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

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.
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
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
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

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
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

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 + - + -
_ + + -
+
_+ _ + _ +
+ _ _
_ + _ + _

+ _ + +
_
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

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

 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

 Precipitation

Protein Solution Unstable protein solution Aggregate (floc) Uniform precipitate

after adding precipitant formation particles
 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.

Salting in Salting out

Protein solubility

0 0.5 2.0 3.0 4.0

•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.
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
Ammonium Sulfate Nomogram
Precipitation: Practical Considerations
Trial Fractionation with Ammonium Sulfate

Percent Percent Percent

saturation enzyme protein Purification
range precipitated precipitated factor

First trial 0 – 40 4 25
40 – 60 62 22 2.8
60 – 80 32 32 1
80 supernatant 2 21

Conclusion: Enzyme precipitated more in 40-60% than in 60-80%; try 45 -70%

Second trial 0 – 45 6 32
45 – 70 90 38 2.4
70 supernatant 4 30

Conclusion: Good recovery, but purification factor not as good as in first trial; if purity
important, try 48 – 65%

Third trial 0 – 48 10 35
48 - 65 75 25 3.0
65 supernatant 15 40
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
 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.
Salt Precipitation: some important considerations

- The effect of protein purity on ammonium sulfate

precipitation of proteins
 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.

340 68 -66 18 kdal

•In general, the larger the protein, the lower the salt concentration required to precipitate it.
 Salt removal by dialysis

•Dialysis membranes are available with pore sizes from very small (1,500 MW cut off)
to very large (50-100 kDa cut off).

•Also available in conical shapes for use in the centrifuge to both desalt and concentrate
protein.