Separasi dan

Purifikasi dalam
Bioteknologi
Jumeri M. Wikarta,
Ph.D

Fermentation broth
Combination of insoluble, gelatinous biomass,
the nutrient fluid, and the soluble metabolites
resulting from the fermentation operation.
Fluidity for stirring or aeration is 3-7% dry
weight of biomass.
Bacterial fermentation for SCP will produce a
broth of 3% WV in which the slurry is 60%
(by volume) wet biomass and 40%
interparticle fluid.

Typical Product Concentrations

Leaving Fermenters

 If the desired product is extracellular, it

is only necessary to filter the biomass
from the broth and isolate the product
from the fluid.
If a product is intracellular, a cell
disruption step must first be employed.
If the product is also water soluble, this
disruption should be performed while the
biomass is still in a slurry form.

RECOVERY UNIT
OPERATIONS
Filtration and centrifugation are unit
operations in which the suspended solids are
separated from the fluid phase.
Drying is the removal of moisture or solvent
from solid particles
Evaporation is the removal of moisture or
solvent from a solution.
In crystallization, the conditions of a solution
are adjusted to change the solubility of one
of the dissolved compounds so that it leaves
the solution as a solid.

Separation and Recovery

Techniques

1 A = 0,1
nm

Microfiltration, ultrafiltration, and reverse

osmosis are membrane processes in which
separation is based on differences in ability
to flow through a thin barrier that separates
two fluids.
Microfiltration is a hydraulically driven
process using a membrane with a pore size
in the 100 to 3000 A range.
Ultrafiltration, the pore size is 10-125 A,
while for reverse osmosis, the pore size is
3-10 A.

 Adsorption, ion exchange, column chromatography,

and affinity chromatography grouped as recovery
techniques in which the removed compound or
solute establishes an equilibrium between sites on
a solid phase material and the solution.
In adsorption, the removed species is bonded to
the solid phase material by polarity or weak
chemical bonds.
Ion exchange recovers material by the interchange
of ions between the liquid and solid phases.
Column chromatography may use adsorptive, ion
exchange or molecular sieve materials to separate
solutes which are first loaded onto a column of
the separation material and then eluted in such a
manner that the individual solutes are collected in
separate fractions.
In affinity chromatography, the removed species is
bound with a high level of selectivity to ligands

 In solvent extraction, the removed compound

establishes an equilibrium distribution between
immiscible solvents, usually water and an organic
liquid.
Electrophoresis and electrodialysis are separation
techniques that separate charged molecules or
ions using an electric field.
Electrophoresis separates charged components by
accentuating small differences in ionic mobility in
an electric field using a moving carrier fluid.
Electrodialysis concentrates components on the
basis of electromigration through ionic membranes.

Perforated bowl or basket type

centrifuge
Used with a filter
bag of nylon,
terylene, or cotton.
The feed is added
continually.
When the bowl is
filled with the
biomass cake, fresh
washing liquid can
be added to
displace the residual
broth fluid retained
in the cake.

Tubular bowl centrifuge

A continuous
feed of material
passes through the
machine. As soon
as the bowl is full
of the biomass
cake, the
centrifuge is
stopped, stripped
down, the cake
removed, the
machine cleaned
and
the sequence

Multi chamber solid bowl centrifuge

Allows three or more
time the capacity of
the tubular bowl.
The disassembly and
cleaning of this
centrifuge is more
difficult.

Nozzle disk centrifuge

Operate continuously
since the solids are
automatically removed
from the bowl.
Discharges a continuous
stream of concentrated
solid-slurry.

Desludger or intermittent discharge

centrifuge
Has an
intermittent
discharge of
solids with lower
water content
than the nozzle
centrifuge.

Scroll centrifuges

Uses a screw within a rotating

bowl to allow continuous removal
of the solid and the liquid
portions.

Characteristics of Different Types of

Centrifuges

Recovery and
purification of
citric acid

Isolation
procedure for
sodium
clavulanate

Purification
procedure for
micrococcal
nuclease

Recovery processes
for human insulin,
human
growth hormone and
human leukocyte
interferon synthesized
from E. coli

Why purify Proteins?

Characterize
Function
Activity
Structure

Study protein regulation and protein

interactions
Use in assays
Produce Antibodies
Perform structural analysis by X-Ray
and Crystallography

How to purify Proteins?

Biochemists exploit the ways individual
proteins differ from one another, such as
solubility, size, or charge.

Specific
binding

Classical Protein Purification Methods

The Oldest: Ammonium Sulfate
Fractionation

Dialysis Getting Rid of (NH4)2SO4 or

Change Buffers

Isolation of Proteins from

Cells
Many different proteins exists within one cell

Many steps needed to extract protein of

interest, and separate from many
contaminants
Before purification begins, protein must
be released from cell by
homogenization

Compartmentalization provides an opportunity

for a purification step

Protein profile for compartments of gram-negative prokaryotes

Cell Disruption
Chemical: alkali, organic solvents,
detergents
Enzymatic: lysozyme, glucanases,
chitinase
Physical: osmotic shock,
freeze/thaw
Mechanical: sonication,
homogenization,
wet
milling, French press

Chemical Disruption
Detergents such
as Trition X-100 or
NP40 can
permeabilize cells
by solubilizing
membranes.
Detergents can be
expensive,
denature
proteins, and
must be removed

h Press
Cells are placed
in a stainless
steel container.
A tight fitting
piston is
inserted and
high pressures
are applied to
force cells
through a small

Homogenization
Cells are placed in a
closed vessel (usually
glass).
A tight fitting plunger is
inserted and rotated
with a downward force.
Cells are disrupted as
they pass between the
plunger and vessel wall.
Also, shaking with glass
beads works, BUT:
Friction = Heat

Sonication
A sonicator can be
immersed directly into
a cell suspension. The
sonicator is vibrated
and high frequency
sound waves disrupt
cells.

Differential centrifugation

Protein purification
Proteinmixtureappliedtocolumn
Solvent(buffer)appliedtotop,
flowedthroughcolumn
Differentproteinsinteractwith
matrixtodifferentextents,flow
atdifferentrates
Proteinscollectedseparatelyin
differentfractions

Column Chromatography

Molecules can be separated on the basis of:

1.SIZEGel filtration (Size

exclusion )
2.CHARGEIon exchange
3.SPECIFIC BINDINGAffinity

How We Get Proteins Out of

Cells

Recycling Preparative
HPLC

Recycling Preparative
HPLC

Gel Filtration
Separates molecules according to differences in
size as they pass through a gel filtration medium
pack in a column
A key position in the purification of thousands
enzymes, polyssacharides, nucleic acids, protein,
and other biological macromolecules.
Molecules do not bind to the chromatography
medium so buffer composition does not directly
affect resolution
The liquid inside the pores is sometimes reffered
to as the stationary phase and this liquid is in
equilibrium with the liquid outside the particles,
reffered to as the mobile phase

Gel Filtration
Can be performed in the presence of essential
ions or cofactor, detergent, urea, at high or low
ionic strength, at 37C or in the cold room
according to the requirements of the experiment
The sample zone moves down the bed as eluent
is added to the top
The small molecules which diffuse into the gel
beads are delayed in their passage down the
column compare with the large molecules which
cant diffuse into the gel and move continuously
down the column in the flowing eluent
The large molecules thus leave the column first
followed by the smaller molecules in the order in
their size

el filtration chromatography

Size Exclusion/Gel-filtration

Size-exclusion chromatography

Size-exclusion
chromatography
Absorbance at 280 is used to identify
protein-containing fractions. You can
also perform an enzyme specific assay.

Columns Connected to
Fraction Collectors

Elution profile (chromatogram) of gel filtration

Resolution in gel filtration

Factors influence :
- Sample volume
- The ratio of sample
volume
to column volume
- Column dimension
- Particle size
- Particle size distribution
- Packing density
- Pore size of the particles
- Flow rate
- Viscosity of the sample
and
buffer

Resolution/ degree of
separation

Rs 1.5 two component are present in

equal proportions
Rs can be increased by :
- Using longer column
- Gel medium with smaller particles and/or greater
selectivity
- Small sample volume
- Low flow rate

Separation time
Range from a few minutes to many
hours
According to the difficulty of the
separation, the gel medium, the
column length, and the flow rate
Improve the resolution also lead to
longer separation time

Sample volume

Resolution depend on
the ratio of sample
volume to column
volume
Large sample to
column volume ratio
giving lower resolution

Reccomended sample volume as a

per cent of the total bed volume
for good resolution
Medium
Sephadex
Sepharose
Sepharose CL
Sephacryl HR
Superdex prep
grade
Superose prep
grade
Superdex
Superose

Reccomended sample
volume (% Vt)
25
25
25
12
12
12
0.5
0.5

Molecular weight
determination
A gel should be chosen, so that the
samples expected MW falls on the linear
part of the selectivity curve and in the
middle of a suitable range of calibration
standard
If the MW is unknown, a gel with wide
fractionation range (Sephacryl HR) will be
most suitable

Gel filtration
media selection
guide

Gel Filtration Media

Ion-exchange (Adsorption) chromatography

Separation depends upon the reversible

adsorption of charged solute molecules to
immobilized ion exchange groups of
opposite charge.
Probably the most frequently used for the
separation and purification of proteins,
polypeptides, nucleic acids,
polynucleotides, and other charged
biomolecules.
Widespread applicability, its high resolving
power, its high capacity, and the simplicity
and controllability of the method.

Ion-exchange
chromatogra
phy

Ion Exchange

Interaction based on overall

charge (less specific than
affinity)
Cation exchange
Anion exchange

Ion-Exchange
chromatography
-

If pH mobile phase =7.2

Then charge of the proteins:
(+)

+
+

+
+

(-)

(-)

(+)

+
+
+

+
+
+
+
+

+ +

+
+
Anion exchange column = + charged

Functional groups used on ion

exchangers

Stepwise elution for ion exchange

Affinity chromatography

Separates proteins on the basis of a reversible

interaction between a protein (or group of
proteins) and a specific ligand coupled to a
chromatography matrix.
Can be used to separate active biomolecules from
denatured or functionally different forms, to
isolate pure substances present at low
concentration in large volumes of crude sample
and also to remove specific contaminants
High selectivity, hence high resolution, and
usually high capacity for the protein(s) of interest.
Purification can be in the order of several
thousand-fold and recoveries of active material
are generally very high.

Affinity
chromatogra
phy

Affinity Chromatography
Uses specific binding properties of molecules/proteins
Stationary phase has a polymer that can be covalently linked
to a compound called a ligand that specifically binds to
protein

Affinity chromatography
separation by biological
binding interactions
Example: GST Glutathione
GST-tagged proteins bind
to
gluthatione on beads
Non-specifically or
weakly
bound proteins washed
off

thrombinsite

proteinofinterest
apply sample

GST

porous
bead

glutathione

GST-tagged proteins
eluted
with glutathione

wash

elute

Protein purification by chromatography

What sort of equipment do

we need?

How to pack the column?

A purification table for a

hypothetical enzyme

Separating and visualizing proteins

SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis

1. Heat sample with SDS and -mercaptoethanol

SDS = Detergent (anionic)
- Denatures proteins
- Coats proteins
- Each protein has similar mass/charge ratio
-mercaptoethanol/DTT
- reduces disulfide bonds
2. Separate on polyacrylamide gel
- polymer of acrylamine/bis-acrylamide
- TEMED, ammonium persulfate catalyst for polymerization
- Protein migrates through gel matrix in electric field.

Estimating the molecular

weight of protein

Protein Properties
Proteins denatured by heating them in a sample buffer
containing SDS
The proteins no longer have any secondary, tertiary, or
quaternary structure
Resultant proteins take on a rod-like shape and a
uniform negative charge- to-mass ratio proportional to
their molecular weights

The equipment of SDSPAGE

Preparing gel