1. Disruption using lytic agents
Disruption process utilizing chemicals or enzymes as lytic agents
are also used commonly, but tend to be expensive and also require
removal of the lytic agent
Chemicals as lytic agents
EDTA: Treatment with EDTA is used to release periplasmic proteins
from gram-negative bacteria as it disrupts the outer membrane of
the bacteria by binding Mg2+ and Ca2+ ions that cross-link the
lipopolysaccharide (LPS) molecules.
Antibiotics: The common class of antibiotics such as penicillin or
cycloserine inhibits peptidoglycan synthesis in growing cells, which
are not able to maintain their osmotic pressure and hence disrupt.
The assembly of peptidoglycon is also inhibited by Chaotropic agents
such as gaunidine hydrochloride and urea, that disrupt water structure
Note: Methods for gram-ngative bacteria and growing cells only

2. Disruption using lytic agents
Chemicals as lytic agents
Organic solvents and detergents: They cause dissolution of the lipids
in the periplasmic membrane and the outer membrane. Detergents
can be invariably used for solubilization of membrane proteins.
Detergents like Triton X-100 is commonly used but other detergents
like cholate and SDS are also used.
Organic solvents like toluene, trichloroethane, chloroform and ether
were found efficient in autolysis of yeast.
Alkaline lysis: Effective but harsh. Alkali added to the cell suspension
reacts with the cell walls and produces saponification of lipids in the cell
walls.

3. Disruption using lytic agents
Enzymes as lytic agents
Lytic enzymes: Enzymes hydrolyse the walls of microbial cells,
and when sufficient wall has been removed, the internal osmotic
pressure bursts the periplasmic membrane allowing the intracellular
components to be released.
The best known lytic enzyme for bacteria is lysozyme (a carbohydrase)
from hen egg white, which catalyzes the hydrolysis of β-1,4-glycosidic
bonds in the pepetidoglycon layer of bacterial cell wall.
Gram-positive bacteria more susceptible to enzymatic lysis
than gram-negative.
Glucanase used for yeast lysis
Note: Combined mechanical, nonmechanical and lytic disruption provide
efficient methods

4. Disruption of Animal and Plant Tissues
Absence of cell walls makes the disintegration of
mammalian tissue rather easy
Use of domestic homogenizer or industrial meat grinder
for cutting tissues
Colloid mill blender-type homogenizer for pilot or
industrial scale for finer grinding
Plant cell wall is rigid. Homogenization carried-out in cold
buffer with waring blender
Frozen and ground to dry powder
Phenolic compounds including tannins mix with the extract
and cause inactivation-use amberlite or PVP to remove
phenols

5. Extraction
Liquid-Liquid Extraction: Used to separate inhibitory fermentation
products such as ethanol, solvents, organic acids and antibiotics
Extraction requires the presence of two liquid phases
A multistep alternating aqueous-organic two phase systems are
used for antibiotic recovery
Solvents such as amylacetate or isoamylacetate are used
Provide both concentration and subsequent purification

6. Extraction
The extraction of compound from one phase to the other is based
on solubility differences of the compound in one phase relative to
other. When the compound is distributed between two immiscible
liquids, the ratio of the concentrations in the two phases is known
as the distribution coefficient: Yl
Kd = XH
Yl and XH are the concentrations of the solute in light and heavy
phases, respectively. The light phase will be organic solvent and
heavy phase will be fermentation broth

7. Extraction of penicillin
Typical penicillin broth contains 20 – 35 g antibiotic/liter
pKa values of penicillin 2.5 – 3.1
Near pH 2.0 – 3.0 neutralization renders them extractable
by organic solvents because of more solubility in organic
solvent
Subsequent back extraction with aqueous phosphate buffer
(pH 5 – 7.5) increases penicillin concentration
Repeat the process
The penicillin is finally recovered as sodium penicillin
precipitate from a butanol-water mixture
Centrifugal Podbielniak extractors are used for the process

8. Pricipitation
The distribution of charged and hydrophobic residues at the surface
of the protein molecule is the feature that determines solubility in various
solvents
The solubility behaviour of the protein can be changed drastically as the
solvent properties of water are manipulated, causing the protein to
precipitate out from the medium
+ _ _ + _
+ _ + _
_ _ _
+ _ _
_ _ _ _
Hydrophobic
patch

9. Precipitation: some important considerations
The hydrophobic patches consist of the side
chains of Phe, Tyr, Trp, Leu,Ile, Met, and Val.
Acidic: Glu, Asp
Basic: His, Lys, Arg

10. Interacting forces keeping protein soluble in water
1. The polar interaction between protein and solvent
2. The ionic interaction between protein and salt ion
3. The repulsive force between protein and protein
4. The repulsive force between protein and small aggregate

11. Modes of Precipitations
Protein precipitants include inorganic cations and anions NH4+, K+, Na+,
SO42-, PO43-, Cl-, Br-, NO3- etc for salting out
Bases or acids, H2SO4, HCl, NaOH for isolectric precipitation
Organic solvents such as ethanol, acetone, methanol, n-propanol
Non-ionic polymers like PEG and polyelectrolytes like PEI, PAA, carboxy
methyl cellulose
Heat and pH induced perturbations

12. Precipitation
Protein Solution Unstable protein solution Aggregate (floc) Uniform precipitate
after adding precipitant formation particles

13. Salting In and Salting Out
All proteins require some counter-ions (i.e. salt) to be soluble in aqueous media. Therefore, protein solubility increases with  salt concentration at low ionic strength.
At higher ionic strength, protein solubility generally decreases with  salt concentration due to reducing the activity of water and neutralization of surface charge.
Protein solubility
Salting in Salting out
Each protein has a distinct solubility profile as a function of salt concentration defined by:
Log S(mg/ml) = A - m(salt concentration)
where A is constant dependent on temp. & pH and m is constant dependent on the
salt employed.

14. Precipitation Salt precipitation
Saturated concentration of ammonium sulphate
for protein solution ~ 4.05 M
Protein fractionation by salt: e.g.,
0 – 30%; 30 – 60%; 60 – 80%
Grams ammonium sulfate to be added to 1 liter of protein solution
a) At M1 molar, to take it to M2 molar
g = 533(M2 – M1)/4.05 – 0.3 M2
b) At S1% saturation, to take it to S2% saturation
g = 533(S2 – S1)/100 – 0.3 S2
Note: After salt precipitation the salt is removed by dialysis or desalting columns for further application in purification

15. Ammonium Sulfate Nomogram

16. Precipitation: Practical Considerations
Trial Fractionation with Ammonium Sulfate
Percent Percent Percent
saturation enzyme protein Purification
range precipitated precipitated factor
First trial –
40 –
60 –
80 supernatant
Conclusion: Enzyme precipitated more in 40-60% than in 60-80%; try %
Second trial –
45 –
70 supernatant
Conclusion: Good recovery, but purification factor not as good as in first trial; if purity
important, try 48 – 65%
Third trial –
65 supernatant

17. Salt Precipitation: some important considerations
Most effective salts are those with multiple-charged anions such as
sulfate, phosphate and citrate
For cations, monovalent ions are used NH4+ > K+ >Na+
Potassium salts are ruled out on solubility grounds except potassium
phosphate which however, produces higher density in the solvent than
protein aggregate- difficulty in centrifugation
Sodium sulfate not highly soluble at lower temperature, citrate cannot be
used below pH 7.0, produces strong buffering action
Phosphates are less effective
Finally one salt has all the advantages and no disadvantage (except if
required to operate at high pH): Ammonium sulphate

18. Salt Precipitation: some important considerations
Salt never precipitates all the protein, but just reduces its solubility
If the starting material has a enzyme concentration of 1 mg/ml; reduction
in solubility to 0.1mg/ml means 90% precipitation
On the other hand if the starting material has a concentration of 0.1 mg/ml
no precipitation will occur
So precipitation is not an absolute property of the enzyme concerned, but
will depend on both the properties of other proteins present (coprecipitation)
and the protein concentration in the starting solution
The addition of salts increase the density of the medium and thus brings
densities close to the densities of protein aggregates in the solution.
thus high speeds and longer times are required for centrifugation.

19. Salt Precipitation: some important considerations
- The effect of protein purity on ammonium sulfate
precipitation of proteins

20. Salt precipitation
Different types of salts effect the solubility of proteins to different extents. Most widely used in protein fraction are sulfate salts, particularly ammonium sulfate (NH4)2SO4.
kdal
In general, the larger the protein, the lower the salt concentration required to precipitate it.

21. Salt removal by dialysis
Dialysis membranes are available with pore sizes from very small (1,500 MW cut off) to very large ( kDa cut off).
Also available in conical shapes for use in the centrifuge to both desalt and concentrate protein.