Downstream Processing (release of intracellular products)

Question 1

What methods can be used to disintegrate/disrupt cells?

Answer 1

Physical Methods
Chemical Methods
Enzymatic Methods

Question 2

Name the physical methods that are used to disrupt cells

Answer 2

1. Ultrasonication
2. Heat Shock (thermolysis)
3. Osmotic shock
4. High pressure homogenisation
5. Impingement
   6.Grinding with glass beads

Question 3

Name the chemical methods that are used to disrupt cells

Answer 3

1. Alkalies
2. Organic solvents
3. Detergents

Question 4

Name the enzymatic methods that are used to disrupt cells

Answer 4

1. Lysozyme
2. Glucanase, mannanase, proteases

Question 5

Name a disadvantage of ultra sonication

Answer 5

due to high cost, it is not suitable for large-scale use in industries

Question 6

How are cells subjected to osmotic shock?

Answer 6

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.

Question 7

What is osmotic shock used for?

Answer 7

for the release of hydrolytic enzymes and binding proteins from Gram-negative bacteria.

Question 8

What are the advantage and disadvantage of heat shock?

Answer 8

Advantage: relatively easy and cheap.

Disadvantage: can be used only for a very few heat-stable intracellular products.

Question 9

What is high pressure homogenisation?

Answer 9

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.

Question 10

What species is impingement method used for?

Answer 10

E coli

Question 11

What is the advantage of impingement technique?

Answer 11

it can be effectively used for disrupting cells even at a low concentration.

Question 12

What is the principle of impingement?

Answer 12

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.

Question 13

What factors influence the efficiency of cell breakage using the technique of ‘grinding with glass beads?

Answer 13

cell breakage-size and quantity of the glass beads, concentration and age of cells, temperature and agitator speed.

Question 14

State a condition in which alkali extraction method could be used

Answer 14

extracting recombinant growth from E coli using sodium hydroxide at ph 11

Question 15

Name some organic solvents that could be used for cell breakage

Answer 15

ethanol, methanol, isopropanol, butanol, toluene

Question 16

Name detergents (both ionic and non-ionic) that could be used for extracting intracellular products

Answer 16

Ionic: CTAB, SLS
Non-ionic: Tween, Triton 100

Question 17

State a disadvantage of using detergents in downstream processing and state a method to overcome the problem

Answer 17

Problem: Affect purification step due to high salt concentration

Solution: Ultrafiltration or ion-exchange chromatography for purification

Question 18

State one advantage of cell disruption using enzymatic method

Answer 18

lysis of cells occurs under mild conditions in a selective manner.

Question 19

State species on which lysozyme could be used.

Answer 19

Gram-positive bacteria (with high content of cell wall mucopeptides)

Gram-negative bacteria (lysosome in associatation with EDTA)

Question 20

How does lysozyme act on gram-negative bacteria?

Answer 20

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.

Question 21

Apart from lysozyme, what other enzymes could be used for cell disruption? What species do these enzymes act on?

Answer 21

Glucanase, mannanase, along with other proteases can be used to lyse yeast cell walls.

Question 22

Name the ionic detergents that are used in cell membrane disruption to release intracellular components.

Answer 22

CTAB (cationic cetyl trimethyl ammonium bromide)

SLS (anionic sodium lauryl sulfate)