Downstream Processing (release of intracellular products) Flashcards
What methods can be used to disintegrate/disrupt cells?
Physical Methods
Chemical Methods
Enzymatic Methods
Name the physical methods that are used to disrupt cells
- Ultrasonication
- Heat Shock (thermolysis)
- Osmotic shock
- High pressure homogenisation
- Impingement
6.Grinding with glass beads
Name the chemical methods that are used to disrupt cells
- Alkalies
- Organic solvents
- Detergents
Name the enzymatic methods that are used to disrupt cells
- Lysozyme
- Glucanase, mannanase, proteases
Name a disadvantage of ultra sonication
due to high cost, it is not suitable for large-scale use in industries
How are cells subjected to osmotic shock?
This method involves the suspension of cells (free from growth medium) in 20% buffered sucrose. The cells are then transferred to water at about 4°C.
What is osmotic shock used for?
for the release of hydrolytic enzymes and binding proteins from Gram-negative bacteria.
What are the advantage and disadvantage of heat shock?
Advantage: relatively easy and cheap.
Disadvantage: can be used only for a very few heat-stable intracellular products.
What is high pressure homogenisation?
This technique involves forcing of cell suspension at high pressure through a very narrow orifice to come out to atmospheric pressure. This sudden release of high pressure creates a
liquid shear that can break the cells.
What species is impingement method used for?
E coli
What is the advantage of impingement technique?
it can be effectively used for disrupting cells even at a low concentration.
What is the principle of impingement?
a stream of suspended cells at high velocity and pressure are forced to hit either a stationary surface or a second stream of suspended cells (impinge literally means to strike or hit). The cells are disrupted by the forces created at the point of contact. Micro fluidizer is a device developed based on the principle of impingement.
What factors influence the efficiency of cell breakage using the technique of ‘grinding with glass beads?
cell breakage-size and quantity of the glass beads, concentration and age of cells, temperature and agitator speed.
State a condition in which alkali extraction method could be used
extracting recombinant growth from E coli using sodium hydroxide at ph 11
Name some organic solvents that could be used for cell breakage
ethanol, methanol, isopropanol, butanol, toluene
Name detergents (both ionic and non-ionic) that could be used for extracting intracellular products
Ionic: CTAB, SLS
Non-ionic: Tween, Triton 100
State a disadvantage of using detergents in downstream processing and state a method to overcome the problem
Problem: Affect purification step due to high salt concentration
Solution: Ultrafiltration or ion-exchange chromatography for purification
State one advantage of cell disruption using enzymatic method
lysis of cells occurs under mild conditions in a selective manner.
State species on which lysozyme could be used.
Gram-positive bacteria (with high content of cell wall mucopeptides)
Gram-negative bacteria (lysosome in associatation with EDTA)
How does lysozyme act on gram-negative bacteria?
lysozyme in association with EDTA can break the cells.
As the cell wall gets digested by lysozyme, the osmotic effects break the periplasmic membrane to
release the intracellular contents.
Apart from lysozyme, what other enzymes could be used for cell disruption? What species do these enzymes act on?
Glucanase, mannanase, along with other proteases can be used to lyse yeast cell walls.
Name the ionic detergents that are used in cell membrane disruption to release intracellular components.
CTAB (cationic cetyl trimethyl ammonium bromide)
SLS (anionic sodium lauryl sulfate)
