Disruption using lytic agents

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
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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.
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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 ß-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

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• 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

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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
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• 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
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• 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

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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

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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.

• 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.

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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
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Ammonium Sulfate Nomogram
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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
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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 gt K
  gtNa
• 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

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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.

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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.

340 68 -66 18 kdal

• In general, the larger the protein, the lower the
  salt concentration required to precipitate it.

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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.