2. Stabilization of enzyme
Enzyme Stabilization is gaining importance due
to the wide range of enzyme applications. The
techniques that have been attempted to achieve
enzyme stabilization can be divided into broad
categories of aqueous and non-aqueous
stabilization. Some of them are:
1. Immobilization
2. Nanostructures
3. Modification of chemical structure
4. Protein engineering
5. Freeze drying
3. Stabilization of enzyme: Immobilization
An immobilized enzyme is an enzyme which is
attached to an inert, insoluble material such as
calcium alginate (produced by reacting a mixture
of sodium alginate solution and enzyme solution
with calcium chloride).
This can provide increased resistance to
changes in conditions such as pH or
temperature.
4. Stabilization of enzyme: Immobilization
It also allows enzymes to be held in place
throughout the reaction, following which they are
easily separated from the products and may be
used again - a far more efficient process and so
is widely used in industry for enzyme catalyzed
reactions.
Several hundred enzymes have been
immobilized in different forms and approximately
a dozen immobilized enzymes, for example
penicillin G acylase, lipases, proteases,
invertase, etc. have been used as catalysts in
various large scale processes.
5. Stabilization of enzyme: Immobilization
Adsorption on glass, alginate beads or matrix:
Enzyme is attached to the outside of an inert
material.
• This method is the slowest among all.
• As adsorption is not a chemical reaction, the active site of the
immobilized enzyme may be blocked by the matrix or bead,
greatly reducing the activity of the enzyme.
Entrapment:
The enzyme is trapped in insoluble beads or
microspheres, such as calcium alginate beads.
However, this insoluble substances hinders the
arrival of the substrate, and the exit of products.
6. Stabilization of enzyme: Immobilization
Cross-linkage:
The enzyme is covalently bonded to a matrix
through a chemical reaction.
This method is the most effective method among all.
As the chemical reaction ensures that the binding site does not
cover the enzyme's active site, the activity of the enzyme is only
affected by immobility.
However the inflexibility of the covalent bonds precludes the self-
healing properties exhibited by chemoadsorbed self-assembled
monolayers.
Use of a spacer molecule like poly(ethylene glycol) helps reduce
the steric hindrance by the substrate in this case.
7. Stabilization of Enzyme: Nanostructure
A nanostructure is an object of intermediate size
between molecular and microscopic (micrometer
-sized) structures.
Nanotextured surfaces have one dimension on the nanoscale,
i.e., only the thickness of the surface of an object is between 0.1
and 100 nm.
Nanotubes have two dimensions on the nanoscale, i.e., the
diameter of the tube is between 0.1 and 100 nm; its length
could be much greater.
Finally, spherical nanoparticles have three dimensions on the
nanoscale, i.e., the particle is between 0.1 and 100 nm in each
spatial dimension. The terms nanoparticles and ultrafine particles
(UFP) often are used synonymously although UFP can reach into
the micrometre range. The term 'nanostructure' is often used
when referring to magnetic technology.
8. Stabilization of Enzyme: Chemical Modification
This is in general the reaction of a specific
residue at a rate much greater than that of other
residues of the same kind, such that it can be
labeled specifically and thus identified.
Usually this procedure is used to identify a
residue at the active site of an enzyme, or other
specific site on a protein such as an effector site
or a site where binding to another protein or
nucleic acid occurs.
Active-site-directed chemical modification:
9. Stabilization of Enzyme: Chemical Modification
It can also be described as a combination
of reversible competitive inhibition and
irreversible chemical modification.
Active-site-directed chemical modification contd.
10. Stabilization of Enzyme: Freeze Drying
Freeze-drying (also known as lyophilization or
cryodesiccation) is a dehydration process
typically used to preserve a
perishable material or make
the material more convenient
for transport.
Freeze-drying works by freezing
the material and then reducing the surrounding
pressure and adding enough heat to allow the
frozen water in the material to sublime directly
from the solid phase to the gas phase.
11. Freeze Drying: Application
Pharmaceutical and biotechnology
Pharmaceutical companies often use freeze-
drying to increase the shelf life of products,
such as vaccines and other injectables.
By removing the water from the material and
sealing the material in a vial, the material can
be easily stored, shipped, and later reconstituted
to its original form for injection.
12. Freeze Drying: Application
Freeze-dried coffee,
a form of instant coffee.
Food Industry
Instant coffee is sometimes freeze-dried,
despite the high costs
of the freeze-driers used.
The coffee is often dried
by vaporization in a hot
air flow, or by projection
on hot metallic plates.
Freeze-dried fruit is used in some
breakfast cereal.
13. Freeze Drying: Application
Food Industry
Freeze-drying is used to preserve food and make
it very lightweight.
• freeze-dried ice cream.
• popular and convenient for hikers because the
reduced weight allows them to carry more food
and reconstitute it with available water.
However, the freeze-drying process is used more
commonly in the pharmaceutical industry.
14. Freeze Drying: Application
Technological Industry
In chemical synthesis, products are often freeze-
dried to make them more stable, or easier to
dissolve in water for subsequent use.
In bioseparations, freeze-drying can be used
also as a late-stage purification procedure,
because it can effectively remove solvents.
Furthermore, it is capable of concentrating
substances with low molecular weights that are
too small to be removed by a filtration membrane.
15. Freeze Drying: Application
Technological Industry
Freeze-drying is a relatively expensive process.
Therefore, freeze-drying is often reserved for
materials that are heat-sensitive, such as
proteins, enzymes, microorganisms, and blood
plasma.
The low operating temperature of the process
leads to minimal damage of these heat-sensitive
products
16. Stabilization of enzyme
All these methods have their own advantages
and disadvantages. None is suitable for all
enzymes. There are some simple techniques
that can help enhance stability of enzymes:
• Enzymes denature very quickly at
temperatures above body temperature. Thus,
they should be stored at low temperatures.
• Several enzymes lose their function on being
stored for very long (even under favorable
conditions). Thus, it is suggested that the
enzyme solution should be prepared just
before use.
17. Enzyme Turnover Number
Turnover number (also termed kcat) is defined as
the maximum number of molecules of substrate
that an enzyme can convert to product per
catalytic site per unit of time.
kcat = Vmax/[E]T
Carbonic anhydrase has a turnover number of
400,000 to 600,000 s−1, which means that each
carbonic anhydrase molecule can produce up to
600,000 molecules of product (bicarbonate ions)
per second.
