The document discusses protoplasts, which are plant cells that have had their cell walls removed, leaving the cell membrane and organelles. It describes methods for isolating protoplasts from plant tissues using either mechanical or enzymatic methods. The enzymatic method uses enzymes like pectinase and cellulase to break down the cell wall. Protoplasts have various applications including isolating cell organelles and studying cell structures. The document also discusses immobilizing enzymes by binding them to inert matrices, which has benefits like reusability and stability. Methods of immobilization include adsorption, covalent binding, and entrapment in gels.

1. PROTOPLAST AND IMMOBILIZATION
BY
Dr.U.Srinivasa,
M.Pharm, Ph.D.

2.  Definition :
 Protoplast is a cell without a cell wall are called
protoplast.
 They contain all the normal cell organelles plus
the nucleus.
 The cell wall of a plant cell can be decomposed
and removed by the treatment of the lytic
enzymes like cellulose and pectinase

3. Isolation of protoplast
 Protoplasts can be isolated from all types of
actively growing young and healthy tissues.
METHODS OF ISOLATION :
1.Mechanical method
2. Enzymatic method
3. Combination of above methods

4.  During the process of isolation, the
cells are first separated by
mechanical method and subsequently
protoplasts are isolated by enzymatic
method

5. Mechanical method
 The cells are first placed in a suitable
plasmolyticum .This treatment makes the
protoplasms of these plasmolysed cells
shrink away from their cell walls( this
makes the removal of cell wall easy)
 Further ,they are cut with a knife.

6.  Then the protoplasts are released from the cells
through the cell wall , and then the tissue is
again deplasmolysed.

7.  Advantages :
 It is suitable method for the isolation of protoplasts from
vacuolated cells. Eg onion bulbs, scales, radish roots
 Dis advantages :
 Poor yield
 Unsuitable for the isolation of protoplasts from
meristematic cells
 Unsuitable for the isolation of protoplasts from less
vacuolated cells

8. Enzymatic method
 Protoplasts can be isolated from aIt is widely
used method.The isolation of protoplasts
requires digestion of cell wall and middle
lamellae. This is affected by the use of
enzymes
 variety of tissues including leaves, roots, in vitro
shoot culture and cell suspension culture

9. Steps
 1.Sterilization of leaves
 2. Peeling of the epidermis
 3. Enzymatic treatment
 4. Isolation and cleaning of the protoplasts

10.  Sterilization of leaves –
 Fully expanded leaves are sterilized by the
following procedure
 A. dipping in 70% ethyl alcohol for about a
minute and then with 2% solution of sodium
hypochlorite for 20-30 minutes
 B. rinsing with sterile distilled water three
times.

11.  Peeling of the epidermal layer :
 The lower epidermis is carefully peeled off
and the stripped leaves are cut into small
pieces.
 This operation must be carried out under
aseptic conditions
 Mesophyll protoplasts can be isolated from
the peeled leaf segments while epidermis
yields epidermal protoplasts

12.  Enzymatic treatment :
 There are two methods
 1.Direct method ( 1 step)
 2.Sequential method (2 steps)

13.  Direct method :
 Here simultaneous treatment with macerase
( pectinase ) and cellulose enzymes is carried
out.
 0.5% macerase and 2% cellulose enzyme in
13% sorbitol or mannitol at pH 5.4

14. • Sequential method :
• 1 step – Sample is treated with macerase
( pectinase ) enzyme for isolation of cells
2 step – Isolated cells are treated with cellulose
enzyme for protoplast isolation
In both cases , peeled leaf segments are placed
with the lower surface downwards in a petridish
containing enzyme mixture

15. Purification
 The isolated protoplasts are usually
associated with a range of cell debris and
broken cell organelles .
 Methods used for purification :
 1. Sedimentation
 2. Flotation

16. Applications
 Suitable for the isolation of cell organelles
and chromosomes
 Suitable for the isolation of mutants
 To affect genetic transformations through
DNA or organelle uptake
 Study of cell wall formation, membrane
transport , ultra structures

17. Immobilization of Enzyme
Definition
“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".
Immobilization is a process of aggregate formation and
adhesion on a matrix under controlled conditions.

18. Advantages
 1) No purification of enzyme after production
 2) High enzyme activity (high reactor activity)
 3) Enhanced operational stability
 4) Low enzyme cost
 5) Application of multienzyme reaction is
possible

19. How to stabilize enzyme?
• Addition of substrate analogues
• Addition of sugar alcohols (e.g. sorbitol)
• Addition of cofactors (e.g. calcium ion)
• Immobilization: reuse & long-term
stability !

20. Methods of immobilization
1.Direct intercellular binding due to natural affinity .
Eg: Adhesion, adsorption,agglutination
2.Covalent bonding on inert matrices
3.Embedding
4.Cross linking with biopolyfunctional reagents
5. Purely physical retensions in diverse pore size
eg: entrapment, microencapsulation

21. Agents used
 Agarose gel
 Alginate gel
 Chitosan
 Polyacrylamide
 Polyurethane foam
 Polyethylene oxide

22. Enzyme Immobilization

23. Immobilization by Binding
• Adsorption:
• Electrostatic interactions (van der Waals forces, ionic
and hydrogen bonds betweeen the cell surface and
the support materials
• cell wall composition: determined by distribution of
carboxyl and amino groups of the peptide amino acids
of cell wall surface (ex) yeast cells are negative
charge, thus choose a positively charged support
• Advantages: ability to regenerate the support
• Disadvantsges: low stability (desorption of cells due
to changes of pH and/or ionic strength)

24. • Covalent-binding methods
• Advantages:
• Free of diffusional limitations
• High operational stability
• Uniform binding
• Disadvantages:
• Toxicity of the coupling agents(loss of activity and
cell viability, not acceptable in food and
pharmaceutical fields)
• Hard to regenerate the supports