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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
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IOP Publishing
Introduction to Pharmaceutical Biotechnology, Volume 2
(Second Edition)
Enzymes, proteins and bioinformatics
Ahmed Al-Harrasi, Saurabh Bhatia and Ajmal Khan
Chapter 4
Immobilization of enzymes
4.1 Introduction
In modern science, biologically active natural compounds, mainly enzymes, have
gained significant attention in medicine and various biotechnological processes where
they are being employed commercially. At present a number of ailments related to
lysosomal enzyme activities have been recognized. These diseases could ideally be
cured by supplementation or replacement of defective or deficient enzyme.
Additionally, enzymes with very specific activity are usually involved in the catalysis
of several reactions. Enzymes have various significant clinical applications as
bioreactors, biosensors and in other enzyme-based reactions, however, their use
is restricted for a number of reasons, such as high cost, low availability and quick
inactivation under physiological conditions followed by fast clearance, therefore there
is the requirement for a greater amount of enzyme during the course of treatment.
Moreover, possible antigenicity, inactivation by different endogenous natural inhibitors and, lastly, the challenge to achieving higher levels for therapeutic activity, are
limitations on the practical clinical use of enzymes. Regardless of these limitations,
enzymes have a vast range of possible applications covering therapeutic, analytical and
industrial aims. The application potential of an enzyme is established by the distinctive
enzyme properties of precise specificity for the substrate and reaction catalyzed. In
order to be practically suitable, an enzyme must have excellent catalytic behavior, with
specificity, stability and reusability. According to research, the majority of enzymes
involved in particular reactions become comparatively less active during the last phase
because of the accumulation and detrimental effect of the reaction product.
Immobilized enzymes are currently the subject of significant interest because of their
benefits over soluble enzymes. In addition to their use in industrial processes,
immobilization methods are the basis for making a number of biotechnology products
with applications in diagnostics, bioaffinity chromatography and biosensors [1].
doi:10.1088/978-0-7503-5387-8ch4 4-1 ª IOP Publishing Ltd 2024. All rights,
including for text and data mining (TDM), artificial intelligence (AI) training, and similar technologies, are reserved.
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Earlier, only single immobilized enzymes were employed, however, since the 1970s
more complex systems including two-enzyme reactions with co-factor regeneration
and living cells were explored. The enzymes can be attached to the support by
interactions ranging from reversible physical adsorption and ionic linkages, to
stable covalent bonds. Although the choice of the most suitable immobilization
method depends on the nature of the enzyme and the carrier, in the last few years
immobilization technology has gradually become a matter of rational design [1]. As a
result of enzyme immobilization, some important features, such as catalytic activity or
thermal stability, are changed. These properties have been demonstrated and exploited
[1]. The concept of stabilization has been an important driving force for immobilizing
enzymes. Furthermore, true stabilization at the molecular level has been established,
e.g. proteins immobilized through multipoint covalent binding [1].
Enzymes exist as bioproteins and thus are environmentally sensitive to different
pH and temperature. Therefore, stability remains a challenge for the effective cure of
problems associated with enzymes and enzyme technology. The major focus of
immobilization is characteristically the economic application of enzyme systems.
Additional merits of the immobilized enzyme system are ease of control and
uniformity of conversion. The solution to qualifying the economic concerns is the
evaluation of the cost of the constituents of the immobilized system against the value
of its performance. During immobilization, the enzyme utilizes the carrier phase for
stealth, and safe homing. The immobilization matrix or support facilitates exchange,
but remains separate from the bulk phase in which substrate, effect or inhibitor
molecules are present and isolated. This entails limiting soluble protein catalysts or the
individual buoyancy of cells in reactor systems, which can require constant changes of
fresh solution. Usually the immobilized system is placed in an appropriate column
through which the charge is allowed to pass constantly. The definition may be
expanded to cover intact cells and biocatalysts under the category of guest. The
process of immobilization, i.e., the restrictive localization of enzymes, may in the
immediate future have great potential in the development of biosensors, bioelectronic
sensors in fermentation technology and enzyme therapeutics [2]. The carrier phase,
also known as the enzyme phase, is classically water insoluble but can be combined
with as hydrophilic porous polymeric matrix, e.g. cellulose, agarose and acrylamide. In
other words, immobilization is a general term that designates the retention of a
biologically active catalyst within a reactor or analytical system with the help of a
suitable carrier support. A unique procedure is planned by using immobilization
techniques through which fluid can pass without any difficulty, converting the
substrate into the product under controlled enzymatic reaction, and all together
allowing the easy removal of the catalyst from the product as it leaves the reactor. The
immobilization, also referred to as forced homing, of enzymes is achieved by covalent
coupling using adsorption or physical entrapment of enzymes within the enzyme phase
(polymeric matrix). The activity of immobilized enzymes is usually dependent on the
type of matrix and enzyme phase. One of the most common immobilization matrices
which utilizes anchoring on the surface of an insoluble polymeric matrix is cellulose or
polyacrylamide via covalent bond formation. The enzyme phase is available in the
form of fine particulate, membranous or monolithic spheres. The enzyme in turn may
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
be bound to another enzyme via cross-linking. This may render the overall composite
as an insoluble, but active, polymeric enzyme system. Once a support or carrier is
employed for the purpose of enzyme immobilization, the stability of that carrier during
its use is equally important. A carrier (membrane or matrix) that is not stable at a
suitable pH, ionic strength or under the solvent conditions of the process, may be
disrupted or dissolved releasing the enzyme component. This obviously suggests that it
is not essential for a carrier to be insoluble and durable at all pHs, ionic strengths or
solvent conditions. The different procedures most frequently employed for enzyme
immobilization using solid supports can be categorized into two main classes on the
basis of the approaches used to restrain biocatalysts: those with catalysts within the
support and those which hold the biocatalyst on the surface of the support. Each class
is further divided on the basis of their usage of covalent and noncovalent coupling
procedures for surface immobilization, with cross-linking entrapment and encapsulation techniques for immobilization in or within a support. The different types of
supports used in immobilization are shown in figure 4.1 and table 4.1.
4.2 Types of immobilization
On-surface immobilization involves [4]:
• Covalent coupling with polymers employing their functional group for
anchoring, moreover, the groups must be non-functional or non-essential
for biological activity [5].
• Adsorption on an inert support or ion-exchange resins.
• Complexation and chelation.
In the procedure of within-support immobilization, the enzyme is immobilized by
cross-linking by multifunctional reagents, followed by entrapment within a structure
of known/defined geometry. Entrapment can be achieved by:
• encapsulation in microcapsules, hollow fibers, liposomes, etc.
• occlusion within a cross-linked gel.
Immobilization frequently causes obvious changes in the apparent parameters of the
enzyme related to the reaction it catalyzes. These parameters include the reaction
rate, effect of the inhibitor (which may be lost completely in the case of an
immobilized enzyme), Michaelis–Menten kinetics, and the optimum temperature
and pH corresponding to maximum activity. The degree of variation among these
parameters may not only be related to the technique of immobilization selected, but
also to some extent to the enzyme reaction. Iso et al have demonstrated the
microencapsulation of lipase (Pseudomonas fluorescens), carried out using a
(W/O)/W two-phase emulsion technique [6].
4.2.1 Surface immobilization by covalent coupling
Immobilization of enzymes by covalent bonding to an insoluble polymer has the
supposed benefit of irreversible binding of the enzyme to the support matrix. Many
of the bonds usually employed are, however, not 100% stable in use. If insufficient
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 4.1. Schematic or immobilization techniques.
care is taken to confirm that the enzyme can survive the reaction conditions,
considerable loss of enzyme activity will occur. Even under promising conditions,
60% loss of activity is not uncommon [7]. The covalent bond between a biocatalyst
and a support matrix results in a complex which is significantly stable throughout
the functional use of the enzyme. However, the process subjects the delicate threedimensional structure of the biopolymer to strong disruptive physical and chemical
forces or stresses. Thus, where the superior form and stability of an immobilized
enzyme are more important than the irreversible loss of activity that is certainly
associated with immobilization procesess, such enzymes are preferred over their
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 4.1. Different types of supports used in immobilization [3].
Classification of supports
Organic
Natural polymers
• Polysaccharides: cellulose, dextrans, agar, agarose, chitin, alginate
• Proteins: collagen, albumin
• Carbon synthetic polymers
• Polystyrene
• Other polymers: polyacrylate polymethacrylates, polyacrylamide, polyamides, vinyl and allyl-
polymers
Inorganic
Natural minerals Bentonite Silica processed materials: glass (nonporous and controlled pore)
Metals Controlled pore metal oxides
soluble form. The functional groups fo und on enzyme proteins, by which a covalent
bond with polymeric or other supports could be established, should be non-essential
for enzymatic action. Moreover, the reactions which include comparatively mild
conditions and principally utilize an aqueous medium are favored. This additionally
requires broad information on the amino acids and their active groups involved in
polymeric covalent bonding, the prospect of chemical amelioration or specific
chemical modification on activity, the protection of active group(s) and the threedimensional structure of the enzyme under consideration. The protein functional
groups which could be employed in covalent coupling include:
• COOH: β and α of aspartic acid and glutamic acid, respectively.
• NH
lysine: N-terminal of polypeptide.
2
• OH: Phenol ring on tyrosine.
• SH: Group on cysteine.
The polymeric supports which have been considered as supports for covalent
coupling based immobilization include [1]:
• Aldehyde and acetal groups of polymers.
• Amide group of polypeptides and polyamides (nylon).
• Amino and related groups of amino-ethyl coated polysaccharides and silica
gel, poly(d-aminoglycine), etc.
• Carboxylic acid and related groups of poly(glutamic acid), maleic anhydride
co-polymers, poly(acrylamide) and carboxymethyl cellulose.
• Hydroxyl groups of polysaccharides, PVA, polymethyl methacrylate
(PMMA) and inorganic glasses.
The covalent coupling procedures usually employed for conjugation of enzymes with
a solid/matrix support are cyanobromide, cyclic trans 2,3-carbonates and acid
anhydrides. The approaches employed for covalent anchoring via support activation
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