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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5864_Библиотеки_им_академика_М_И_Перельмана.pdf
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
that the enzyme is retained, whereas the substrate and product molecules can easily
pass through. During this procedure, also known as lattice entrapment, the enzyme
(or cell) is not exposed against strong binding forces or structural distortions. During
an immobilization process, the level of deactivation might be possible. This happens
due to the fluctuations in pH or temperature or the addition of solvents. The
matrices employed for entrapment of enzymes include polyacrylamide gel, collagen,
gelatin, starch, cellulose, silicone and rubber. Enzymes can be entrapped in
numerous ways. There are different approaches to entrapping enzymes such as gel
[33]orfiber entrapping [34, 35] and microencapsulation [36, 37].
4.6.6.1 Enzyme i nclusion in gels
This is achieved by enzyme entrapment inside gels (figure 4.4). The poly(vinyl
alcohol) bearing styrylpyridinium groups (PVA-SbQ), a soluble pre-polymer bearing
photo-cross-linkable group, has been used extensively to entrap enzymes, and
numerous bioassays based on this immobilization matrix have been demonstrated
[38, 39]. The sol–gel process was established on the capability to form solid metal or
semi-metal oxides through the aqueous process of hydrolytically labile precursors.
Enzymes can also be entrapped in an agarose gel [39]. In contrast to synthetic
polymers, e.g. polyacrylamide, this matrix is biocompatible, nontoxic, provides a
natural microenvironment to the enzyme and also offers adequate accessibility to
electrons to shuttle between the enzyme and the electrode. The entrapment
approaches are easy to perform, and allow the deposition of enzymes, mediators,
and additives in the same sensing layer. Furthermore, the activity of the enzyme is
conserved during the immobilization process, as the biological element is not altered
[39]. Biosensors based on physically entrapped enzymes are frequently described by
increased operational and storage stability.
Figure 4.4. Immobilization of enzymes by entrapment. (a) Inclusion in gels. (b) Inclusion in fibers.
(c) Inclusion in microcapsules.
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4.6.6.2 Enzyme i nclusion in fibers
Entrapment involves inclusion of an enzyme in a polymer network (gel lattice) such
as an organic polymer, a silica sol–gel or a membrane device such as a hollow fiber
or a microcapsule. Entrapment requires the synthesis of the polymeric network in
the presence of the enzyme. The last step involves cross-linking of enzyme aggregates
or crystals, using a bifunctional reagent, to prepare carrier-free macroparticles [40].
The enzymes are trapped in the fibers of the matrix.
4.6.6.3 Enzyme i nclusion in microcapsules
During this procedure enzymes are trapped inside a microcapsule matrix (figure 4.4).
The hydrophobic forms of the matrix polymerize to yield a microcapsule containing
enzyme molecules [41]. The major limitation for entrapment of enzymes is the
leakage from the matrix. Most of the researchers suggested utilizing this method of
entrapment for immobilization of whole cells. Entrapped cells can be used in largescale production of amino acids such as l-isoleucine, l-aspartic acid, l-malic acid and
hydroquinone [41].
4.6.6.4 Micro-encapsulation
Micro-encapsulation is a kind of entrapment in which spherical particles in a liquid
or suspension are enclosed in a semipermeable membrane [42]. The membrane may
be polymeric, lipoidal, lipoprotein-based or non-ionic in nature. The three steps of
microencapsulation are:
• Building superior membrane reactors.
• Formation of the emulsion.
• Stabilization of the emulsion to yield microcapsules [42].
Currently microencapsulation is employed for immobilization of enzymes and
mammalian cells. For example, cultured pancreatic cells can be immobilized by
microencapsulation; immobilized hybridoma cells have also been evidenced by this
technique [43].
4.6.7 Covalent binding
Enzyme immobilization can be accomplished by formation of covalent bonds
between the chemical groups of enzymes and the chemical groups of the support
(figure 4.4). This procedure is extensively used. However, covalent binding is
frequently linked with loss of some enzyme activity. Pretreatment (to form preactivated support) of the inert support is typically required before it binds to
an enzyme. The following are the common procedures of covalent binding.
Immobilizations of proteins by procedures based on the formation of covalent
bonds are among the most extensively used. A benefit of these procedures is that,
because of the stable nature of the bonds developed between enzyme and the matrix,
the enzyme is not released into the solution upon use. However, in order to attain
high levels of bound activity, the amino acid residues vital for catalytic activity must
not be involved in the covalent linkage to the support; this may prove a challenging
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
condition to accomplish in some cases. A simple method that occasionally increases
the activity yield is to perform the coupling reacti on in the presence of substrate
analogs [44]. Covalent methods for immobilization are used when there is a strict
obligation for the absence of the enzyme in the product. An extensive range of
reactions have been established depending on the functional groups accessible on
the matrix [45 ]. Coupling procedures can generally be categorized in two main
classes:
• Activation of the matrix by addition of a reactive function to a polymer.
• Alteration of the polymer backbone to synthesize an activated group.
The activation procedures are usually intended to make electrophilic groups on the
support which, in the coupling step, react with the strong nucleophiles on the
proteins. The basic principles governing the course of covalent coupling to the
matrices are similar to those used for the chemical alteration of proteins. The most
often used reactions include the following side chains of the amino acids: lysine
(ε-amino group), cysteine (thiol group), and aspartic and glutamic acids (carboxylic
group).
• Cyanogen bromide activation: The inert support materials such as cellulose,
sepharose and sephadex enclosing glycol groups are activated by CNBr,
which at that time binds to enzymes and immobilizes them (figure 4.5).
• Diazotation: A number of the support materials (aminobenzyl cellulose,
amino derivatives of polystyrene, aminosilanized porous glass) are exposed
to diazotation on treatment with NaNO
and HCI. They, in return, bind
2
covalently to tyrosyl or histidyl groups of enzymes (figure 4.5).
• Peptide bond formation: Enzyme immobilization can also be achieved by the
development of peptide bonds between the amino (or carboxyl) groups of the
support and the carboxyl (or amino) groups of enzymes (figure 4.5). The
support material is initially chemically treated to form active functional
groups.
• Activation by bi- or polyfunctional reagents: A number of reagents, e.g.
glutaraldehyde, can be used to build bonds between the amino groups of
the enzymes and the amino groups of the support (such as aminoethylcellulose, albumin, amino alkylated porous glass). This is represented in figure 4.6.
Figure 4.5. A general representation of the immobilization of enzymes by covalent binding.
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 4.6. Immobilization of enzymes by covalent binding: (a) cyanogen bromide activation, (b) diazotation,
(c) peptide bond formation and (d) activation by bifunctional agent.
4.6.8 Cross-linking
A characteristic feature of the immobilization of enzymes by cross-linking is the lack
of a solid support. The enzyme molecules are immobilized by building cross-links
between them, and by the participation of polyfunctional reagents. In fact, these
reagents react with the enzyme and make bridges which further form the support to
hold enzyme molecules (figure 4.7). There are a number of reagents used for crosslinking. These include glutaraldehyde, diazobenzidine, hexamethylene diisocyanate
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Figure 4.7. Immobilization of enzyme molecules by cross-linking.
and toluene di-isoth iocyanate. Glutaraldehyde is the most frequently used crosslinking reagent [38]. It successfully reacts with lysyl residues of the enzymes and
finally yields a Schiff’s base. The cross-links developed between the enzyme and
glutaraldehyde are irreversible and can tolerate extreme pH and temperature.
Glutaraldehyde cross-linking has been effectively utilized to immobilize a number
of industrial enzymes such as glucose isomerase and penicillin amidase [38]. The
method of cross-linking is very simple and cost-effective, but the drawback is that it
includes the possibility of enzyme denaturation by the polyfunctional reagent.
4.7 Choice of immobilization technique
Selection of a particular procedure for immobilization of enzymes is based on a trial
and error approach to select the perfect one. The enzyme catalytic activity, stability,
regenerability and cost are the most important factors that decide a technique.
4.7.1 Immobilization of l-amino acid acylase
Japanese researchers [46] discovered the first enzyme immobilized on a matrix,
known as l-amino acid acylase. Later, a number of immobilization procedures were
attempted by these researchers, but only three showed promising results. These were:
• Covalent binding to iodoacetyl cellulose.
• Ionic binding to DEAE-sephadex.
• Entrapment with n polyacrylamide.
4.7.2 Stabilization of soluble enzymes
Enzyme stabilization is essential for any biomedical or industrial application of
enzymes. In several applications, the objective is to deliver a prolonged active
lifetime under normal environmental conditions with traditional substrates at low
concentrations in buffered solutions [47]. However, as enzymes are being employed
for more and more applications, there is a need to use them under extreme
environmental conditions (i.e., high temperatures), in high substrate concentrations
and in nontraditional solvent systems [47]. Certain enzymes cannot be immobilized
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
as they should be used in soluble form, for example, enzymes used in liquid detergents,
some diagnostic reagents and food additives. With the aid of a number of additives or
by chemical modifications these enzymes can be stabilized. The stabilized enzymes
have longer half-lives, although they cannot be recycled. Several significant procedures
for enzyme stabilization are briefly described in the following [47]:
• Solvent stabilization: Some solvents at low concentrations stabilize the
enzymes, whereas at high concentrations the enzymes become denatured,
e.g. acetone (5%) ethanol (5%) can stabilize benzyl alcohol dehydrogenase.
• Substrate stabilization: By adding substrates the active site of an enzyme can
be stabilized, e.g. starch stabilizes α-amylase; glucose stabilizes glucose
isomerase.
• Stabilization by polymers: This type of stabilization can be achieved by the
addition of polymers, such as gelatin, albumin and polyethylene, particularly
at increased temperatures.
• Stabilization by salts: Metalloenzymes stability of can be attained by
supplementing salts, e.g. Ca, Fe, Mn, Cu and Zn. Proteases are commonly
stabilized by the addition of calcium.
• Stabilization by chemical modifications: During this procedure, enzymes can
be stabilized by appropriate chemical modifications without loss of biological
activity. There are different types of chemical modifications:– Addition of
polyamino side chains, such as polytyrosine.
– Acylation of enzymes by adding groups, e.g. acetyl, propionyl and
succinyl.
• Stabilization by rebuilding: Ideally, enzyme stability is dependent on the
hydrophobic interactions in the core of the enzyme. It was thus suggested that
enzymes can be stabilized by increasing hydrophobic interactions. For this
reason, the enzyme is initially unfolded and later rebuilt in one of the
following ways (see figure 4.8):– The enzyme can be chemically treated
(e.g. using urea and a disulfide) and then refolded.
– The refolding can be done in the presence of low molecular weight
ligands.
– For certain enzymes, refolding at higher temperatures (around 50 °C)
stabilizes them.
• Stabilization by site-directed mutagenesis: Site-directed mutagenesis has been
fruitfully used to synthesize more stable and functionally more efficient
enzymes such as subtilisin E.
4.8 Immobilization of cells
A single-step reaction procedure is suitable for the immobilization of an individual
cell, but is not appropriate for multienzymes or for reactions demanding co-factors.
During this procedure whole cells or cellular organelles can be immobilized, yielding
a system which acts as a source for various enzymes, and hence is referred to as a
multi-enzyme system. Furthermore, immobilized cells rather than enzymes are
often favored even for single reactions, owing to the cost factor in isolating enzymes.
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Figure 4.8. Stabilization of an enzyme by refolding.
For those enzymes which are dependent on the spacial arrangement of the
membrane for their activity, cell immobilization is ideal. There are several significant
applications of immobilized enzymes, for example, they are traditionally used for the
treatment of sewage. The procedures used for the immobilization of cells are almost
the same as those employed for the immobilization of enzymes, with appropriate
modifications. Techniques such as entrapment and surface attachment are frequently used. Gels, and to some extent membranes, are also used.
4.8.1 Immobilization of viable cells
By means of gentle immobilization it is possible to preserve the viability of the cells.
These immobilized cells are mainly useful for fermentation. Occasionally, mammalian cell cultures are made to function as immobilized viable cells.
4.8.2 Immobilized non-viable cells
In most cases, immobilized non-viable cells are considered over enzymes or even
viable cells. This is primarily because of the expensive isolation and purification
procedures. An excellent example is the immobilization of cells producing glucose
isomerase for the large-scale production of high-fructose syrup.
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4.8.3 Drawbacks of immobilizing eukaryotic cells
Prokaryotic cells (mainly bacterial) are generally used for immobilization.
According to various reports it is also possible to immobilize eukaryotic plant and
animal cells. Owing to the presence of cellular organelles, the metabolism of
eukaryotic cells is slow. Consequently, for the large-scale production of biochemicals, prokaryotic cells are considered. However, for the synthesis of complex
proteins (e.g. immuno-globulins) and for proteins that experienced post-translational modifications, eukaryotic cells may be used.
4.8.4 The effect of immobilization on enzyme properties
Cell immobilization is often linked to modifications in enzyme properties, particularly the kinetic properties of enzymes. Some of these are as follows:
• A considerable reduction in enzyme specificity. This may be because of
conformational changes that take place when the enzyme becomes
immobilized.
• The kinetic constants (K
m
and V
) of an immobilized enzyme differ from
max
those of the innate enzyme. This is because of the conformational modifications of the enzyme which may further affect the affinity between the enzyme
and substrate.
4.8.5 Immobilized enzyme reactors
Immobilized enzyme cells are exploited for large-scale production in the form of
enzyme reactors [43]. They are generally of two types: batch reactors and continuous
reactors. The frequently used enzyme reactors are depicted in figure 4.9.
Batch reactors. In these types of reactors, the immobilized enzymes and substrates
are employed, and the reaction is allowed to start under constant stirring. As the
reaction is finished, the product is isolated from the enzyme (typically by denatura-
tion). Soluble enzymes are frequently used in batch reactors. It is somewhat
challenging to isolate the soluble enzymes from the products, therefore, there is the
drawback of their reuse. Special procedures have been developed for the recovery of
soluble enzymes, which may occasionally result in loss of enzyme activity [48].
Stirred tank reactors. The most modest form of batch reactor is the stirred tank
reactor (figure 4.9). It is composed of a reactor fixed with a stirrer that permits good
mixing, and a suitable temperature and pH control. However, in this system there
may be loss of some enzyme activity. The basket reactor is a modification of the
stirred tank reactor. In this method, the enzyme is retained over the impeller blades.
Both the stirred tank reactor and basket reactor have a well-mixed flow pattern [48].
Plug flow type reactors. These reactors are alternatives to flow pattern type
reactors. The flow rate of fluids can be regulated by a plug system. Plug flow type
reactors are available in the form of packed-bed or fluidized-bed reactors (figure 4.9).
These reactors are mainly useful when flow type reactors furnish insufficient product
formation. Moreover, plug flow reactors are also beneficial for obtaining kinetic
data on the reaction systems [48].
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Figure 4.9. Immobilized enzyme/cell reactors: (a) batch stirred tank reactor, (b) packed-bed reactor,
(c) fluidized-bed reactor and (d) continuous stirred tank reactor.
Continuous reactors. During this procedure the substrate is supplemented constantly, whereas the product is eliminated. Immobilized enzymes can also be
employed for continuous operation. Continuous reactors have certain merits over
batch reactors. These entail regulation over the product formation,
suitable operation of the system and easy automation of the whole procedure.
There are two main types of continuous reactors: the continuous stirred tank reactor
(CSTR) and plug reactor (PR) [49]. A schematic depiction of CSTR is shown in
figures 4.9 and 4.10. CSTR is suitable for good quality product formation.
Membrane reactors. A number of membranes with an array of chemical
compositions can be used. Polysulfone, polyamide and cellulose acetate are the
most often used materials for membranes. The biocatalysts (enzymes or cells) are
usually retained on the membranes of the reactor. The substrate is added into
reactor while the product passes out. Good mixing in the reactor can be attained
using a stirrer (figure 4.10). In a continuous membrane reactor, the biocatalysts are
held on membrane layers over which substrate molecules are passed (figure 4.10). In
a recycle model membrane reactor, the contents, i.e., the solution containing
enzymes, co-factors, and substrates, along with freshly released product, are recycled
by using a pump (figure 4.10). The product passes out of the system and can be
recovered [43–45].
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Figure 4.10. Membrane reactors: (a) batch membrane reactor, (b) continuous membrane reactor and
(c) recycle membrane reactor (coloured lines indicate membranes).
4.8.6 Applications of immobilized enzymes and cells
Immobilized enzymes and cells are extensively utilized for industrial, analytical and
therapeutic applications, including their utilization in food production and research
in biochemistry, microbiology and other allied sciences [50]. A summary of
industrial applications of immobilized cells is provided in table 4.2.
4.9 Manufacture of commercial products
A list of significant immobilized enzymes and their industrial applications is
mentioned in table 4.2. Further details on the production of L-amino acids and
high-fructose syrup are mentioned below.
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