Enzyme Technology
Methods of immobilisation
There are four principal methods available for
immobilising enzymes (Figure 3.1):
- adsorption
- covalent binding
- entrapment
- membrane confinement
Figure 3.1.
Immobilised enzyme systems. (a) enzyme non-covalently adsorbed to an insoluble
particle; (b) enzyme covalently attached to an insoluble particle; (c) enzyme
entrapped within an insoluble particle by a cross-linked polymer; (d) enzyme
confined within a semipermeable membrane.
Carrier matrices for enzyme immobilisation by adsorption
and covalent binding must be chosen with care. Of particular relevance to their
use in industrial processes is their cost relative to the overall process costs;
ideally they should be cheap enough to discard. The manufacture of high-valued
products on a small scale may allow the use of relatively expensive supports and
immobilisation techniques whereas these would not be economical in the
large-scale production of low added-value materials. A substantial saving in
costs occurs where the carrier may be regenerated after the useful lifetime of
the immobilised enzyme. The surface density of binding sites together with the
volumetric surface area sterically available to the enzyme, determine the
maximum binding capacity. The actual capacity will be affected by the number of
potential coupling sites in the enzyme molecules and the electrostatic charge
distribution and surface polarity (i.e., the hydrophobic-hydrophilic balance) on
both the enzyme and support. The nature of the support will also have a
considerable affect on an enzyme's expressed activity and apparent kinetics. The
form, shape, density, porosity, pore size distribution, operational stability
and particle size distribution of the supporting matrix will influence the
reactor configuration in which the immobilised biocatalyst may be used. The
ideal support is cheap, inert, physically strong and stable. It will increase
the enzyme specificity (kcat/Km) while reducing product
inhibition, shift the pH optimum to the desired value for the process, and
discourage microbial growth and non-specific adsorption. Some matrices possess
other properties which are useful for particular purposes such as ferromagnetism
(e.g., magnetic iron oxide, enabling transfer of the biocatalyst by means of
magnetic fields), a catalytic surface (e.g., manganese dioxide, which
catalytically removes the inactivating hydrogen peroxide produced by most
oxidases), or a reductive surface environment (e.g., titania, for enzymes
inactivated by oxidation). Clearly most supports possess only some of these
features, but a thorough understanding of the properties of immobilised enzymes
does allow suitable engineering of the system to approach these optimal
qualities.
Adsorption of enzymes onto insoluble supports is a very simple method
of wide applicability and capable of high enzyme loading (about one gram per
gram of matrix). Simply mixing the enzyme with a suitable adsorbent, under
appropriate conditions of pH and ionic strength, followed, after a sufficient
incubation period, by washing off loosely bound and unbound enzyme will produce
the immobilised enzyme in a directly usable form (Figure 3.2). The driving force
causing this binding is usually due to a combination of hydrophobic effects and
the formation of several salt links per enzyme molecule. The particular choice
of adsorbent depends principally upon minimising leakage of the enzyme during
use. Although the physical links between the enzyme molecules and the support
are often very strong, they may be reduced by many factors including the
introduction of the substrate. Care must be taken that the binding forces are
not weakened during use by inappropriate changes in pH or ionic strength.
Examples of suitable adsorbents are ion-exchange matrices (Table
3.1), porous
carbon, clays, hydrous metal oxides, glasses and polymeric aromatic resins.
Ion-exchange matrices, although more expensive than these other supports, may be
used economically due to the ease with which they may be regenerated when their
bound enzyme has come to the end of its active life; a process which may simply
involve washing off the used enzyme with concentrated salt solutions and
re-suspending the ion exchanger in a solution of active enzyme.
Figure 3.2. Schematic
diagram showing the effect of soluble enzyme concentration on the activity of
enzyme immobilised by adsorption to a suitable matrix. The amount adsorbed
depends on the incubation time, pH, ionic strength, surface area, porosity, and
the physical characteristics of both the enzyme and the support.
Table 3.1 Preparation of
immobilised invertase by adsorption (Woodward
1985)
|
Support type
|
% bound at
|
DEAE-Sephadex
anion exchanger
|
CM-Sephadex
cation exchanger
|
pH 2.5
|
0
|
100
|
pH 4.7
|
100
|
75
|
pH 7.0
|
100
|
34
|
Immobilisation of enzymes by their covalent coupling to
insoluble matrices is an extensively researched technique. Only small amounts of
enzymes may be immobilised by this method (about 0.02 gram per gram of matrix)
although in exceptional cases as much as 0.3 gram per gram of matrix has been
reported. The strength of binding is very strong, however, and very little
leakage of enzyme from the support occurs. The relative usefulness of various
groups, found in enzymes, for covalent link formation depends upon their
availability and reactivity (nucleophilicity), in addition to the stability of
the covalent link, once formed (Table 3.2). The reactivity of the protein
side-chain nucleophiles is determined by their state of protonation (i.e., charged
status) and roughly follows the relationship -S− > -SH > -O− >
-NH2 > -COO− > -OH >> -NH3+where the charges may be estimated from a knowledge of the pKa values of the ionising groups
(Table 1.1) and the pH of the
solution. Lysine residues are found to be the most generally useful groups for
covalent bonding of enzymes to insoluble supports due to their widespread
surface exposure and high reactivity, especially in slightly alkaline solutions.
They also appear to be only very rarely involved in the active sites of enzymes.
Table 3.2 Relative
usefulness of enzyme residues for covalent coupling
Residue
|
Content
|
Exposure
|
Reactivity
|
Stability
of couple
|
Use
|
Aspartate
|
+
|
++
|
+
|
+
|
+
|
Arginine
|
+
|
++
|
-
|
±
|
-
|
Cysteine
|
-
|
±
|
++
|
-
|
-
|
Cystine
|
+
|
-
|
±
|
±
|
-
|
Glutamate
|
+
|
++
|
+
|
+
|
+
|
Histidine
|
±
|
++
|
+
|
+
|
+
|
Lysine
|
++
|
++
|
++
|
++
|
++
|
Methionine
|
-
|
-
|
±
|
-
|
-
|
Serine
|
++
|
+
|
±
|
+
|
±
|
Threonine
|
++
|
±
|
±
|
+
|
±
|
Tryptophan
|
-
|
-
|
-
|
±
|
-
|
Tyrosine
|
+
|
-
|
+
|
_+
|
+
|
C terminus
|
-
|
++
|
+
|
+
|
+
|
N terminus
|
-
|
++
|
++
|
++
|
+
|
Carbohydrate
|
- ~
++
|
++
|
+
|
+
|
±
|
Others
|
- ~
++
|
-
|
-
|
- ~
++
|
-
|
The most commonly used
method for immobilising enzymes on the research scale (i.e., using less than a
gram of enzyme) involves Sepharose, activated by cyanogen bromide. This is a
simple, mild and often successful method of wide applicability. Sepharose is a
commercially available beaded polymer which is highly hydrophilic and generally
inert to microbiological attack. Chemically it is an agarose
(poly-{b-1,3-D-galactose-a-1,4-(3,6-anhydro)-L-galactose}) gel.
The hydroxyl groups of this polysaccharide combine with cyanogen bromide to give
the reactive cyclic imido-carbonate. This reacts with primary amino groups (i.e.
mainly lysine residues) on the enzyme under mildly basic conditions (pH 9 -
11.5, Figure 3.3a). The high toxicity of cyanogen bromide has led to the
commercial, if rather expensive, production of ready-activated Sepharose and the
investigation of alternative methods, often involving chloroformates, to produce
similar intermediates (Figure 3.3b). Carbodiimides (Figure
3.3c) are very useful
bifunctional reagents as they allow the coupling of amines to carboxylic acids.
Careful control of the reaction conditions and choice of carbodiimide allow a
great degree of selectivity in this reaction. Glutaraldehyde is another
bifunctional reagent which may be used to cross-link enzymes or link them to
supports (Figure 3.3d). It is particularly useful for producing immobilised
enzyme membranes, for use in biosensors, by cross-linking the enzyme plus a
non-catalytic diluent protein within a porous sheet (e.g., lens tissue paper or
nylon net fabric). The use of trialkoxysilanes allows even such apparently inert
materials as glass to be coupled to enzymes (Figure 3.3e). There are numerous
other methods available for the covalent attachment of enzymes (e.g., the
attachment of tyrosine groups through diazo-linkages, and lysine groups through
amide formation with acyl chlorides or anhydrides).
(a) cyanogen bromide
[3.1]
(b)
ethyl chloroformate
[3.2]
(c) carbodiimide
[3.3]
(d) glutaraldehyde
[3.4]
(e) 3-aminopropyltriethoxysilane
[3.5]
Figure
3.3. Commonly used methods for the covalent immobilisation of enzymes.
(a) Activation of Sepharose by cyanogen bromide. Conditions are chosen to
minimise the formation of the inert carbamate. (b) Chloroformates may be used to
produce similar intermediates to those produced by cyanogen bromide but without
its inherent toxicity. (c) Carbodiimides may be used to attach amino groups on
the enzyme to carboxylate groups on the support or carboxylate groups on the
enzyme to amino groups on the support. Conditions are chosen to minimise the
formation of the inert substituted urea. (d) Glutaraldehyde is used to
cross-link enzymes or link them to supports. It usually consists of an
equilibrium mixture of monomer and oligomers. The product of the condensation of
enzyme and glutaraldehyde may be stabilised against dissociation by reduction
with sodium borohydride. (e) The use of trialkoxysilane to derivatise glass. The
reactive glass may be linked to enzymes by a number of methods including the use
thiophosgene, as shown.
It is clearly important that the immobilised enzyme retains
as much catalytic activity as possible after reaction. This can, in part, be
ensured by reducing the amount of enzyme bound in non-catalytic conformations (Figure
3.4). Immobilisation of the enzyme in the presence of saturating
concentrations of substrate, product or a competitive inhibitor ensures that the
active site remains unreacted during the covalent coupling and reduces the
occurrence of binding in unproductive conformations. The activity of the
immobilised enzyme is then simply restored by washing the immobilised enzyme to
remove these molecules.
Figure 3.4. The effect of covalent
coupling on the expressed activity of an immobilised enzyme. (a) Immobilised
enzyme (E) with its active site unchanged and ready to accept the substrate
molecule (S), as shown in (b). (c) Enzyme bound in a non-productive mode due to
the inaccessibility of the active site. (d) Distortion of the active site
produces an inactive immobilised enzyme. Non-productive modes are best prevented
by the use of large molecules reversibly bound in or near the active site.
Distortion can be prevented by use of molecules which can sit in the active site
during the coupling process, or by the use of a freely reversible method for the
coupling which encourages binding to the most energetically stable (i.e., native)
form of the enzyme. 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.
Entrapment
of enzymes within gels or fibres is a convenient method for use in processes
involving low molecular weight substrates and products. Amounts in excess of 1 g
of enzyme per gram of gel or fibre may be entrapped. 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. The entrapment process may be a purely physical caging or involve
covalent binding. As an example of this latter method, the enzymes' surface
lysine residues may be derivatised by reaction with acryloyl chloride (CH2=CH-CO-Cl) to give the acryloyl amides. This product may then be
copolymerised and cross-linked with acrylamide (CH2=CH-CO-NH2) and bisacrylamide
(H2N-CO-CH=CH-CH=CH-CO-NH2) to form a gel. 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.
Membrane confinement of enzymes may be
achieved by a number of quite different methods, all of which depend for their
utility on the semipermeable nature of the membrane. This must confine the
enzyme while allowing free passage for the reaction products and, in most
configurations, the substrates. The simplest of these methods is achieved by
placing the enzyme on one side of the semipermeable membrane while the reactant
and product stream is present on the other side. Hollow fibre membrane units are
available commercially with large surface areas relative to their contained
volumes (> 20 m2 L−1) and permeable only to substances of
molecular weight substantially less than the enzymes. Although costly, these are
very easy to use for a wide variety of enzymes (including regenerating coenzyme
systems, see Chapter 8) without the additional research and development costs
associated with other immobilisation methods. Enzymes, encapsulated within small
membrane-bound droplets or liposomes (see Chapter 7), may also be used within
such reactors. As an example of the former, the enzyme is dissolved in an
aqueous solution of 1,6-diaminohexane. This is then dispersed in a solution of
hexanedioic acid in the immiscible solvent, chloroform. The resultant reaction
forms a thin polymeric (Nylon-6,6) shell around the aqueous droplets which traps
the enzyme. Liposomes are concentric spheres of lipid membranes, surrounding the
soluble enzyme. They are formed by the addition of phospholipid to enzyme
solutions. The micro-capsules and liposomes are washed free of non-confined
enzyme and transferred back to aqueous solution before use.
Table 3.3 presents a
comparison of the more important general characteristics of these methods.
Table 3.3 Generalised
comparison of different enzyme immobilisation techniques.
Characteristics
|
Adsorption
|
Covalent
binding
|
Entrapment
|
Membrane confinement
|
Preparation
|
Simple
|
Difficult
|
Difficult
|
Simple
|
Cost
|
Low
|
High
|
Moderate
|
High
|
Binding force
|
Variable
|
Strong
|
Weak
|
Strong
|
Enzyme leakage
|
Yes
|
No
|
Yes
|
No
|
Applicability
|
Wide
|
Selective
|
Wide
|
Very wide
|
Running Problems
|
High
|
Low
|
High
|
High
|
Matrix effects
|
Yes
|
Yes
|
Yes
|
No
|
Large diffusional
barriers
|
No
|
No
|
Yes
|
Yes
|
Microbial
protection
|
No
|
No
|
Yes
|
Yes
|
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This page was established in 2004 and last updated by Martin
Chaplin on
24 August, 2018
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