyme, enzyme-free products.
 Retention of enzyme, enzyme-free products.
 Cost efficiency.
 Enhanced stability.
 Use as controlled release agents.
 The ability to stop the reaction rapidly by
removing the enzyme from the reaction
Solution (or vice-versa).
 Allows development of multi-enzyme reaction

5. MATRIX/SUPPORT USED IN
IMMOBILIZATION TECHNIQUE
 The matrix/support used should be cheap and easily
available.
 Their reaction with medium and cells should be as
minimum as possible.
 The matrix/supports are grouped into three major
categories:
1. Natural Polymers: Alginate, chitosan and chitin,
starch, cellulose, collagen.
2. Synthetic Polymers: DEAE cellulose, PVC, PEG.
3. Inorganic materials: silica, glass, charcoal,
activated carbon.

6. METHODS OF IMMOBILIZATION

7. ADSORPTION
 It is the oldest and simplest method of cell
immobilization.
 Involves the physical binding of biocatalyst on the
surface of carrier matrix.
 Carrier may be organic or inorganic.
 Types of adsorbents: Cellulose, Polystyrene resin,
Glass, Alumina, Silica gel.
 The process of adsorption involves the weak
interactions like Vander Waal, ionic interactions or
hydrogen bonds.

8. Methods of immobilization by
Adsorption
1) Static Process: This is most efficient technique but
requires maximum time. In this technique, enzyme is
immobilized by allowing it to be in contact with the carrier
without agitation.
2) Dynamic Process: This process typically involves the
admixing of enzyme with the carrier under constant
agitation using mechanical shaker.
3) Reactor loading: This process is employed for the
commercial production of immobilized enzymes. The
carrier is placed into the reactor and enzyme solution is
transferred to the reactor with agitation of the whole content
in the reactor.
4) Electro-Deposition: In this technique, carrier is placed in
the vicinity of one of the electrode in an enzyme bath and
electric current is applied leading to migration of enzyme
towards the carrier. This results in deposition of enzyme on

9. Advantages Disadvantages
 Little or no
conformation change
of the enzyme.
 Simple and cheap.
 No reagents are
required.
 Can be Recycled,
Regenerated &
Reused.
 Efficiency is less.
 Slow method.
 Desorption of the
enzyme protein
resulting from changes
in temperature, pH,
and ionic strength.

10. CROSS LINKING
 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.
 It is used mostly as a means of stabilizing adsorbed
enzymes and also for preventing
leakage from polyacrylamide gels.
 The most common reagent used for
cross-linking is glutaraldehyde.
 No matrix or support is involved.

11. Advantages Disadvantages
 Very little
desorption(enzyme
strongly bound).
 Best used in
conjunction with other
methods.
 Cross linking may
cause significant
changes in the active
site.
 Not cost effective.

12. COVALENT BONDING
 Involves the formation of covalent bond between
enzyme and support that forms a stable complex
 Most widely used method for immobilization.
 The most common technique is to activate a
cellulose-based support with cyanogen bromide,
which is then mixed with the enzyme.
 The protein functional groups which could be
utilized in covalent coupling are: amino,
carboxylic, phenol ring, and indole group.
 Carriers/support used: cellulose, agarose etc.

13. Methods of covalent bonding
1. Diazoation: bonding between amino group of
support and thyrosil or histidyl group of
enzyme.
2. Peptide bond: between amino/carboxyl
groups of support and enzyme.
3. Poly functional reagents: Use of bi-
functional or multi-functional reagent
(gluteraldehyde) which forms the bonding
between amino groups of support and
enzymes.

14. Advantages Disadvantages
 The strength of
binding is very strong,
so, leakage of enzyme
from the support is
absent or very little.
 This is a simple, mild
and often successful
method of wide
applicability
 Enzymes
are chemically
modified and so
many are
denatured during
immobilization.
 Only small
amounts of
enzymes may be
immobilized (about
0.02 grams per
gram of matrix).

15. ENTRAPMENT
 The entrapment method of immobilization is based
on localization of an enzyme within the lattice of
polymer matrix, gels or capsule.
 Retains enzyme and allows the penetration of
substrate.
 Polymers like polyacrylamide, collagen, cellulose
acetate, calcium alginate or carrageenan are used
as the matrices.
 It can be classified into lattice and micro capsule
type.
 Polymers like polyacrylamide, collagen, cellulose

16. Methods of Entrapment
1. Inclusion in gels: Enzymes trapped in gel. Eg: Poly
acrylamide gel, Poly vinyl alcohol gels.
2. Inclusion in fibers: Enzymes supported on fibre format.
Eg: Cellulose and Poly -acryl amide gels.
3. Inclusion in micro capsules: Enzymes trapped on
microcapsules made by monomer mixtures of polyamine,
polybasic acid chloride monomers.

17. Advantages Disadvantages
 Easy to perform
 Loss of enzyme
activity upon
immobilization is
minimized.
 The enzyme can leak
into the surrounding
medium.
 Another problem is the
mass transfer
resistance to
substrates and
products.
 Substrate cannot
diffuse deep into the
gel matrix.

18. ECAPSULATION
 Enclosing cells in a semi permeable membrane
capsule.
 Capsule is made up of nitro cellulose or nylon.
 Effectiveness depends upon stability of enzymes.

19. Advantages Disadvantages
 Cheap and simple
method
 Large number of cells
can be immobilized by
encapsulation.
 Pore size limitation.
 Only small substrate
molecules are
allowed to cross the
membrane.

20. APPLICATIONS OF PLANT CELL
IMMOBILIZATION
Enhanced production of secondary metabolites:
Eg: capsaicin, L- DOPA, methyl xanthines etc.
Biotransformation: Bioconversion of β methyl
digitoxin has been achieved using Digitalis lanata
immobilized cell cultures upto 70 days.
Other uses:
1. It is used in synthetic seed technology.
2. It can be used for transport of protoplasts.
3. Immobilized plant cells can be cultured as single
cells for prolonged period.

21. THANK YOU