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Immobilization of Enzyme

Immobilized enzyme:
o Since enzymes are globular proteins and are soluble in water, so it is
difficult to separate enzymes for reuse in a batch process.
oEnzymes can be immobilized from surface or inside from a insoluble
matrix.
oFrom soluble form they can be immobilized by retaining them with a semi
permeable membrane.
oA main advantage of immobilized enzyme is that it can be reused since it
can easily be separated from the reaction solution and an be easily retained
in a continues flow reactor.
o Immobilized enzymes may show selectively altered chemical or physical
properties and it may stimulate the realistic natural environment where the
enzyme came from the cell.
Immobilization techniques:
Immobilization techniques can be classified by two
methods:
1.Chemical method (covalent bond formation
dependent)
2.Physical method (non-covalent bond formation
dependent)
1). Chemical method:
oA) Covalent attachment: the covalent attachment of enzyme
molecules via non-essential amino acids residue to water
insoluble, functionalized supports are the most widely used
methods for immobilizing enzymes.
oFunctional groups of the non essential amino acid residue that
are suitable for the immobilization process are free α-, β-, or y-
carboxyl groups, α- or β-amino groups, and phenyl, hydroxyl,
sulfhydryl, or imidazole groups.
B)Cross-linking Using Multifunctional Reagents:
o Water-insoluble enzymes can be prepared by using multifunctional
agents that are all bifunctional in nature and have low molecular
weight, such as glutaraldehyde.
o Enzymes can be reacted with multifunctional reagent alone so that
they are cross-linked intermolecularly by the reagent to form a
waterinsoluble derivative.
o Another method is to adsorb enzymes on a water-insoluble,
surface-active support followed by intermolecular cross-linking
with multifunctional reagents to strengthen the attachment.
o Multifunctional reagents can be also used to introduce functional
groups into water-insoluble polymers, which then react covalently
with water-soluble enzymes.
2). Physical methods:
oThis method is the simplest way to immobilize enzymes. Enzymes can
be adsorbed physically on a surface-active adsorbent by contacting an
aqueolls solution of enzyme with an adsorbent. Commonly employed
adsorbents are alumina, anion-exchange resins, calcium carbonate,
carbon, cationexchange resins, celluloses, clays, collagen, colloid-ion,
conditioned metal, glass plates, diatomaceous earth, and
hydroxyapatite.
Advantages & disadvantages of physical
methods
1. The procedure of immobilization is simple.
2. It is possible to separate and purify the enzymes while being immobilized.
3. The enzymes are not usually deactivated by adsorption.
4. The adsorption is a reversible process. Immobilized Enzyme 53
However, adsorption techniques also have several disadvantages:
1. The bonding strength is weak.
2. The state of immobilization is very sensitive to solution pH, ionic strength,
and temperature.
3. The amount of enzymes loaded on a unit amount of support is usually low.
A) Entrapment:

oEnzymes can be entrapped within cross-linked polymers by forming a


highly cross-linked network of polymer in the presence of an enzyme.
o This method has a major advantage in the fact that there is no
chemical modification of the enzyme, therefore, the intrinsic
properties of an enzyme are not altered.
oHowever, the enzyme may be deactivated during the gel formation.
Enzyme leakage is also a problem. The most commonly employed
cross-linked polymer is the polyacrylamide gel system.
B) Microencapsulation:

oEnzymes can be immobilized within semipermeable membrane microcapsules.


This can be done by the interfacial polymerization technique.
oOrganic solvent containing one component of copolymer with surfactant is
agitated in a vessel and aqueous enzyme solution is introduced. The polymer
membrane is formed at the liquid-liquid interface while the aqueous phase is
dispersed as small droplets.
o One example of this process is the polyamide nylon system, in which 1, 6-
diaminohexane is the water-soluble di-amine and 1,10-decanoyl chloride is the
organic-soluble di-acid halide. The organic solvent for this system is a
chloroformcyclohexane mixture (1:4 v/v) containing usually 1 percent (v/v)
Span85 surfactant. The immobilized enzyme produced by this technique provides
an extremely large surface area.
DOWNSTREAM
PROCESSING/BIO SEPARATION
Downstream processing
Introduction:
• After successful fermentation or enzyme reactions, desired products must be
separated and purified. This final step is commonly known as downstream
processing or bioseparation.
• The fermentation products can be the cells themselves (biomass), components
within the fermentation broth (extracellular), or those trapped in cells
(intracellular), examples of which are listed in table:

Types Products Concentration


Cell itself Yeast, single cell proteins 30g/L
Extracellular Alcohols, organic acids, 100-20g/L
amino acids,
enzymes,antibiotics
Intracellular Recombinant DNA proteins 10g/L
Examples of bioprocessing products and their typical concentrations
• if the product of our interest is the cell, cells are separated from the
fermentation -broth and then washed and dried.
• In the case of extracellular products, after the cells are separated,
products in the dilute aqueous medium need to be recovered and
purified.
• The intracellular products can be released by rupturing the cells and
then they can be recovered and purified. The downstream
processing for enzyme reactions will be similar to the process for
extracellular products.
Some of the unique characteristics of bioseparation products
can be listed as follows:
1. The products are in dilute concentration in an aqueous medium.
2. The products are usually temperature sensitive.
3. There is a great variety of products to be separated.
4. The products can be intracellular, often as insoluble inclusion
bodies.
5. The physical and chemical properties of products are similar to
contaminants.
6. Extremely high purity and homogeneity may be needed for
human health care.
EXTRACELLULAR TECHNIQUES
A) Solid liquid separation:
o The first step in downstream processing is the separation of solubles from the
fermentation broth. The selection of a separation technique depends on the
characteristics of solids and the liquid medium.
o The solid particles to be separated are mainly cellular mass with the specific
gravity of about 1.05 to 1.1, which is not much greater than that of the broth.
Shapes of the particles may be spheres, ellipsoids, rods, filaments, or flocculents.
The separation of solid particles from the fermentation broth can be
accomplished by filtration or centrifugation.
o Typical sizes for various cells vary widely such as:
Bacterial cells 0.5-1µm
Yeast cells 1-77µm
Fungal hyphae Diameter 5-15µm
Length 50-500µm
Suspension animal cell 10-20µm
Plant cell 20-40µm
B) Filtration:

o Filtration separates particles by forcing the fluid through a filtering medium


on which solids are deposited.
oFiltration can be divided into several categories depending on the filtering
medium used, the range of particle sizes removed, the pressure differences,
and the principles of the filtration, such as conventional filtration,
microfiltration, ultrafiltration, and reverse osmosis.
Biological feed before filtration may require pretreatment such as:
1. Heating to denature proteins
2. Addition of electrolytes to promote coagulation and flocculation
3. Addition of filter aids (diatomaceous earths or perlites) to increase the
porosity and to reduce the compressibility of cakes
C) Centrifugation:

Centrifugation is an alternative method when the filtration is


ineffective, such as in the case of small particles.
Centrifugation requires more expensive equipment than
filtration and typically cannot be scaled to the same capacity as
filtration equipment. Two basic types of large-scale centrifuges
are the tubular and the disk centrifuge as shown schematically
INTRACELLULAR TECHNIQUES
Cell rupturing:
o Once the cellular materials are separated, those with
intracellular proteins need to be ruptured to release their
products. Disruption of cellular materials is usually difficult
because of the strength of the cell walls and the high osmotic
pressure inside.
o Cells can be ruptured by physical, chemical, or biological
methods
A) Physical Methods:
Physical methods include mechanical disruption by milling,
homogenization, or ultrasonication
B) Chemical Methods:
Chemical methods of cell rupture include the treatment of cells with
detergents (surfactants), alkalis, organic solvents, or by osmotic shock. The
use of chemical methods requires that the product be insensitive to the harsh
environment created by the chemicals. After cell disruption, the chemicals
must be easily separable or they must be compatible with the products.
C) Biological Methods:
Enzymatic digestion of the cell wall is a good example of biological cell
disruption. It is an effective method that is also very selective and gentle, but
its high cost makes it impractical to be used for large-scale operations.
RECOVERY/CONCENTRATION OF
DILUTE AQUEOUS SOLUTION
A) EXTRACTION:
i) SINGLE-STAGE
ii) MULTI-STAGE

B) ADSORPTION:
i) Conventional adsorption
ii) Ion exchange
iii) Affinity adsorption
PURIFICATION
TECHNIQUES:
A) PRECIPITATION
B) CHROMATOGRAPHY
C) ELECTROPHORESIS
D) MEMBRANE SEPARARTION :
i) Conventional filtration
ii) By polymeric membranes
iii) Pressure driven membrane separation(see table 10.2)
THANK YOU

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