by M. K. Goel, 1994
Methods used for the immobilization of enzymes fall into four main categories:
Physical adsorption of an enzyme onto a solid is
prabably the simplest way of preparing immobilized enzymes. The method
relies on non-specific physical interaction between the enzyme protein and
the surface of the matrix, brought about by mixing a concentrated solution of
enzyme with the solid.
A major advantage of adsorption as a general method of insolubilizing
enzymes is that usually no reagents and only a minimum of activation steps
are required. As a result, adsorption is cheap, easily carried out, and
tends to be less disruptive to the enzymic protein than chemical means of
attachment, the binding being mainly by hydrogen bonds,
multiple salt linkages, and Van der Waal's forces. In this respect, the
method bears the greatest similarity to the situation found in biological
membranes in vivo and has been used to model such systems.
Because of the weak bonds involved, desorption of the protein resulting from changes in temperature, pH, ionic strength or even the mere presence of substrate, is often
observed. Another disadvantage is non-specific further adsorption of other proteins or other substances as the immobilized enzyme is used.
This may alter the properties of the
immobilized enzyme or, if the substance adsorbed is a substrate for the enzyme,
the rate will probably decrease depending on the surface mobility of enzyme and
Adsorption of the enzyme may be necessary to facilitate the covalent reactions
described later. Stabilization of enzymes temporarily
adsorbed onto a matrix has been achieved by cross-linking the protein in a
chemical reactiion subsequent to its physical adsorption.
Confining enzymes within the lattices of polymerized gels is another method for immobilization. This allows the free diffusion of low molecular
weight substrates and reaction products. The usual method is to polymerize the
hydrophilic matrix in an aqueous solution of the enzyme and break up the
polymeric mass to the desired particle size.
As there is no bond formation between the enzyme and the polymer matrix,
occlusion provides a generally applicable method that, in
theory, involoves no disruption of the protein molecules. However, free
radicals generated on the course of the polymerization may affect the
activity of entrapped enzymes. Another disadvantage is that only low molecular
weight substrates can diffuse rapidly to the enzyme, thus making
the method unsuitable for enzymes that act on macromolecular substrates,
such as ribonuclease, trypsin, dextranase, etc.
The broad distribution in pore size of synthetic gels of the polyacrylamide
type inevitably results in leakage of the entrapped enzyme, even after
prolonged washing. This may be overcome by cross-linking the entrapped protein
with glutaraldehyde. Alternatively, ultrafiltration membranes of
well-defined pore size may be used to occlude the enzyme.
Immobilization of enzymes has been achieved by intermolecular
cross-linking of the protein, either to other protein molecules or to
functional groups on an insoluble support matrix.. Cross-linking an enzyme
to itself is both expensive and insufficient, as some of the protein material
will inevitably be acting mainly as a support, resulting in relatively low
enzymic activity. Generally, cross-linking is best used in conjunction with
one of the other methods. Preventing leakage from
polyacrylamide gels has already been mentioned, but it is used much more widely
as a means of stablilizing adsorbed enzymes.
Since the enzyme is covalently linked to the support matrix, very little desorption is likely using this method. Marshall (1973), for example, reported that carbamyl
phosphokinase cross-linked to alkylamine glass with glutaraldehyde lost only
16% of its activity after continuous use in a column at room temperature for
The most intensely studied of the insolubilization
techniques is the formation of covalent bonds between the enzyme and the
support matrix. When trying to select the type of reaction by which a given
protein should be insolubilized, the choice is limited by the
fact that the binding reaction must be performed under conditions that do
not cause loss of enzymic activity, and the active site of the enzyme must be
unaffected by the reagents used.
The functional groups of proteins suitable for covalent binding under mild
conditions include (i) the alpha amino groups of the chain and the epsilon
amino groups of lysine and arginine, (ii) the alpha carboxyl group of the chain
end and the beta and gamma carboxyl groups of aspartic and glutamic acids,
(iii) the phenol ring of tyrosine, (iv) the thiol group of cysteine, (v) the
hydroxyl groups of serine and threonine, (vi) the imidazile group of
histidine, and (vii) the indole group of tryptophan.
A small number of reactions have been designed to couple with functional
groups on the protein other than the amino and phenolic residues. Aminoethyl
cellulose has been coupled to the carboxylic acid residues of enzymic protein
in the presence of carbodiimide, and thiol residues of a protein have been
oxidatively coupled to the thiol groups of a cross-linked copolymer of
acrylamide and N-acryloyl-cystein.
It is possible in some cases to increase the number of reactive residues of an
enzyme in order to increase the yield of insolubilized enzyme and to provide
alternative reaction sites to those essential for enzymic activity. As with
cross-linking, covalent bonding should provide stable, insolubilized enzyme
derivatives that do not leach enzyme into the surrounding solution. The
wide variety of binding reactions, and insoluble carriers with functional
groups capable of covalent coupling, or being activated to give such groups,
makes this a generally applicable method of insolubilization, even if very
little is known about the protein structure or active site of the enzyme to
When immobilizing an enzyme on a surface it is most important to choose a
method of attachment aimed at reactive groups outside the active catalytic and binding site of that
enzyme. Considerable knowledge of active sites of particular enzymes will
enable methods to be chosen that would avoid reaction with the essential groups
therein. Alternatively, these active sites can be protected during
attachment as long as the protective groups can be removed without loss of
enzyme activity. In some cases, this protective function can be fulfilled by a
substrate of the enzyme or a competitive inhibitor; this also contributes towards retention
of tertiary structure of the enzyme.
The surface on which the enzyme is immobilized has several vital
roles to play such as retaining of tertiary structre in the enzyme by
hydrogen bonding or the formation of electron transition complexes.
Retention of tertiary structure may also be a vital factor in maximizingthermal stability in the immobilized state. In this respect it is
wise to follow closely the new findings in the chemical nature of soluble
thermostable enzymes. The microenvironment of surface and the immobilized enzyme has an anionic or cationic nature of the surface that can cause a displacement in the optimum
pH of the enzyme of up to 2 pH units. This may be accompanied by a general
broadening of the pH region in which the enzyme can work effectively.
Immobilization by cross-linking the protein enzyme in order to insolubilize it
or merely immobilize it in desired location has many possibilities and is
relatively cheap. Several aldehydes and other cross-linking agents are
now available for this purpose. Extension of this approach to a process
where an enzyme is an integral component of a copolymer could permit
designing for reversible polymerization.
In enzyme reactor, the highest specific activity, in terms of weight of
enzyme and support employed, is desirable. It is considered an added bonus if
the support is employed to fulfil another function. The most important of
these functions is separation since, if this occurs simultaniously with
reaction, unfavorable equilibria may be displaced. One approach is to use a
molecular sieve as the support and, in packed reactors to pulse the reactor bed
alternatively by passage of substrate solution and water, so that bands of
unused substrate and product are progressing down the column. It so happens
that these enzymes for which this would be useful are also those which in
some cases benefit in having the enzyme immobilized on a porous support.
For an industrial reactor it is preferable to use
supports that are non-biodegradable such as glass, silica, Celite, Bentonite,
alumina, or titanium oxide, if possible. Even the linkages between enzyme
and support can be non-biodegradable, as with the titanium procedure. As far
as the biodegradability of the enzyme, in some cases, when in active use,
enzymes will partially protect themselves, notably all those that effect
oxidation with production of hydrogen peroxide as a by product.
Many types of reactors have been proposed: packed beds with downward flow,
suspended particles in a fluid ben with upward flow of substrate, a simple
stirred reactor, tubular reactors, membrane reactors, and many others. In some
of these the physical nature of the surface becomes a major problem. Thus some
supports that form excellent packed beds fail to do so when coated with enzyme
and particles which ideally self-suspend in a fluid bed then aggregate during
use requiring more power to pump through substrate allied to a lowered
catalytic activity from a decreased surface area. Many problems were
encountered with porous glass supports until it was realized that the enzyme was not simply
losing activity but the glass itself could dissolve. This has now been overcome by the
zirconium treatment of the glass surface. Additives to enzymes intended to
preserve enzyme activity are also rarely fully disclosed. This often causes
confusion as to the weight of protein in the enzyme product and can even
interfere with the coupling procedure or decrease its efficiency.
It is important to understand the changes in physical and chemical
properties which an enzyme would be expected to undergo upon
insolubilization if the best use is to be made of the various insolubilization
techniques available. Changes have been observed in the stability of
enzymes and in their kinetic properties because of the microenvironment imposed
upon them by the supporting matrix and by the products of their own action.
The stability of the enzymes might be expected to either increase
or decrease on insolubilization, depending on whether the carrier provides a
microenvironment capable of denaturing the enzymic protein or of stabilizing
it. Inactivation due to autodigestion of proteolytic enzymes should be reduced
by isolating the enzyme molecules from mutual attack by immobilizing them on
a matrix. It has been found that enzymes coupled to inorganic carriers were
generally more stable than those attached to organic polymers when stored at
4 or 23 ° centigrade. Stability to denaturing agents may also be changed
Changes in activity of enzymes due to the actual process
of insolubilization have not been studied very much. There is usually a
decrease in specific activity of an enzyme upon insolubilization, and this
can be attributed to denaturation of the enzymic protein caused by the coupling
process. Once an enzyme has been insolubilized, however, it finds itself in
a microenvironment that may be drastically different from that existing in
free solution. The new microenvironment may be a result of the physical and
chemical character of the support matrix alone, or it may result from
interactions of the matrix with substrates or products involved in the
The Michaelis constant has been found to decrease by more than one order of
magnitude when substrate of opposite charge to the carrier matrix was used.
Again, this only happened at low ionic strengths, and when neutral substrates
were used. The electrostatic potential was calculated by insertion of the
Maxwell-Bottzmann distribution into the Michaelis-Menton equation using the
changes in Michaelis constant, and good agreement was obtained with the value
for the electrostatic potential calculated from the pH-activity shifts.
The diffusion of substrate from the bulk solution to the micro-environment
of an immobilized enzyme can limit the rate of the enzyme
reaction. The rate at which substrate passes over the insoluble particle
affects the thickness of the diffusion film, which in turn determines the
concentration of substrate in the vicinity of the enzyme and hence the rate
The effect of the molecular weight of the substrate can also be large. Diffusion of large molecules will obviously be limited by steric
interactions with the matrix, and this is reflected in the fact that the
relative activity of bound enymes towards high molecular weight substrates
has been generally found to be lower than towards low molecular weight
substrates. This, however, may be an advantage in some cases, since the
immobilized enzymes may be protected from attack by large inhibitor molecules.
- Physical adsorption onto an inert carrier,
- Inclusion in the lattices of
a polymerized gel,
- Cross-linking of the protein with a bifunctional reagent,
- Covalent binding to a reactive insoluble support.