protoplast isolation and immobiliz by dr.u.srinivasa

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