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IMMOBILIZATION
• Enzymes have extensive applications in food industries such as
baking, dairy products, starch conversion and beverage
processing (fruit, vegetable juices, beer and wine).
• In textile industries, they have found a special place due to their
effect on end products.
• In industries such as paper and pulp making and detergents, the
use of enzymes has become a necessary processing strategy.
• Some of the major class of industries such as health care &
pharmaceuticals and chemical manufacturing have been
increased due to the catalytic nature of enzymes.
• Another major application of enzymes is in waste management
especially for solid wastes treatment and waste water purification.
• Past few years have marked the significance of enzymes in
production of biofuels such as biodiesel, bioethanol, biohydrogen
and biogas from biomass conversion.
• However, all these desirable characteristics of enzymes
and their widespread industrial applications are often
obstructed by their lack of long-term operational
stability, shelf life and by their recovery & reusability.
• Enzyme immobilization is one of the strategies to
overcome these problems.
• Immobilization is defined as the physical confinement
of the viable biocatalysts, including enzymes and whole
cells, to a certain defined region of space known as the
carrier or support material to limit its free movement
while preservation of their viability and catalytic
functions.
• The first discovery of this process was by Nelson and Griffin
1961, when they find that invertase is immobilized
(adsorbed) on charcoal and able to hydrolyze sucrose.
Both (c) and (d) may be reduced by use of 'spacer' groups between the enzyme
and support, effectively displacing the enzyme away from the steric influence
of the surface.
ADVANTAGES AND DISADVANTAGES OF
THE COVALENT BINDING METHOD
Advantages:
1- The enzyme doesn't leak from the carrier.
2- The enzyme can be easily interacts with the substrate because
it is in the surface of the carrier.
3- The enzyme stability is often increased due to its strong
binding with the carrier.
Disadvantages:
1- The yield activity of the enzyme is low due to using of toxic
materials during immobilization.
2- The optimal immobilization conditions are difficult to be find.
3- Renewable of the carrier and recovery of the enzyme is
impossible.
ENTRAPMENT
• The entrapment method of immobilization is based on the
localization of an enzyme within the lattice of a polymer matrix
or membrane.
• The entrapment process may be a purely physical caging or
involve covalent binding.
• Amounts in excess of 1 g of enzyme per gram of gel or fibre may
be entrapped.
• It is done in such a way as to retain protein while allowing
penetration of substrate.
• Entrapment of enzymes within gels or fibres is a convenient
method for use in processes involving low molecular weight
substrates and products.
• However, the difficulty which large molecules have in
approaching the catalytic sites of entrapped enzymes
precludes the use of entrapped enzymes with high
molecular weight substrates.
• Enzymes may be entrapped in cellulose acetate fibres
by, for example, making up an emulsion of the enzyme
plus cellulose acetate in methylene chloride, followed by
extrusion through a spinneret into a solution of an
aqueous precipitant.
• Entrapment is the method of choice for the
immobilisation of microbial, animal and plant cells,
where calcium alginate is widely used.
• It can be classified into: lattice, microcapsule,
membrane, and reversed micelle types.
1- Lattice-Type
• Entrapment involves
entrapping enzymes within the
spaces of a cross-linked water-
insoluble polymer.
• Some synthetic polymers such
as polyarylamide, calcium
alginate, polyvinylalcohol and
natural polymers such as starch
and agar have been used to
immobilize enzymes using this
technique.
• a) Polyacrylamide: This polymer which is used in
electrophoresis analysis. The disadvantage of this
method is the toxicity pf acryl amide.
Large diffusional
No No Yes Yes
barriers
• Additional cost.
• It sometime affects stability and activity of enzyme.
• This approach can not be used when one of the
substrates is insoluble.
• Some immobilization methods restricts the diffusion
of substrate.
CLASSIFICATION OF SUPPORT
Organic Inorganic
Natural polymers Natural minerals
Polysaccharides: cellulose,
Bentonite, silica
dextrans, agar, agarose, chitin,
alginate Processed materials
Proteins: collagen, albumin Glass (non-porous and
Carbon controlled pore), metals,
Synthetic polymers controlled pore metal oxides
Polystyrene
Other polymers: polyacrylate,
polymethacrylates,
polyacrylamide, polyamides,
vinyl and allyl-polymers
• Natural organic carriers have many functional groups
which stabilize biocatalysts.
• This class of carriers includes: alginate, κ-carrageenan,
chitosan, sawdust, straw, charcoal, plant fibres, corncob,
bagasse, rice, husks of sunflower seeds, diatomite and
mycelium.
• These supports are hydrophilic, biodegradable,
biocompatible, and inexpensive because they are mostly
waste from the food industry.
• However, the possibility of their application in
bioremediation processes is limited because of low
resistance to biodegradation, sensitivity to organic
solvents, and stability in a narrow pH range.
• Synthetic organic carriers have numerous functional
groups with diversified characters.
• This class includes polypropylene, polyvinyl chloride,
polystyrene, polyurethane foam, polyacrylonitrite and
polyvinyl alcohol.
• Their advantage is the possibility to regulate their structure
at the macromolecular level, the selection of the proper
molecular weight, the spatial structure and the manner and
arrangement order of each active functional group in the
chain.
• Furthermore, synthetic supports can be formed into
various shapes (tubes, membranes, coatings, carriers of
various shapes from spherical to oval), and they are easily
available and relatively inexpensive.
• Moreover, during synthesis, the porosity, pore diameter,
polarity and hydrophobicity of the carrier may be controlled.
• Porous supports should have a controlled pore distribution in
order to optimize capacity and flow properties.
• Inorganic carriers (natural and synthetic) have a high
chemical, physical and biological resistance.
• They are represented by magnetite, volcanic rocks,
vermiculite, porous glass, silica-based materials, ceramics and
nanoparticles.
• A significant disadvantage of these carriers is the presence of
a small number of functional groups, which prevents sufficient
bonding of the biocatalyst.
• For that reason they are used in the formation of hybrid
carriers, combining natural polymers and synthetic
nanoparticles.
MATRICES FOR ENZYME
IMMOBILIZATION
Surface-Bound Enzymes
The physical and chemical properties of the matrices
used for enzyme immobilization are very important as
they are major governing factors of chemical,
biochemical, mechanical and kinetic properties of
immobilized enzymes. The matrix can be biopolymer,
synthetic organic polymer, hydrogels, smart polymer
or inorganic solid.
BIOPOLYMERS
• They are water-insoluble polysaccharides (e.g., cellulose, starch,
agarose, chitosan, and proteins such as gelatin and albumin).
• The first industrial application of a biopolymer came in 1960,
using immobilized aminoacylase from Aspergillus oryzae onto
DEAE-Sepahdex (modified cellulose with diethylaminoethyl) by
ionic adsorption for production of amino acids.
• The process was performed in a fixed-bed reactor under
continuous operation.
• Since then, several other commercial enzymes were immobilized
onto DEAE-Sephadex.
• In case of immobilized enzyme onto DEAE-Sephadex, a retention
of 70 % enzyme activity was observed with respect to the soluble
enzyme.
• Agarose is an excellent matrix which has been extensively
used.
• In addition to its high porosity which leads to a high
capacity for proteins, some other advantages of using
agarose are hydrophilic character, absence of charged
groups (which prevents nonspecific adsorption of substrate
and products), and commercial availability.
• However, an important limitation of agarose and other
porous supports is the high cost.
• An approach to avoid this problem is the use of reversible
methods of immobilization that allow matrix regeneration
and reuse.
SYNTHETIC ORGANIC POLYMERS
• The various synthetic organic polymers are being used
for enzyme immobilization are; Eupergit-C (acrylic
resins), Sepa beads FP-EP, Amberlite XAD-7 (porous
acrylic resins) etc.
• Eupergit-C has surface diameter of 170 mm and pore
diameter of 25 nm.
• It is prepared by using compounds: N,N′-methylene-bi-
(methacrylamide), methacrylamide, allyl glycidyl ether
and glycidyl methacrylate.
• It is hydrophilic in nature with chemical and mechanical
stability over a pH range of 0–14.
• It has a high density of oxirane (Ethylene oxide) moieties
on its surface, which is responsible for multi-point
enzyme binding via covalent bonds at either neutral or
alkaline pH thus giving a long term operational stability
in wide pH range.
• After enzyme attachment, the remaining epoxy groups of
oxirane are blocked by mercaptoethanol, ethanolamine
or glycine to prevent non-specific interactions.
• Eupergit-C has been used for several industrial enzymes.
• Major success has been achieved in case of Penicillin
amidase with a reusability of more than 800 washes.
• However, due to diffusional limitations by Eupergit-C,
kinetic properties of immobilized enzymes have been
greatly interpolated.
• Sepa beads consists of functionalized
polymethacrylate-based resin with oxirane groups,
similar to Eupergit C.
• Amberlite XAD-7 is another synthetic matrix used to
immobilize enzymes by simple adsorption, for
example lipases from Humicola lanuginosa, Candida
antarctica and Rhizomucor miehei.
• Sepa-beads are best immobilizing matrix with
respect to Eupergit-C and Amberlite XAD-7 due to
excellent operational stability, reusability and
kinetic properties of immobilized enzymes.
INORGANIC SOLIDS
• They include alumina, silica, zeolites and mesoporous
silicas.
• They are known to be the cheapest matrix being used for
the immobilization of several industrial or nonindustrial
enzymes.
• Immobilized enzymes which are specifically used in
organic solvents have been very helpful for various
industrial processes, most importantly in packed bed
reactors based on packed granules.
• The immobilized enzyme is stable for several months under
dried conditions and stability.
• Silica has been found to be an excellent matrix for enzyme
immobilization due to their small size (dia. 2–40 nm) and high
surface area ranging from 300–1500 m2 g–1 with very high
stability at elevated temperatures.
• Its surface can be easily functionalized with any moiety, due to
which it can attach any enzyme.
• Immobilized enzymes are either present on the surface or
inside silica based on immobilization protocols.
• Epoxide hydrolase from Aspergillus niger has been
immobilized onto silica by covalent linkage leading to an
immobilized enzyme with retention of 90 % of enzyme activity
with respect to soluble enzyme.
• Immobilized enzymes have a shelf life of thousand times
greater than that of soluble enzymes.
• Inorganic solids have been used in the generation of
protein-coated microcrystals (PCMCs).
• They are prepared by mixing enzyme solutions with a
concentrated salt solution (potassium sulfate) followed by
its drop-wise addition to water-miscible solvent (isopropyl
alcohol) generating micron-sized crystals containing
enzyme on their surface.
• PCMCs have very high stability in the absence of any
solvent for a long period with applicability for wide range
of solvents.
• This method of immobilization causes the least damage to
the three-dimensional structure of the enzyme.
SMART POLYMER
• The most studied example of smart polymer is thermostable
biocompatible polymer [poly-N-isopropylacrylamide
(polyNIPAM)].
• PolyNIPAM exhibits the unique property of existing in two states;
the solution state when the temperature is lower than 32 °C, and
the polymer state when the temperature is 32 °C.
• Enzyme immobilization is done by mixing the enzyme solution
with solution state of polyNIPAM, i.e. <32 °C followed by an
increase in temperature, which precipitates immobilized enzyme
and subsequently filtration to get immobilized enzyme.
• This method of immobilization causes the least damage to
enzyme conformation.
• The enzyme is attached by covalent linkage with polyNIPAM
involving its vinyl groups with NH2 groups present on enzyme
surface.
• Penicillin G amidase has been successfully immobilized onto
polyNIPAM leading to an immobilized enzyme with a higher
stability and activity almost similar to soluble enzyme.
• Recently, a thermostable polymer has been made using 2-(2-
methoxyethoxy) ethyl methacrylate and oligo(ethylene glycol)
methacrylate (OEGMA).
• The formed polymer resembles poly(ethylene glycol) in terms of
low toxicity and it is anti-immunogenic.
• It is similar to polyNIPAM in terms of existing in solution and
polymer states as a function of temperature.
• Solution state temperature can vary from 26–90 °C depending on
the amount of OEGMA used.
Entrapment Carriers
• Enzymes can be immobilized by enclosing them inside the
matrices.
• Sol-gel is a metal alkoxides that has been used for the
entrapment of several enzymes.
• Enzyme immobilization into silica sol-gel is prepared by
hydrolytic polymerization of tetraethoxysilane followed by
drying.
• The immobilization method involves drying, which is the
determining factor in the morphology of sol-gel.
• For example, drying by evaporation produces xerogels with
nano-cages and pores while drying in the presence of CO2
produces aerogel with a delicate structure with phenomenal
porosity.
• Silica aerogel is known to be the lightest solid on earth with a
density of 0.001.
• Furthermore, the addition of Celite to sol-gel has led to
increased thermal stability of immobilized lipase.
• When higher alkyl groups are used for sol-gel preparation in
the presence of additives (e.g., isopropyl alcohol, crown ethers,
surfactants and KCl), an increase in Vmax by a factor of 10 has
been observed.
• The addition of silica quartz fiber has improved the mechanical
properties of immobilized enzyme.
• Sol-gel immobilized lipases have been used for the synthesis
of biodiesel by esterifying oil from sunflower seed.
• However, they cannot be used at high substrate concentrations
due to diffusion constraints.
• Experiments are still ongoing that aim to improve the activity
of immobilized enzymes via entrapment by various additives.