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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5344_Библиотеки_им_академика_М_И_Перельмана.pdf
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
are: diazotization, Schiff’s base formation, glutaraldehyde treatment, thiodisulfide
and imido ester formation. With the help of these coupling agents and certain
procedures, covalent bonds are developed between functional groups on enzymes
and respective counter groups on polymeric support with a net result of an
immobilized enzyme system formation.
4.2.2 Adsorption
Considering the functionality at the level of operation, adsorption appears to be a
most economical and simple process, however, the forces involved are extremely
complex. So far, practically no material has been reported which could be employed
for adsorptive immobilization with its own surface contribution [1]. The nature of
the carrier and the enzyme protein surface determines the bonds that exist between
the enzyme protein and carrier, such as ionic, hydrogen, covalent, coordinated
covalent or hydrophobic, or even a combination of any of these [1]. Immobilization
can be achieved by coupling an enzyme to either the external or internal surface of a
carrier. When an enzyme is immobilized externally, it is necessary that the particle
size of the carrier must be small enough to offer a substantial surface for binding.
These particles possibly have diameters ranging between 500 Å mm and 1 mm
diameter [1]. The external surface binding employed during immobilization of the
enzyme is beneficial as it does not include conditions such as pore diffusion. The
drawbacks, however, include a comparatively small surface area for binding,
exposure of enzymes against microbial attack, physical abrasion of the enzyme
and inhibitory effects due to turbulence related to the bulk solution. Finally, the
reduced particles cause a high pressure drop (gradient) in continuous packed-bed
reactors [1]. While the internal surface participation of a porous carrier during
enzyme immobilization has been a noticeable shortcoming, many adavatages can be
gained from this approach. The main shortcoming of internal immobilization relates
to pore diffusion. Therefore, the process calls for proper optimization of pore
diameter, surface area, surface charges and other related parameters.
4.2.3 Complexation and chelation
Numerous techniques for the immobilization of enzymes have been developed. By
choosing a suitable carrier material and coupling procedure, it is typically possible to
obtain immobilized derivatives with good activity and stability [8]. However, the
majority of these comprise irreversibly bonded enzymes, which means that the carrier
cannot be regenerated and reused. This issue can be avoided by immobilization based
on ion exchange, hydrophobic interaction or thiol disulfide interexchange [8].
4.2.3.1 Metal link chelation
This method provides an immediate coupling without chemical derivatization or
activation of a support or matrix. The procedure is principally based on the
chelation properties of transition metals, specifically titanium and zirconium, which
are attractive due to their nontoxic nature [9]. A scheme based on titanium chloride–
cellulose demonstrates the process of transition metal association–chelate formation.
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Titanium metal has the feature of octahedral co-ordination with ionic molecules
which function as ligands. In the majority of cases these ligands are water molecules
or chloride ions. In the event where the chloride ion behaves as a ligand, its surplus
electron density is employed in the development of a partial-covalent bond with the
metal ion (Ti), thus the whole positive charge of titanium is minimized by a unit.
4.2.3.2 Chloroaquo complexes of titanium in HCI solution
These ligands may be substituted by other water-based ligands or other electron
donating groups. The strength of association depends on the chemical nature of the
ligand [10]. Hydroxyl ions are active ligands for transition metals; consequently they
may well have the potential to create complexes with polysaccharides, where new
ligating hydroxyl ions may be substituted for pre-existing ones if any are present [10].
Certain polysaccharides, e.g. cellulose, contain a vicinal diol group which is not
involved in glycosidic linkage and is therefore accessible for free chelation by
transition metals. The chelate is an outcome of the substitution of two ligands from
the titanium ion by polysaccharide hydroxyl groups. For steric reasons the number
of ligands which can be substituted by cellulose hydroxyl groups is limited [10].
4.2.3.3 Hydrous transition metal oxides
During this procedure hydrous metal oxides are used as the support for enzyme or
cell immobilization [10]. The oxides can be prepared by precipitation after
hydrolysis of the corresponding chloride. The method of immobilization is mainly
based on chelation. Therefore the precipitation is achieved to obtain hydrous oxide
in the presence of the enzyme, which may result in effective and efficient
immobilization. However, all safety measures needed to account for potential
detrimental conditions should be taken in to consideration [10]. The metal chlorides
which may be employed for transformation to hydrous oxides include cobalt(ii),
copper(ii), iron(ii), manganese(ii), tin(ii), zinc(ii), chromium(ii), vanadium(iii), tin(iv)
and zirconium(iv). The complexes retain a considerable portion of the activity
profile of the chelated immobilized enzyme. Due to the low operational stability of
enzymes immobilized on metal hydrous oxide, a cross-linking step based on
glutaraldehyde treatment has been recommended. This method of modification
could avert protein loss into the solution during operation.
4.2.4 Within-support immobilization
The entrapment or encapsulation of enzymes can be achieved via their inclusion in a
matrix that is based on highly cross-linked polymers, encapsulation in microcapsules
or in distinct non-aqueous phases [11]. The characteristic feature of these procedures, mainly matrix inclusion and encapsulation, is that the enzyme is not attached
to the matrix, thus any issues, such as the steric blockades related to covalent or
electrostatic binding, are not encountered. The procedure offers scope for developing biocatalysts in various physical shapes and forms. While beads are most
common, fibers, sheets, emulsions and gels have been used in bioreactors [11]. In
conclusion, immobilization by means of entrapment methods is an attractive option
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where the difference in size of the catalyst and substrate should be characteristically
large. Practically, membrane reactors are well adapted for enzymes which act on a
low molecular weight substrate, whereas three-dimensional gel is favored for
immobilizing cellular/particular biocatalysts and therefore is the scheme of choice
for immobilization of live cells [11]. Usually entrapment is achieved by dissolving an
enzyme in a solution that is required either for dissolution or for preparation of the
enzyme phase (carrier), and then treating this solution so that a distinctive and
discrete biphasic system is created in the form of dispersion.
4.2.5 Cell immobilization
Enzyme immobilization has now become a mature technology and a number of
immobilization procedures have been developed. The groundbreaking attention has
moved onto the development of immobilization of whole plant/animal cells, which
offers another route to highly specific enzyme processes aimed at multistage
processing and production of fine chemicals against first generation biocatalysts.
In the last few decades, experience and expertise in the immobilization of microbial
cells has improved such that a range of gentle procedures are now accessible to
produce fine chemicals, fabrics and membranes incorporating a high number of
viable cells. Hydrogels are being studied for mammalian cell immobilization. Their
material features can be designed or structured for biocompatibility, selective
permeability, mechanical and chemical stability, and other necessities as specified
by the application, including uniform cell distribution and a given membrane
thickness or mechanical strength [12]. These aqueous gels are a good choice for
analytical and tissue engineering applications and can be employed for immobilization in therapies for various diseases as well as to generate bioartificial organs [12].
4.2.5.1 Distinctive features of plant and animal cells
In cell immobilization, currently most research is focused on using the prokaryotic
bacterial cells as a subject [13]. The differences between prokaryotic and eukaryotic
cells are clearly distinguishable and involve size, form and function. Prokaryotes
lack of organelles, have a different cell wall composition and are typically one tenth
the size or one thousandth the volume of plant and animal cells. Eukaryotic cells
have different subcellular, membrane bound organelles such as nuclei, mitochondria, lysosomes, vacuoles and various other plastids. As a result of these major
differences the metabolic functions of eukaryotes proceed more slowly than those of
bacteria. Eukaryotic cells can express a superior amount of genetic information in a
number of ways, to produce distinct, differentiated (i.e., specific) cells which
frequently function co-operatively to develop tissues, organs or even whole
organisms. As a result, they develop complex mechanisms for both intracellular
and intercellular control and informative transfer, involving hormones, cell mediators and specific cell surface receptors. They are hence capable of more sophisticated responses than the basic survival-related reactions of bacteria. The whole
complexity of their metabolism, affecting the gene expression in individual cells,
presents problems in the maintenance and use of eukaryotes, even when successful
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immobilization has been achieved. However, these different levels of control could
clearly offer valuable opportunities for the manipulation of cellular activity.
Unfortunately, at present these mechanisms are so poorly understood at the
molecular level, that bacteria continue to be considered for any applications where
either cell type could be employed.
4.2.5.2 Products from eukaryotes
There is developing interest in the large-scale culture of plant and animal cells for the
production of high value pharmaceuticals, flavors, hormones and immunological
products, despite the difficulties associated with such cells. The complication of
organization and regulation of biosynthetic pathways resulting in secondary
products among eukaryotes is likely to be beyond the practical or economic limits,
whereas the practical and economic limits of genetic manipulation of bacteria are yet
to arise. In addition, certain operational and post-translational modifications, such
as glycosylation, are unlikely to be accomplished by incorporating gene sequences
into prokaryotes. It might also be claimed that the most satisfactory source of
material for replacement therapy in the treatment of humans will be attained from
genetically engineered human cell lines, and that the best way of synthesizing plant
and animal cell products is by direct genetic manipulation of these cells and their use
in an immobilized form.
4.2.5.3 Limitations of immobilization of eukaryotic cells
In contrast to enzymes, immobilized cells are less reactive to high substrate
concentrations, surfactants, solvents and rapid changes in physical factors.
Therefore, the process intensity and flexibility of response are likely to be lower.
The substrate is the major nutrient of the cell and in a single-pass reactor, cells at the
end of the process will either be exposed to nutrient starvation, or lower conversion
must be accepted. The sensitivity of higher cells against environmental factors, e.g.
osmotic shock, oxygen, shear, gas balance, pH, temperature and toxic chemicals, is a
clear drawback to the procedures which might be employed for immobilization.
Nevertheless, these issues are to some extent offset by the capacity of cells to
regenerate after immobilization, if maintained under suitable conditions. The
hormonal reactions of eukaryotes allow additional levels of control and flexibility
compared to microorganisms, which often only respond to nutrient levels or gross
environmental changes. To achieve the maximum merits of immobilization, cells
must be reused and thus must be retained in a healthy condition. The rich nutrient
media in which both plant and animal cells must be maintained are highly
susceptible to microbial contamination and so all operations must be carried out
in an aseptic environment. This further restricts immobilization methods and adds to
the cost and complexity of the techniques. Moreover, stored medium can deteriorate
and may become increasingly unreliable when continuous operation of a process is
considered. It can be seen that some of these limitations are in fact related to the cells
rather than the immobilization process. If synthesis by eukaryotic cells is selected, as
discussed above, the advantages ensuing from immobilization can be very high, as
long as satisfactory process control is available. However, it is obvious that the
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complexity involved in using cultured plant and animal cells will in general limit
their application in the production of high value compounds.
4.2.6 Commercial production of enzymes
Enzyme production still requires attention in the modern biotechnology industry.
The arrival of genetic engineering has allowed the industrial scale production of
enzymes and other proteins which are synthesized naturally in only minute
quantities [14]. This progress is mainly significant with regard to the synthesis of
enzymes and other proteins of therapeutic importance, which are now accessible in
clinically useful quantities [14]. The level of downstream processing to which any
enzyme is exposed depends on its future application. The production of enzymes in
bulk usually requires little downstream processing, and therefore these are comparatively crude preparations. Enzymes intended for therapeutic applications are
subject to a far higher grade of downstream processing, frequently introducing three
to four chromatographic steps. Although enzymology is one of the oldest recognized
branches of the biochemical sciences, it continues to be an area of constant, active
research. The repeated detection of new enzymes, and better knowledge of
previously discovered enzymes and their functional implications, has suggested
several novel applications for these catalytic activities [14].
4.3 Genetic engineering for microbial enzyme production
Enzymes are the large biomolecules that are obligatory for the many chemical
interactions that sustain life. Enzymes produced from genetically modified microorganisms play an important role in food technology [15]. Enzymes accelerate all the
metabolic developments in the body and each carries out a specific task. They are
highly efficient, and can trigger reaction rates 100 million to 10 billion times quicker
than any normal chemical reaction. Owing to developments in recombinant
technology and protein engineering, enzymes have developed as vital molecules
that have been extensively employed for different industrial and therapeutical
purposes. Microbial enzymes are currently gaining much attention with fast
expansion of enzyme technology [15]. Microbial enzymes are poplular due to their
economic feasibility, high yields, consistency, ease of product modification and
optimization, regular supply due to absence of seasonal fluctuations, rapid growth of
microbes on inexpensive media, stability and greater catalytic activity [15].
Microbial enzymes play a significant role in the diagnosis, treatment, biochemical
investigation and monitoring of various dreaded diseases. Amylase and lipase are
two key enzymes that have been closely studied and have great significance in
different manufacturing and therapeutic industries. The expression of a gene always
results in the synthesis of a specific protein in the form of an enzyme, hormone, etc,
for a specific function. We can say that the enzymes are functional proteins that are
synthesized after the expression of specific sequences present in the gene that encode
for that particular protein (enzyme) [15]. Currently genetic engineers are paying
more attention to the production of these therapeutic proteins using rDNA
technology. Due to their many advantages microbes are considered as an excellent
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source for the production of these therapeutic proteins. Recent developments in
rDNA technology have allowed the efficient microbial production of commercial
enzymes [15]. After the identification of an enzyme, especially those are with
potential use in industry, multiple copies can be produced by cloning a
suitable gene and introducing it into a suitable host.
4.3.1 Cloning methods
There are several cloning methods for the commercial production of enzymes, as
mentioned in figure 4.2. cDNA templates are considered the best for the production
of enzymes. cDNA, which can be synthesized from mRNA to establish a cDNA
library, is required for the development of a suitable probe for a particular enzyme.
The specific cDNA clones required can be identified on hybridization with
oligonucleotide probes. This desired gene should be inserted into a suitable host
such as Aspergillus oryzae for the expression of a desirable enzyme. This approach
can be utilized to produce high quality industrial enzymes with better yield. There
have been several reports based on enzyme synthesis using cloning methods:
• The fungal enzyme lipolase, present in Humicola languinosa, was recently
shown to eliminate fat stains in fabrics [16]. However, commercial production
of this fungal enzyme from this organism is not possible as it synthesizes in
very low amounts. Thus the gene that encodes for this lipolase was initially
Figure 4.2. Cloning for the industrial production of enzymes.
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identified and then separated, cloned and fi nally incorporated into A. oryzae
to produce multiple copies [16]. By following this approach, commercial
production of this enzyme can be positively attained. One of the most
appreciated features of lipolase is its stability and resistance against degradation by the proteases that are usually employed in the detergent industry.
These features make lipolase a good candidate for applications in the fabric
and detergent industry.
• Rennet (major component is chymosin) is a milk clotting enzyme that can be
isolated from different sources, in particular the stomach of a young calf.
Generally, rennet is considered as combination of various enzymes, in
particular chymosin, which is added to raw milk in the cheese industry to
produce cheese. Usually this process involves curd cutting and fractional
exclusion of the whey. Afterwards the prepared cubes are allowed to ripen in
the whey until they change into slim blocks. Chymosin is extensively utilized
commercially, in particular in cheese manufacturing [3]. Its commercial
preparation also includes other proteases; bovine pepsin is usually present
in varying concentrations. To meet the market requirements commercial
production should be high. Unfortunately, using earlier or existing
approaches it is not possible to produce the high amounts of rennet for the
market demand. Therefore, using a cloning method, in particular by exploring the genes responsible for the production of chymosin, can be considered
as a potential approach for its industrial production.
4.4 Protein studies for modification of commercial enzymes
By protein engineering and site-directed mutagenesis, it is now possible to modify
the structure of a protein/enzyme. In enzyme research, most of the modifications in
the enzymes are carried out to enhance enzyme stability, which can further improve
its catalytic function. Modification also allows the development of degradation and
oxidation resistant enzymes. Moreover, structural modification of enzymes can
change their substrate preference and improve tolerance against alkali and organic
solvents [15]. There are number of approaches available to achieve the aforementioned objective, one of the most common is site-directed mutagenesis. Enzymes can
be redesigned by using site-directed mutagenesis. This requires the systematic
alteration of protein structure by rDNA technology and chemical synthesis of
DNA fragments to allow the superficial modification of proteins by site specific
mutagenesis of their genes. Thus mutated genes can be utilized to produce enzymes
with improve yield and novel properties. Kinetic investigation of mutant enzymes
with high-resolution structural data from protein x-ray crystallography can be
utilized to draw the relationships between structure and function [17]. In particular,
the strength and nature of enzyme–substrate interactions and their roles in catalysis
and specificity may be examined [17].
During mutagenesis a number of amino acids present in the target enzyme at a
particular site are allowed to modify to produce an enzyme with desirable features,
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e.g. structural modification of phospholipase A2 by using fundamental knowledge of
protein engineering to resist elevated concentrations of acid. This modification allows
its efficient utilization as a food emulsifier. Commercial preparation of phospholipase
A2 is mainly produced from the pancreas for the further production of lysolecithin.
This allows its extensive utilization as an excellent emulsifier for the food, cosmetics
and pharmaceutical industries. Most eukaryotic proteins are N-terminally altered by
one or more processing enzymes. Enzymes acting on the very first amino acid of a
polypeptide include different peptidases, transferases and ligases [18].
The tyrosyl–tRNA synthetase from Bacillus stearothermophilus is being scientifically examined by site-directed mutagenesis. A fine-structure inspection is revealing
the subtle roles of hydrogen bonding in catalysis and specificity [17]. Alteration of
the residues that hydrogen-bond with ATP and tyrosine display how the energetics
must be examined in terms of an exchange reaction with solvent water. Based on this
knowledge, and structural data, an enzyme of vastly improved enzyme–substrate
affinity has been engineered [17].
4.5 Enzyme and cell immobilization
Usually, enzymes in free solutions (i.e., in soluble or free form) react with substrates
to yield final products. This type of enzyme utilization is inefficient, mainly for largescale production, as enzymes are not stable and they cannot be recovered for reuse.
Enzymes or cells immobilization emerged as a technique of confining/anchoring
the enzymes or cells in or on an (inert) support for their stability and functional
reuse. By means of this technique, the enzymes produced are more effective and
economical for large-scale use. A number of researchers referred to immobilization
as ‘the goose that lays the golden egg’ in enzyme technology. Immobilized enzymes
retain their structural conformation necessary for catalysis. There are various
benefits to immobilized enzymes:
• They are stable and more efficient in function.
• They can be reused repeatedly.
• Their products are enzyme-free.
• They are ideal for multi-enzyme reaction systems.
• The control of enzyme function is easy.
• They are appropriate for large-scale and therapeutic use.
• They reduce effluent disposal problems. There are, however, certain draw-
backs related to immobilization:
• There is a possibility of loss of biological activity of an enzyme
• Immobilization is a costly process that frequently requires sophisticated
equipment.
Immobilized enzymes are usually chosen over immobilized cells owing to their
ability to produce the products in a pure form. However, there are numerous benefits
to using immobilized multi-enzyme systems, e.g. organelles and whole cells, over
immobilized enzymes.
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4.6 Immobilization methods
The frequently used procedures for immobilization of enzymes are adsorption,
entrapment, covalent binding and cross-linking.
4.6.1 Adsorption methods
Adsorption includes the physical binding of enzymes (or cells) on the surface of an
inert support. The support materials may be inorganic (e.g. alumina, silica gel,
calcium phosphate glass) or organic (starch, carboxymethyl cellulose, DEAEcellulose, DEAE-sephadex). Adsorption of enzyme molecules on the inert support
involves weak forces, e.g. van der Waals forces and hydrogen bonds (figure 4.3).
Consequently, the adsorbed enzymes can be removed by minor changes in pH, ionic
strength or temperature. This is a shortcoming for the large-scale utilization of
enzymes.
4.6.2 Nonspecific adsorption
The immobilization procedure of nonspecific adsorption is generally based on
physical adsorption or ionic binding [19, 20]. During physical adsorption the
enzymes are attached to the matrix via hydrogen bonding, van der Waals forces,
or hydrophobic interactions; while in ionic bonding the enzymes are bound via salt
linkages. The types of forces present in noncovalent immobilization can be reversed
by varying the conditions that affect the strength of the interaction (e.g. pH, ionic
strength, temperature, or polarity of the solvent). Immobilization by adsorption is a
gentle, easy to perform process, and generally conserves the catalytic activity of the
enzyme. Such procedures are consequently economically attractive, but may suffer
Figure 4.3. Immobilization of enzymes by absorption: (a) by van der Waals forces and (b) by hydrogen
bonding.
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from limitations, e.g. enzymes escape from matrix when the interactions are
comparatively weak.
4.6.3 Ionic binding
One method for the reversible immobilization of enzymes is to use protein–ligand
interactions based on the principles employed in chromatography. For instance, one
of the first claims of chromatographic principles in the reversible immobilization of
enzymes was the use of ion-exchangers [21, 22]. The procedure is simple and
reversible, but it is generally challenging to discover conditions under which the
enzyme remains both firmly bound and fully active. Currently, the use of immobilized polymeric-ionic ligands has permitted the modification of protein–matrix
interactions and has thus enhanced the properties of the derivative. Some patents
have been published on the use of polyethyleneimine to bind a rich variety of
enzymes and whole cells [22]. However, issues may rise from the use of an extremely
charged support when the substrates or products themselves are charged; the kinetics
can be distorted resulting in partition or diffusion phenomena. Consequently,
enzyme features, e.g. pH optimum or pH stability, may change [23, 24]. Although
this could be a problem it could also be valuable to change the optimal conditions of
a certain enzyme against more alkaline or acidic conditions, depending on the
application [25].
4.6.4 Hydrophobic adsorption
Another approach is the use of hydrophobic interactions. During this procedure, it is
not the development of chemical bonds but rather an entropically driven interaction
that occurs. Hydrophobic adsorption has been employed as a chromatographic
principle for more than three decades. It relies on well-known experimental variables
such as pH, salt concentration and temperature [26]. The power of interaction relies
on both the hydrophobicity of the adsorbent and the protein. The hydrophobicity of
the adsorbent can be controlled by the degree of substitution of the support and by
the size of the hydrophobic ligand molecule. The effective reversible immobilization
of β-amylase and amyloglucosidase to hexyl-agarose carriers has been demonstrated
[27, 28]. Several other examples of strong reversible binding to hydrophobic
adsorbents have also been demonstrated [29, 30].
4.6.5 Affinity binding
The basis of affinity between complementary biomolecules has been applied to
enzyme immobilization. The outstanding selectivity of the interaction is a key
benefit of the procedure. However, the method often needs the covalent binding of a
costly affinity ligand (e.g. antibody, or lectin) to the matrix [31, 32].
4.6.6 Entrapment method
By means of physical entrapment enzymes can be easily immobilized inside a
polymer or a gel matrix. The size of the matrix pores is maintained in such a manner
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