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 EnzymeDefinition“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?• 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 immobilization1.Direct intercellular binding due to natural affinity . Eg: Adhesion, adsorption,agglutination2.Covalent bonding on inert matrices3.Embedding4.Cross linking with biopolyfunctional reagents5. Purely physical retensions in diverse pore size eg: entrapment, microencapsulation
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)
• 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