Immobilized Enzymes Video Lecture - Biotechnology Engineering (BT)

You would recall that in our previous lecture
we had discussed what are immobilized enzymes,
why should we immobilize enzymes and what
are the requirements of immobilization of
enzymes and the three major requirements – a
soluble enzyme and the carrier and method
of immobilization. We were looking into their
characteristics. The soluble enzyme, as we
all know, required for immobilization must
have certain properties that is it should
possess high specific activity and also desirable
functional properties that are required for
use of immobilization enzyme.
Besides that the carrier must also have certain
properties like porosity that means large
surface area so that heavy enzyme loading
can be given to the immobilized preparation
and we were talking about methods of immobilization.
A large numbers of methods of immobilization
are reported in literature. Most of them can
be classified simply into three major categories
one is carrier binding methods. In most methods
we take either a prefabricated carrier or
a carrier for conditioning either activation
or some conditioning and couple that to the
enzyme with a physico-chemical interaction.
It could be a covalent bond, it could be some
non-covalent interactions and those methods
fall under carrier binding methods. Typical
examples are adsorption; covalent binding
and we will be discussing them in detail.
The other method is cross linking.
The enzymes themselves posses a large number
of functional groups which can be made to
react to each other, thereby aggregating the
molecules and making them insoluble in water.
The aggregates will be insoluble and they
can be used as immobilized preparation which
can be readily separated from the lipid phase.
Just to repeat the cross linking refers to
linking more than one enzyme molecule through
their functional residues present on their
surface. Most of the enzyme molecules will
have a large number of functional residues.
They may be hydrophobic groups; they may be
electrostatic charges or other functional
residues. They can be linked under certain
conditions, under ambient condition so that
their inactivation is not allowed and on those
conditions aggregates of the enzymes can be
made which can function as immobilized enzymes.
The third type of method is entrapment methods
where the enzymes are entrapped within a matrix.
It could be a gel, it could be a micro capsule
and there by you could provide them an environment
which is very analogues to a living cell and
then use them as immobilize preparations.
You can see that a large number of methods
are available. Under each category also there
are number of methods and we need to choose
a suitable method for a particular purpose.
There are no general rules. It often goes
by trial and error and some available information.
Out of the methods available, which we will
discuss subsequently, if we have to choose
an immobilization method to use for a specific
application there are four distinct parameters
which should be looked into. The first and
probably the foremost is yield of immobilization.
That means during immobilization procedure
what is the loss of enzyme activity because
there is no method which can give you hundred
percent recovery of the enzyme activity that
you have put into the system.
There will be some enzyme which will be deactivated
or lost during the immobilization and the
loss of enzyme activity has to be kept minimum
for all practical purposes. When you consider
the yield of an immobilization, it is basically
a term which can be considered on the basis
of material balance. That means if you take
a soluble enzyme, add to a carrier and allow
it by any method of immobilization we get
two streams - one will be residual enzyme
activity in the supernatant which is left
uncoupled and immobilized enzyme preparation.
This is the total picture of all the species
that will be available and the loss of enzyme
activity, soluble enzyme activity let us say
as E0, residual enzyme activity as Er and
the immobilize enzyme activity as Ei, then
the loss of enzyme activity is
E0-Er-Ei/E0
That is the loss or rather fractional loss
of enzyme activity. Of the total enzyme activity,
the activity is distributed into two fractions-the
immobilized enzyme as well as the uncoupled
enzyme in the residue and these two theoretically
should combine and form the total value of
E0 but very often at the end of a method there
is some balance left and the balance is the
loss due to deactivation during immobilization.
The yield of the immobilization is defined
as
r = Ei/E0-Er
Very often there is confusion in reporting
these terms and that’s why I have tried
to put them in the form of an expression.
While we talk of a yield we must keep into
mind the residual activity in the supernatant.
Its not in the total enzyme because part of
the enzyme has been recovered in the supernatant
which if desired can be recycled. Therefore
the yield will be the activity which is recovered
in the immobilized form divided by E0-Ei in
the residue.
When you talk of Ei that is the activity of
immobilized enzyme you are measuring in terms
of the activity only. So that means it is
active. Right in the beginning I made it very
clear that when we talk of quantification
of enzyme it is just not a physical quantity
like mass or concentration. It is the functional
property. Its quantity is functional property
and the activity represents functional activity.
We are not measuring Ei in terms of protein
contents. I am measuring all these terms E0,
Er and Ei in terms of catalytic activity.
So it is functional. This loss, if it is a
let us a physical quantity like protein concentration
the balance will be perfect. You cannot account
for any loss of protein in the system. It
is only the loss of activity because of the
conformational changes. The other factor which
is important to be considered is enzyme loading.
That means that becomes almost similar parameter
like what you considered in case of a soluble
enzyme that is specific activity. When we
talk about specific activity higher the specific
activity, enzyme is catalytically better.
Similarly in case of enzyme loading we talk
of international units per gram of carrier
or if you take into account the bulk density
of the carrier you can also say international
units per ml of carrier bed that means you
pack the carrier into a bed, packed bed, and
then consider the volume.
You can express them in either quantity, either
as international units per gram of carrier
loading or sometimes we can also refer them
in terms of volume and thereby it can be compared
directly to the soluble enzyme. So enzyme
loading is another factor and we want a preparation
with as high loading as possible theoretically.
The enzyme loading will also have a direct
linkage with the mass transfer limitations.
We will see subsequently how mass transfer
and catalytic activity are linked. Then the
third parameter which is important to look
at is storage and operational stability.
As I mentioned that we have to look for enzyme
and one of the major advantage of immobilized
enzyme is that they can be reused, they can
be repeatedly used, continuously used and
therefore they must possess a much higher
stability than the soluble enzyme. This stability
is not only in terms of use but also during
storage and this parameter is usually expressed
in terms of half life.
Either half life during storage that means
the time in which the activity loses to fifty
percent of the original activity or during
use in a continuous reactor or in a batch
reactor that is in terms of number of cycles.
You can express in that term. That will be
only restricted when you are talking of a
fibre or a surface. The most commonly used
matrices are beads- the spherical particles
and that’s why gram or volume and they are
packed in a packed bed. When I say per ml
I am referring to a parameter which is analogues
to use in a packed bed. But if you this in
a surface, in fibre or in a film you can also
express in terms of a surface area. That is
always allowed. But for all industrial application
the surface excepting for biosensors and things
like that which are very specific applications,
for industrial catalysis it is used in the
form of particles and used with the packed
bed. We will discuss those aspects subsequently.
Iu/m2 also is a feasible parameter that we
can define. Then the fourth property which
is a very important basis for choosing the
immobilization method is carrier cost and
regenerability because each method has a limitation
of the choice of carrier.
For example in the case of adsorption you
require a carrier. If suppose you want to
adsorb by ionic binding, you need a carrier
which possesses charges. Similarly if you
want a covalent binding you need a carrier
which possesses certain functional residues
which can be coupled to the enzyme. So each
method will have some limitations on the choice
of carrier and therefore the cost of the carrier.
Another parameter which must be taken into
account is regenerability. Very often even
after the immobilized enzyme preparation has
been exhausted during use the carrier can
be recovered and reused. So the cost of the
carrier must be taken into account in terms
of per cycle not the absolute cost. You must
differentiate between the absolute cost and
cost of the carrier per cycle. Because many
of the carriers although expensive, can be
used for a long period and in number of repeated
cycles. Therefore they are still acceptable
and desirable because of other desirable property.
The first and probably the simplest method
of immobilization is by adsorption. The adsorption
method is a very broad, non-specific method
and it involves binding of the enzyme to the
surface of an insoluble carrier that has not
been specifically functionalized for covalent
coupling. I must mark here particularly the
phrase “specifically functionalized for
covalent coupling”.
This probably gives you an idea that this
is a very broad non-specific method. If we
consider all the carrier binding methods excluding
those which involve covalent linkage, rest
of all the bindings, interactions between
the carrier and the enzyme are covered under
the term called adsorption. A very broad based
method which involves a number of non-specific,
multivariant, reversible interactions between
the enzyme and the carrier at mild conditions.
The second point also builts in a number of
phrases. For example the interactions that
are involved are non-specific, very often
more than one interaction is involved in a
particular method, multivariant that means
more than one physical property is employed.
In most cases the methods based on adsorption
are reversible in nature. That means under
certain conditions while you can adsorb the
enzyme on to a carrier you can also create
condition by which you can desorb the enzyme.
The basic property of this reversible interaction
also makes it a weak binding. Weak binding
in the sense while during use, the enzyme
can also dissolve slowly over a long period
of time and if the carrier characteristics
are sturdy they can be regenerated and reused.
But enzyme loss can be there big time, over
a long period of time. Multivariant means
that more than one physico-chemical interaction
is involved in binding. Another feature is
mild conditions and that will apply to most
of the immobilization methods and also to
adsorption. That means the environmental conditions
required for adsorption must be mild and here
I think I like to very specifically define
the term mild. Mild refers here not to the
value one or two it refers to those conditions
under which enzyme is conformationally stable.
The definition of mild condition refers to
those conditions which are environmentally
acceptable to the enzyme. If the enzyme is
stable at 70ºC, 70ºC is acceptable and can
be considered as mild. But if the enzyme is
not stable even at 20ºC you may have to go
to 4 degrees to immobilize and the definition
of mild will remain at 4 degrees. I have purposely
not used the word ambient because you refer
it to a well definite range. The advantage
of the situation is that usually the methods
based on adsorption results in high yields.
We are giving the definition of yield that
means the total amount of activity, which
is resulting in the immobilized preparation
is reasonably high, almost in the range of
say about 70 to 90 % enzyme activity is recovered
in immobilized preparation and because the
conditions are mild they don’t tend to cause
conformational change so that is a very plus
point as compared to other methods in which
yields will be much lower.
A disadvantage is that the preparations do
not have very high operational stability.
The method is reversible in nature, the interactions
are reversible and so operational stability
is usually not very high. Although by various
other manipulations like adsorption, coupling
with cross linking the operational stability
can be improved but intrinsically adsorption
based methods are usually operationally high.
Again when I say the term high it refers in
the comparative form and the comparison here
is with covalent binding. If suppose in the
case of covalent binding half lives are of
the order of few months here it may be of
the order of few days. So that is what it
refers to but allows the regeneration with
carrier. On the other hand although the operational
stability is not very high but the advantage
is that the carrier can be regenerated and
reused. That is the plus point. You have a
large variety of carriers that can be used.
We will look at some of the examples of the
carriers and finally adsorption based methods
have been very often used to study the enzymology
of systems that naturally occur in biological
system because in case of living cells most
of the enzymes are supported on a solid matrix
through adsorption only. There are no covalent
linkages. Either the enzymes are supported
on membrane or they are supported on certain
particulate matters and therefore the methods
based on adsorption are usually very analogues
similar to those that are found in the living
system.
Operational stability is defined in terms
of half life. Consider the use of immobilized
enzyme in a reactor and pack it in a packed
bed, feed in and effluent out. Here X-axis
is operational time and Y-axis is fraction
of conversion. What you notice is that when
you start at zero time the fraction of conversion
will keep on going down as the enzyme activity
is reduced or lost. The fraction of conversion
keeping the residence time constant and over
operational time, this is operational time
not the residence time, residence time is
constant here it will go down. If you consider
as a first order process then the half life
of this that means the time it takes to reach
the fifty percent of the original value is
half life. If we consider
E = E0 e-kdt
You can simply calculate half life as
t1/2 = 0.69/kd
t1/2 is less in the case of adsorption. Other
way, compared to covalent binding. Right.
Because the interaction is reversible in nature
so it keeps leaking. A very crude term could
be the enzyme keeps leaking values, very small
amounts, slowly not in a very fast rate. This
property of leaking of the enzyme from immobilized
preparation has been very advantageously used
in many cases of the enzyme therapy purposes,
where we want to put an immobilized enzyme
preparation onsite at a site where the enzyme
is required and the enzyme must leak continuously
not in a very high concentration and over
a long period of time. Some systems like immobilized
……. has been inserted into the blood stream
in the immobilized forms - microcapsules and
the enzyme leaks slowly and the immobilized
system can remain in blood stream over a few
months. You have to give another shot to keep
the enzyme activity available in the blood
stream. So these methods can have an advantage
that the reversible nature can also be used
in some cases where you want to use the drug
or a therapeutic agent with the requirement
of slow availability over a long period of
time. As far as study of reactor system is
concerned, we are not measuring the enzyme
activity every time. What we are measuring
is the conversion at the effluent. Conversion
is related to the enzyme activity. So that’s
why the graph is in the form of conversion.
Three most common types of adsorption methods
are based on physical adsorption which is
again a most non-specific type of adsorption
where we use hydrogen bonding, electrostatic
interactions or Vanderwaal interactions, a
variety of interactions where there is no
specific reasons and we use a very broad range
of carriers like alumina, celite, bentonite,
glass etc where which by certain conditions
of pH, temperature and salt you can adsorb
the enzyme on to the surface and they can
be used.
The second method is ionic binding where you
take a tailored matrix which has a definite
charge on the surface and ion exchange resins
are the most commonly used matrices. DEAE-Cellulose,
Amberlite etc which are charged particles
and these charged particles based on the electrostatic
interaction can bind the enzyme molecules
at a pH where the net charge is counter. The
enzyme at this stage is counter charged at
a particular condition. The third category
is hydrophobic binding where we predominantly
use hydrophobic interactions on the surface
of the carrier and the hydrophobic interactions
on the surface of the enzyme protein.
Although bulk of the hydrophobic residues
are buried inside the residue of enzyme but
still on the surface there are very small
hydrophobic patches which can be used here
for immobilizing on to the surface of a hydrophobic
carrier; carriers like polypropylene beads.
There is a commercial product by the name
Accurel, which is a microporous carrier consisting
of polypropylene, butyl or hexyl sepharose
etc. There are number of hydrophobic carriers
used and which are available for binding of
enzyme. Many of the carriers which are available
today for adsorption of enzymes are developed
for chromatographic separations. For example
all the carriers like hydrophobic binding
or ionic binding were originally developed
for chromatographic separations where the
function is same. That means you are binding
through ionic interactions and desorbing it
under the conditions of desorption and therefore
the same thing applies here. In fact the preparation
of immobilized enzyme by adsorption is very
much similar to chromatographic separations
on ion exchange resin, although we are only
interested in the first part of it. Only adsorption
part; desorption is not our interest where
as in the case of chromatography both the
parts are important.
Another important group of adsorption methods
is metal salt linkage. Certain metal salts
like titanium tetra chloride and ferric chloride
form a kind of an inorganic bridge on the
surface of the carrier and to which the enzyme
molecules can couple. The linkage that is
formed, what we have in the literature and
what people have used, is the metal salt linkage.
It is not a covalent linkage; it is a physical
interaction and there by the enzyme is held
but has been very successful method from the
practical point of view. We will discuss that
in detail. Most of the adsorption methods
can be characterized by the classical adsorption
isotherms. Depending on the nature of the
carrier and nature of the enzyme that we are
talking about, the isotherms may vary. Three
general types of isotherms are known.
One is linear where q is the concentration
of the enzyme protein on to the surface of
the carrier; y is the corresponding concentration
in the solution at equilibrium. If you take
a combination of enzyme in carrier concentration
and at equilibrium measure the concentration
in the solid phase and the liquid and you
make an isotherm, then you may end up sometime
in some cases a linear relationship where
q is equal to k.y
To make the isotherm, take different ratios
of the carrier and the enzyme activity. That
means enzyme activity to carrier ratio is
varied and at different combinations you allow
equilibrium to be formed and under equilibrium
condition you measure the concentration of
the enzyme in the liquid phase and also in
the solid phase. Each point represents a particular
enzyme to carrier ratio. You can find three
different types of isotherms: linear is simple;
Freundlich is more general in the sense that
it does not fit into any hypothesis but in
general you can vary curve fitting. What ever
kind of isotherm you get, you just raise to
a factor of n and by analysis of your data
you will calculate the value of k and n. You
could make a log log plot and you can get
the value of k and n.
The most established and analytically proven
method is Langmuir isotherm. Here q is the
concentration of the enzyme protein on to
the solid matrix and q0 is the maximum capacity
of the matrix. If you take a very high enzyme
concentration compared to carrier, allow equilibrium
to be established that means the concentration
of the enzyme on to the immobilized preparation
which will represent the saturation behavior
is q0. You will notice that it is analogues
to your Michaelis Menten kinetics. These three
expressions the Langmuir isotherm, Michaelis
Menten kinetics, hyperbolic expression and
also the Mannose equation for cell growth
are almost analogues expressions and that
indicates that there is some inter relationship
or some similarity in their basic physical
interpretation.
Langmuir isotherm is based on a theoretical
development. One can develop on the basis
of number of binding sites on the carrier
molecule for enzyme. Here just like we consider
the enzyme substrate binding where the enzyme
acts as the carrier and the substrate is ligand
here the carrier will be the matrix and the
protein will be the ligand and on those conditions
you can develop it or you can analyze it by
a double reciprocal plot. The analogy is that
the three expressions, the Langmuir adsorption
isotherm, the Michaelis Menten kinetics and
Mannose kinetics for cell growth they are
analogues terms. You have to develop an analogy
and understand the relationship of the corresponding
terms. In the case of enzyme we are talking
of enzyme substrate complex formation. That
means enzyme is the matrix, surface is a ligand.
If we talk in terms of adsorption, the substrate
gets adsorbed on to the enzyme surface.
On the other hand here in the case of adsorption
the enzyme is itself a ligand and the matrix
or the carrier is the surface on which the
ligand adsorbs. In the third case, in the
case of cell growth, the Mannose model of
microbial cell growth it is a substrate which
is a ligand just like enzymes but the matrix
is the cell mass itself and it reflects the
transport of substrate into the cell. So this
is adsorption that is transport and enzyme
catalyses enzyme substrate binding. So these
three phenomena are intrinsically someway
related because they end up in a similar kind
of a kinetic expression. As far as the analysis
is concerned all the three can also be analyzed
by a double reciprocal plot. That is if you
plot 1/q versus 1/y you will get a linear
plot and you will get the value of q0 and
k. The adsorption process for a particular
enzyme and for a particular carrier can be
analyzed quantitatively. Many physical factors
influence the adsorption process. I have listed
some of them here. The first parameter is
pH.
The pH will be a very major parameter which
will affect the binding in the case of ionic
binding. Even for other forms of binding processes
pH plays a key role and very often it has
been noted that the binding by adsorption
is maximum at its isoelectric points. Large
variety of relationships with respect to pH
for the quantity adsorbed has been found.
There is no general ruling but in general
or a common feature can be considered that
at the isoelectric point the pH value will
be most preferable for the adsorption processes.
No. Net charge is zero. It is not that the
proteins are not charged at that pH. The negative
and positive charges are equal. Not that there
is no charge.
The second parameter is salts. Salts have
a very complex behavior. In fact it has almost
opposite behavior. You must have studied also
in the case of bio separations particularly
the concentration of salt is used for increasing
as well as decreasing solubility. Salting
in and salting out both are feasible and of
course the phenomena or the physical interactions
are just opposite. But in most cases the salt
concentration is required to be kept at a
minimum because salts are very good eluents
also. In ion exchange chromatography very
often we use high ionic strength as a medium
for elution of the adsorbed material. Therefore
at high salt concentration adsorption is usually
poor and minimum salt concentration is required
to be kept. Protein concentration will be
important and we have seen from the adsorption
isotherm itself. The definite ratio of protein
to carrier will play a particular equilibrium
behavior and higher the protein concentration
you can reach nearer the saturation level
at equilibrium. Temperature again is a very
significant parameter which must be understood.
If we look at adsorption of a small molecular
weight material or a small molecular weight
ligands on to a surface the general behavior
is that increasing the temperature reduces
the adsorption. That means the quantity of
ligand adsorbed per unit weight of the carrier
for low molecular weight ligands will be less
as you increase the temperature and the reason
attributed to this is because of the movement
of the particles as the temperature increases.
That means the movement of particle increases
and the tendency is for desorbing rather than
adsorption. But in the case of protein adsorption
when we are talking about enzymes, we are
talking about proteins and macromolecules,
adsorption over a narrow range of temperature
offers a reverse phenomena. That means adsorption
increases with temperature. Mind the word
I am using “over a narrow range of temperature
increase”. If you increase to a higher level
desorption also might start. But before desorption
even inactivation might start. But we consider
the adsorption at …… range of temperature.
The primary factors responsible for the increase
in adsorption with temperature are when the
protein binds to a surface there is some unfolding
of the protein molecules, which is an endothermic
process and the increase of temperature resulting
in enhanced adsorption supports the hypothesis
that the binding or adsorption of a protein
molecule or a polymeric material to a surface
is an endothermic process coupled with unfolding
of the confirmation. When I say unfolding
I am not referring to total deactivation but
minor modification in the conformation which
extends its total volume or total surface
area.
The other factor which is important, which
is probably attributing to this increase of
adsorption with the increase in temperature
is the diffusional processes because most
of time we are talking about the carrier being
a porous material and the ligands or the protein
here have to diffuse into the surface, into
the internal pore surface and the diffusion
process gets increased by increase in temperature.
So both the unfolding of the protein molecule
on the surface of the carrier and the diffusional
processes involved in the adsorption in the
internal pores ultimately leads to give you
a phenomenon which represents increase in
adsorption with increase in temperature over
a narrow range. Carrier properties, depending
on the property like surface property of the
carrier particles which were used as matrix,
will also play a role on the adsorption behaviour
depending on the nature may be sometime even
the carriers are pre-conditioned, swollen
to improve the property. Time of adsorption
is another parameter which is significant
and important because we are talking about
the macromolecular adsorption. So the diffusion
is involved and the time required very often
is controlled by diffusional limitations and
the rate of diffusion controls the time required
for adsorption process. This in the case of
a small ligand is not a constant. For adsorption
we follow a number of methods. At least four
major methods are used for adsorption. The
first and probably the most trivial method
is the static process.
That means you bring in an enzyme solution
in contact with a carrier and leave it over
night or over a long period of time. Adsorption
takes place without any hesitation. It takes
a very long time and that is the process very
often many people use in laboratories. The
yields are also much lower particularly for
porous carrier because of the diffusional
limitations. One of the most disadvantageous
situations in the case of static process is
that the loading of the enzyme is not uniform
over the carrier. Even if you allow for a
long period the loading is usually not uniform.
Suppose this is the carrier particle. You
have lot of enzyme molecules on the surface
but on the interior it is very, very rarely
distributed and the loading is not uniform.
The most commonly used method in the laboratories
is dynamic batch process in which you mix
the two components, the enzyme and the carrier
under known conditions or under standard conditions
for adsorption, which favours adsorption and
agitate them, stir them mechanically, under
mild stirring so that the carrier particle
should not get degraded and also the enzyme
molecules should not be influenced. The equilibrium
is allowed to be reached over a period of
time. It usually leads to a fast method and
most common method used in the laboratories.
The third method is the reactor loading process.
In the commercial establishment when we use
adsorption as the method of immobilization
we use reactor loading process. That means
first of all the reactor which is to be used
for bio-conversion of the enzymatic reaction
is packed with the carrier itself and then
the enzyme solution is recirculated constantly
through the carrier bed and over a period
of time the enzyme gets adsorbed on the matrix.
It could not only be packed bed; it could
also be stirred reactor in which the enzyme
is added and the enzyme solution is allowed
to be stirred along with the carrier and after
a period of time when the supernatant enzyme
activity has come to minimum value you stop
the process.
In all these methods which we are talking
about, the final step is washing because very
often you will have that some of the enzyme
will be adhering to the surface of the particles
which is not really immobilized in the proper
sense. It is not adsorbed; some adhering along
with the liquid and so fresh wash of the buffer
at a particular pH should be given so that
all the adhering enzyme is washed out and
only that which is physically bound by adsorption
is retained by the carrier. The final washing
in the case of reactor loading process is
done with the substrate solution itself because
during the operation it has to meet the environmental
conditions which are provided by the substrate
solution, the feed. Under those conditions
it must remain adsorbed and so the final washing
in the case of a reactor loading method is
provided by the feed substrate itself so that
whatever desorption has to take place under
feed substrate condition, takes place and
the enzyme is in the stable form.
The fourth method is electrodeposition process
in which the carrier is placed in proximity
to one of the electrodes and the current is
applied between the two electrodes and the
enzyme particles which are charged usually
they get deposited on the carrier particles
and at the end the carrier is removed which
gets coated by the enzyme particle.
One thing we have to keep in mind, a very
important feature that during the electro
chemical process there will be certain ions
which will be removed from the electrodes
or from the process. If the ions are important
for enzyme activity then they must be provided
in the electrolyte itself. Extra quantity
of those will be provided in the electrolyte
otherwise you will not be having a functional
enzyme preparation in the immobilized form.
The last method based on the adsorption process
is by inorganic bridge formation as I mentioned
or metal salt linkage.
This was originally developed by British group
Barker, Emery and Novais, one of the references
I have sited here, in seventy one. It involves
the metal salt linkage. The basic principle
of the method is the method employs transition
metal salts like titanium tetrachloride, zirconium
tetrachloride or ferric chloride for linking
an enzyme to the carrier. Carriers are usually
cellulose, nylon, glass beads etc. The usual
method is that an activated carrier is prepared
by steeping it in a solution of transition
metal salt for five to ten minutes and mind
it that it is important because longer contact
of the matrix in the transient salt solution
can result in the degradation because the
pH of this solution, like titanium tetra chloride,
will be very, very low, almost one to two.
If you put cellulose into it, it might get
degraded totally. So the time of contact is
very important and it is not used mostly more
than five minutes and if you take a dilute
solution probably you can go higher, followed
by filtering and drying of complex at 45ºC.
Excess unreacted salt is washed with buffer.
The activated carrier is then stirred with
the buffered enzyme solution followed by washing
of the unbound enzyme. The method is very
simple and involves two steps, activation
of the matrix by steeping it in a transition
metal salt solution whereby the transition
metal salts form an octahedral complex.
The chemistry of that can be considered like
this. Titanium tetra chloride will exist in
different forms and the complexes will be
either dominated by water molecules as the
ligand in the octahedral complex form. The
titanium tetra chloride solution will exist
in different forms complexed with certain
ligands. For example in solution water molecule
will be present as the ligand and will complex
with water and chloride ions.
In aqueous form titanium has five water molecules
and one chloride. It can also exist as the
chloro complex; five chloride ions and one
water molecules. It can also exist as total
chloride ions in the case of a co-ordination
complex. So when this solution is brought
in contact with the matrix, any carbohydrate
matrix, cellulose has been a very commonly
used matrix, then what happens is that these
ligands are replaced by the hydroxyl groups
present on the carbohydrate molecule. The
ligand particularly the water molecules which
are present here are replaced by the hydroxyl
groups present on the carbohydrate molecule
and you make a network, titanium treated cellulose
which is complexed along with the length of
the chain on the carbohydrate is formed. When
this activated matrix, we consider this as
an activated complex, is further brought into
contact with the protein or enzyme again some
of the water molecules are replaced by the
functional groups on the enzyme. Wherever
you see water molecules, for example these
ones they may be replaced by enzyme molecules.
Wherever hydroxyl groups are present they
can replace part of them and what ends up
is a matrix which has a coordination link
of titanium tetra chloride salt along with
the enzyme molecule.
One of the major advantages is that you always
end up having a distance between the carrier
surface and the enzyme molecules. Therefore
enzyme molecules and the carrier surface are
separated by these linkages, the metal salt
linkages. That provides you a very big advantage
of removing the steric hindrance. Many of
the enzymes when they are coupled to a solid
matrix may not be able to interact with the
substrate because of steric hindrance. But
in this case because there is distance between
the matrix surface and the enzyme, steric
hindrance is minimized.
The second major advantage which has been
noted in these cases is that if you use a
regenerated carrier after the enzyme has been
leached out, you again immobilize the enzyme
on the regenerated carrier. It gives you a
much increased activity and that is the major
advantage. The major reason has been that
the distance from surface has increased. So
steric hindrances are further decreased and
therefore particularly with those enzymes
which are known to have steric hindrances
during immobilization, the ones which use
much larger polymers as the substrate, for
those systems the method is very significant.
So these are the various methods that we have
discussed today. The various methods that
involve the coupling of enzyme by adsorption
to a matrix and their characteristic features
and as I mentioned that the immobilization
methods must be evaluated on the basis of
their yield, operational stability and carrier
regenerability. We stop here.