PROTOPLAST AND IMMOBILIZATION
BY
Dr.U.Srinivasa, M.Pharm, Ph.D.
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
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
During the process of isolation, the
cells are first separated by
mechanical method and subsequently
protoplasts are isolated by enzymatic
method
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.
Then the protoplasts are released from the cells
through the cell wall , and then the tissue is
again deplasmolysed.
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
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
Steps
1.Sterilization of leaves
2. Peeling of the epidermis
3. Enzymatic treatment
4. Isolation and cleaning of the protoplasts
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.
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
Enzymatic treatment :
There are two methods
1.Direct method ( 1 step)
2.Sequential method (2 steps)
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
• 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
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
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
Immobilization of EnzymeImmobilization 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.
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
How to stabilize enzyme?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 !
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
Agents used
Agarose gel
Alginate gel
Chitosan
Polyacrylamide
Polyurethane foam
Polyethylene oxide
Enzyme ImmobilizationEnzyme Immobilization
Immobilization by BindingImmobilization by Binding
• Adsorption: 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)
• Covalent-binding methodsCovalent-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
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