Recent Patents on Engineering 2008, 2, 195-200 195

Enzyme Immobilization in Biotechnology

Cynthia Spahn and Shelley D. Minteer*
Department of Chemistry, Saint Louis University, 3501 Laclede Ave., St. Louis, MO 63103, USA

Received: July 30, 2008; Accepted: August 27, 2008; Revised: September 2, 2008
Abstract: Enzymes are proteins that catalyze chemical reactions. Unlike more traditional organic and inorganic catalysts,
enzymes are large and fragile molecules, so over the years, scientists and engineers have found it more difficult to
immobilize enzyme catalysts on easily separateable supports for use and re-use in a variety of technologies. Over the last
decade, enzyme immobilization has become more important in industry, medicine, and biotechnology. This review will
detail recent patents for techniques for enzyme immobilization, along with patents for chemical and biotechnological
processes that can employ immobilized enzymes, which allow for the re-use of the enzymatic catalysts. These techniques
include methods varying from physical adsorption and covalent attachment to entrapment in polymers and sol-gels. These
techniques have shown value in the development of biosensors, bioprocessing for the chemical industry and the
pharmaceutical industry, and bioremediation.
Keywords: Enzyme immobilization, entrapment, physical adsorption, crosslinking, covalent binding, biosensors,
biotechnology, biocatalysis.

INTRODUCTION The entrapment method involves entrapping enzyme in

Many technologies have been affected or could be
affected in the near future by the immobilization of enzymes.
The ability of enzymes to catalyze reactions has made them
indispensable to science for decades [1]. The immobilization
of enzymes has proven particularly valuable, because it has
allowed enzymes to be easily reused multiple times for the
same reaction with longer half-lives and less degradation and
has provided a straightforward method of controlling
reaction rate as well as reaction start and stop time. It has
also helped to prevent the contamination of the substrate
with enzyme/protein or other compounds, which decreases
purification costs. These benefits of immobilized enzymes
have made them highly applicable to a range of evolving
biotechnologies [2].
There are a variety of methods used to immobilize
enzymes. Three of the most common being adsorption,
entrapment, and cross-linking or covalently binding to a
support, as is shown in Fig. (1). Regardless of the method of
immobilization, the material in which the enzyme is
immobilized in must be insoluble in the solvent, which is
frequently water. The adsorption method involves the
enzyme being physically adsorbed onto the backbone or
support material, often a polymer matrix (i.e. polymer beads
or membranes). This technique is relatively simple, because
it typically either involves bathing the support in a solution
of the enzyme and incubating to allow time for the physical
adsorption to the surface to occur or allowing a solution of
the enzyme to dry on the electrode surfaces and then rinsing
away enzyme that is not adsorbed [3,4]. Unfortunately this
method is troublesome, because it allows leaching of the
enzyme while reacting, thereby, contaminating the substrate
[5, 6]. Physical adsorption can also denature the enzyme
depending on the surface chemistry of the support material.
*Address correspondence to this author at the Department of Chemistry,
Saint Louis University, 3501 Laclede Ave., St. Louis, MO 63103, USA;
Tel: 314-977-3624; E-mail: minteers@slu.edu
either the lattice structure of a material or in polymer
membranes [7-9]. This usually minimizes enzyme leaching
and improves stabilization, but frequently results in transport
limitations of substrate/analyte to the enzyme active site.
However, this technique allows for the ability to tailor the
encapsulating material to provide the optimal microenviron-
ment for the enzyme (i.e. matching the physico-chemical
environment of the enzyme and immobilization material).
This ideal microenvironment could be optimal pH, polarity,
or amphilicity. This can be done with a variety of materials
including: polymers, sol-gels, polymer/sol-gel composites,
and other inorganic materials [10-13]. Finally, enzymes may
also be immobilized through the cross-linking of proteins to
an insoluble support to prevent the loss of enzyme into the
substrate solution [8, 14-16]. The other common technique
for binding to an insoluble support is covalently binding the
enzyme to a functionalized support (i.e. Eupergit C) [17-19].
However, crosslinking or covalently binding the enzyme to
the support material surface typically decreases the degree of
movement of the enzyme, which can dramatically decrease
the enzyme activity.
The immobilization of enzymes has been shown to
improve the stability and lengthen the half-life of the
enzyme, effectively allowing enzymes to work in a larger
range of environments and possibly in connection with a
larger range of enzymes [14, 20]. The immobilization of
enzymes effectively changes the environment the enzyme is
exposed to and therefore may allow it to remain active at
different temperatures or pHs than would be predicted for
the enzyme when not immobilized [21-23].
Traditionally, enzymes have been utilized in reactions
run in either a batch reactor or a variation of a continuous
flow reactor. Batch reactors are applicable to smaller scale
production due to the fact that they are more labor and time
intensive than a continuous flow reactor but are beneficial to
use with expensive enzymes due to the fact that they easily
contain small amounts and are not wasteful. The use of a

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196 Recent Patents on Engineering 2008, Vol. 2, No. 3
Fig. (1). Schematics of the three most common enzyme immobilization techniques: (A) physical adsorption, (B) entrapment, and (c) covalent
attachment/crosslinking.
continuous flow reactor is beneficial in that it may help
increase the reaction time as well as the efficiency of a
reaction using a biocatalyst [24, 25]. Many variations and
combinations of batch and continuous flow reactors are
possible depending on the application [26, 27].
Current advancements in biotechnology have promoted
the usage of immobilized enzymes for a wide range of
applications. This increase in the number of applications of
immobilized enzymes has allowed for an even wider range
of research relating to the field of enzyme immobilization.
Innovative research has recently been completed relating to
Spahn and Minteer
the invention of improved immobilization techniques, new
methods for the production or breakdown of desired
compounds using biocatalytic reactions, methods of drug
delivery and tumor identification utilizing immobilized
enzymes, the use of immobilized enzymes in biosensors, as
well as improved reactors used to efficiently and cost
effectively carry out reactions catalyzed by these enzymes.
Basic methods of immobilization have been previously
discussed, but variations in the substrate materials that the
enzymes are immobilized on, the methods for physically
causing the binding or adsorption to occur, as well as the
Enzyme Immobilization in Biotechnology
combination of several techniques to optimize immobili-
zation for specific applications provide many outlets for
research and innovation [20, 28-31].
A traditional application of immobilized enzymes has
been for the synthesis of chemicals. Enzymes effectively and
selectively catalyze reactions; therefore, they have the ability
to synthesize one compound from another. The industrial
applications of immobilized enzymes are straightforward;
they provide a reusable method for the production of
chemicals through biocatalyzed reactions. Along with
producing desired compounds, immobilized enzymes also
have the ability to breakdown harmful or unwanted com-
pounds providing an additional field of application in
purification and remediation.
Medicine has also benefited from enzyme immo-
bilization. Immobilized enzymes have proven valuable for
many applications in the field including drug delivery
systems and tumor identification, as well as in sensors for
the management of weight and diabetes [14, 32, 33]. Analyte
sensors are an extremely popular application of enzyme
immobilization at the current time. Immobilized enzymes are
used as biocatalysts to convert previously electrochemically
unreactive compounds into compounds which undergo redox
reactions producing a detectable current [14, 34, 35]. The
most popular biosensors include those to help monitor
glucose levels for diabetics [36].
Enantiometric molecules, or molecules that rotate the
plane of polarized light, are currently a source of frustration
in industries such as pharmaceuticals [37]. Compounds
produced in nature are typically produced as optically pure,
or all rotating polarized light in the same direction. However,
lab synthesized chemicals typically form racemic mixtures
where enantiomers rotate the plane of light in opposite
directions causing problems in application. Reactions using
immobilized enzymes to convert previously racemic mix-
tures into optically pure mixture have become increasingly
popular [37-39].
Finally, research into new designs for bioreactors is
currently popular as well. While immobilized enzymes have
the ability to catalyze reactions repetitively, the methods
through which the reactant is supplied to the biocatalyst as
well as the method through which the product is collected is
important to the successful application of enzymatic
biocatalysts. Developments of bioreactors designed for
specific applications may help increase the efficiency of
usage by placing the enzyme under its own ideal conditions
and simplifying methods of combining multi-enzyme
reactions [24-27, 40, 41].
RELATED PATENTS
In the area of immobilization techniques, several patents
have recently been published pertaining to the immo-
bilization of enzymatic biocatalysts using different
procedures and materials. Two United States patents both
titled, “Irreversible Immobilization of Enzymes into Poly-
urethane Coatings”, introduce a method of immobilizing
enzymes in polyurethane through the synthesis of poly-
urethane in a solution containing the desired enzyme [20,
28]. It was found that this entrapment method of
immobilization has maintained exceptional levels of enzyme
Recent Patents on Engineering 2008, Vol. 2, No. 3 197
activity after immobilization. Furthermore, United States
patent, “Epoxide Polymer Surface”, also introduces a
method of immobilizing biocatalysts. This time onto
materials such as microscope slides by attaching bio-
polymers at appropriate epoxide groups while blocking
binding at other epoxide groups in order to maintain enzyme
function after immobilization [29]. Additionally, US patent,
“Kit for Immobilizing Organic Substance, Organic Subs-
tance-Immobilized Structure, and Manufacturing Methods
Therefor”, introduces a new immobilization technique in
which a biological substance is immobilized on the surface
of a substrate containing aluminum oxide through the
binding of a peptide to the aluminum oxide. This new
immobilization technique maintains initial free enzyme
activity even after immobilization by identifying the portion
of the enzyme that must be available to bind to the substrate
and using a different portion for immobilization, so that the
binding does not occur through the active site, thereby,
limiting access to the active site for catalysis [31]. Lastly, a
United States patent, “Process and Apparatus for Activating
Biological Material,” describes a method of effectively
immobilizing enzymes into a mesoporous material, recom-
mended to be mesoporous silica. Following the immo-
bilization of the enzyme, the material is heated to the
enzymes ideal operating temperature or just above the
temperature, usually somewhere between 50 and 90 Celsius,
resulting in enzyme activity higher than that obtained from
non-immobilized enzymes. However, the immobilization
process does not protect the enzyme from denaturing at tem-
peratures too far above the optimum range [42]. Increasing
the operating temperature of enzymes via immobilization is
important both in the area of chemical production, but also in
the pharmaceutical industry. “Immobilization of mutated
pencillin G acylase enzymes on disulfide/epoxide or glyoxyl
supports, improved stability, and applications” is a patent
that detail the use of penicillin G acylase mutants and
immobilization via covalent attachment to a support as a
method for increasing the stability of pharmaceutically
relevant enzymes to heat and organic solvents [43]. The
enzymes were 300,000 times more stable than the enzyme in
solution [43], which will not only allow for the re-use of the
enzyme, but also allow for the use of the enzyme in more
traditional chemical environment (organic solvents and
higher temperatures).
Chemical Production
With regards to the use of enzymes in the production of
chemicals, the recent Russian patent, “Photobiocatalyst for
Generation of Hydrogen, Method of Preparation Therof, and
Photochemical Hydrogen Generation Process ”, presents a
method for the production of hydrogen, catalyzed by the
enzyme hydrogenase immobilized in mesoporous titanium
dioxide films [44]. This is of particular interest, because of
the relative instability of hydrogenases and the large
application and need of hydrogen production for the
hydrogen economy. A United States patent, “Method for
Preparing Para-hydroxystyrene by Biocatalytic Decar-
boxylation of Parahydroxycinnamic Acid in a Biphasic
Reaction Medium”, introduces a high yield method of
producing para-hydroxystyrene, a material commonly used
in the production of resins, automotive coatings, adhesives,
as well as several other industrial applications, from
198 Recent Patents on Engineering 2008, Vol. 2, No. 3 Spahn and Minteer

parahydroxycinnamic acid. The procedure utilizes an matize N-protected amino acids, and “Enzymatic Resolution
immobilized enzyme to catalyze the decarboxylation of of Propylene Glycol Alkyl (or Aryl) Ethers and Ether
parahydroxycinnamic acid. The procedure also provided a Acetates,” introduces a method of optically purifying glycol
method for the easy removal of the product from the reaction ether acetates through the use of immobilized enzymes
solution [45]. “Enzymatic Production of Biodiesels,” performing enantioselective transesterification [37, 51].
describes the invention of a process utilizing lipase enzymes
Along with the production of chemicals, immobilized
immobilized onto a hydrophobic microporous surface by
enzymes also have the ability to catalyze reactions to dispose
carrier immobilization for the production of biodiesel. This
of harmful chemicals. United States Patent, “Improved
method is an improvement over the previous method of
Nitroreductase Enzymes for Bioremediation,” describes the
biodiesel production, which was through the transesteri-
utilization of the ChrR enzyme, an E. coli nitroreductase
fication of vegetable oils and animal fats by a strong base, enzyme, in the biotransformation of hazardous metals for
because the lipase enzyme can effectively catalyze a reaction
environmental and health protection [52]. This enzyme
with crude vegetable oil, not just refined, and the glycerol
reactor is re-useable and reacts away or mediates the harmful
byproduct of the reaction is easily removed [46]. The overall
contaminates. Similarly, enzyme immobilization has also
method is more environmentally friendly and minimizes the
been valuable in removing contaminants from chemical
costly and timely separation steps. Another United States
reactions. International patent, “Polymer Composition and
patent, “Polypeptide and Biosynthetic Pathways for the Method for Removing Contaminates from a Substrate,”
Production of Stereoisomers of Monation and their Pre-
introduces a process of removing contaminates from a
cusors,” introduces a procedure using several immobilized
substrate solution through the introduction of a polymer film
enzymes to catalyze reactions throughout the biosynthetic
or other material containing an immobilized enzyme which
pathway, specifically using an immobilized protease to
binds the contaminate, allowing it to be effectively removed
hydrolyse lactone [47]. Co-immobilization is of particular
from the solution. For example, a polymer formed by
importance, because as biotechnology continues to evolve, combining sodium hydroxide, sodium dodecyl sulfate,
we will need to use enzymes to produce more complex
diethylenetriaminepentaacetic acid, and polyvinyl alcohol
chemistries, which will require enzyme cascades/pathways
was used to decontaminate an area of depleted uranium.
and not just single enzyme reactors. “Method for Producing
Under some circumstances the polymer gel is simply
Ester-exchanged Palm Oil,” a Japanese patent, outlines a
stripped from the substrate and disposed of; however, in
process in which immobilized lipase is packed into a reactor
other cases the contaminate can be chemically removed by
column, catalyzing a reaction producing ester-exchanged depositing it onto a previously clean substrate [53]. Also,
palm oil. Ester-exchanged palm oil is commonly used in the
European patent,“Affinity Purification of Polypeptide on
food industry due its good taste and the fact that it is not
Protein a Matrix,” describes a process of removing
easily oxidized [48]. The immobilized lipase is used in this
contaminants in Protein A chromatography. In the process,
situation to allow for the re-use of the enzyme, so the process
contaminated proteins are removed by immobilizing them
is more cost-effective. Chinese patent, “Method of
onto silica or glass and then removing them from the
Catalytically Synthesizing Alpha-monolinolenin by Using reaction [54]. In this case, the enzyme is not acting as a
Immobilized Lipase,” reports the invention of a method for
biocatalyst, but this enzyme immobilization membrane is
using lipolytic enzyme immobilized in a dielectric hole
helping to purify samples by immobilizing contaminating
material for the synthesis of alpha-monolinolenin, used in
proteins and/or enzymes. Finally, Chinese patent “Refining
the production of anti-cancer drugs [49]. Immobilized
Crude Oil of Soybean by Immobilized Phospholipase,”
enzymes are becoming much more important in the
outlines a reaction between immobilized phospholipase
pharmaceutical industry. Finally, both United States patents, enzymes and crude soybean oil which reduces the cost and
“Process for the Production of Amino Acids”, and
increases the efficiency of the purification/refining of
“Enzymatic Resolution of Propylene Glycol Alkyl (or Aryl)
soybean oil significantly over previous methods [55].
Ethers and Ether Acetates,” provide methods for increasing
the optical purity of solutions, which is of particular interest Biosensors
in the pharmaceutical industry among other industries.
Biosensors are an extremely promising application of
“Enzymatic resolution of a racemic mixture of stereospecific
GABA-T inhibitors” is another patent that is also focussing immobilized enzymes and several patents have been
published recently in the field. An international and a United
on increasing the optical purity of a solution for
States patent titled, “Methods and Materials for Controlling
pharmaceutical use. Penicillin acylase is used to produce
the Electrochemistry of Analyte Sensors,” have published a
stereospecific inhibitors for
-aminobutyric acid transa-
method of increasing the electrochemically reactive surface
minase (GABA-T) [50]. The penicillin acylase is immo-
of an electrode by utilizing a base substrate with a unique
bilized via covalent attachment to Eupergit C support, so it
can be re-used in the pharmaceutical reactor [50]. Most geometric arrangement. The sensor utilizes the enzyme
glucose oxidase immobilized onto either a thick, porous
pharmaceutical products require stereospecific production
substrate or a macroporous polymer in the detection of the
instead of racemic mixtures; therefore, these types of
amount of glucose present in blood [34, 35]. Again, another
optically pure enzymatic synthesis will become even more
combination of international and United States patents titled,
common in the future for the pharamaceutical industry as
“Analyte Sensors and Methods for Making and Using
well as other industries.“Process for the Production of
Amino Acids,” presents an effective process for producing Them,” provides an analyte sensor consisting of a base layer,
a conductive layer containing a working electrode, a layer
optically pure amino acids utilizing immobilized and free N-
responsible for sensing the analyte, and a layer responsible
acetylamino acid racemases, enzymes that effectively race-
Enzyme Immobilization in Biotechnology
for analyte regulation, composed of a hydrophilic comb,
copolymer. The immobilized enzyme is found between the
analyte regulating layer and the working electrode, in some
cases even immobilized onto the working electrode through
covalent bonding. Enzymes have been immobilized into a
variety of materials including a porous metallic substrate and
a macroporous polymer substrate. Possible applications of
this sensor design are as implants for the detection of glucose
in humans or animals [14]. A United States patent, “Method
and CMOS-based Device to Analyze Molecules and
Nanomaterials Based on the Electrical Readout of Specific
Binding Events on Functionalized Electrodes,” presents the
invention of a method of detecting nucleic acid hybridization
through the detection of polarized change by biosensors
utilizing enzymes immobilized on the electrode in order to
increase the polarization change to a detectable level [56] A
final United States Patent, “Medical Apparatus for Breath
Detection,” utilizes immobilized enzymes in a breathalyzer-
like biosensor which is used to detect the acetone levels in
the user’s breath. Acetone levels may be a helpful metabolic
indicator for individuals who are either overweight or
diabetic [32].
Bioreactors
Finally, a collection of patents have recently been filed
for the invention of enzyme reaction systems. An
international patent was filed titled, “Method for Producing
a Useful Substance by Use of an Immobilized Enzyme,”
introducing a fixed-bed column reactor useful for reactions
requiring two liquid phases such as the hydrolysis of fats and
oils. The patent recommends the use of the enzyme lipase
immobilized onto an inorganic carrier, such as silica gel or
porous glass, by physical absorption [57]. “Enzymatic
Modification in a Continuously Regenerated Packed Bed
Column,” a second international patent, describes a system
designed so that enzyme and substrate flow in opposite
directions in a two column reactor. Unmodified substrate is
pumped in at one end and modified substrate is removed at
the other. At the same time, immobilized enzyme is
circulated in the opposite direction [58]. A United States
patent, “Immobilized-Enzyme Microreactor Devices for
Characterization of Bimolecular Analytes and Associated
Methods,” also describes a microreactor designed for integ-
ration into a separation channel, micro-pipette tip, or
integrated circuit which utilizes an immobilized enzyme. The
selection of the enzyme is dependent of the desired use of
the microreactor; however, the patent did recommend that
the enzyme be immobilized onto a polymerized sol-gel
through covalent bonding [59]. Lastly, “Hybrid Immobilized
Catalytic System With Controlled Permeability,” a United
States patent, was published presenting a system for use in
an enzymatic bioreactor which consists of two layers, one
layer being where the enzyme is immobilized in alginate-
chitosan microcapsules and the other being a permeable
layer controlling access to the enzyme or other biocatalyst,
therefore potentially protecting from the surrounding
environment. The two layers can be produced on disks,
films, microfibers, microcapsules or nanocapsules, micro-
spheres and nanospheres [60].
Recent Patents on Engineering 2008, Vol. 2, No. 3 199
CURRENT & FUTURE DEVELOPMENTS
As the need for greener and more efficient industrial
processes increases, more industrial processes will employ
immobilized enzymes. Over the coming years, research will
continue on developing enzyme immobilization strategies
and unique immobilizing architectures for increasing the
activity and stability of the enzyme in industrial streams.
Focus will likely move away from strategies that reduce
enzyme movement (i.e. cross-linking), because these
strategies typically reduce enzyme activity. Instead, research
will focus on developing ideal chemical and physical
microenvironments for immobilizing the enzyme while
maintaining optimal activity. Strategies will also be studied
to increase immobilized enzyme stability in organic solvents,
contaminants, harsh pH environments, and wide temperature
ranges.
CONCLUSIONS
Overall, this review of recent patents pertaining to
immobilized enzymes in biotechnology illustrates the diverse
application of this research. Inventions have recently been
patented in disciplines as different as cancer treatment and
materials engineering, but still relating to immobilized
enzymes for use in biotechnology. This demonstration
provides evidence that increasing research efforts will
continue in the field of enzyme immobilization for biotech-
nology. As methods for the immobilization of enzymes
continue to improve and become commercially widespread,
the availability of immobilized enzymes to industry has the
opportunity to increase significantly. This increase in the
availability of immobilized enzymes would allow for a
growth in the application of immobilized enzymes through-
out the chemical and medical fields. Continuing research on
drug delivery and tumor location analysis, along with the use
of immobilized enzymes in biosensors, makes an escalating
use of immobilized enzymes in medicine inevitable. Finally,
the ongoing development of bioreactors employing immo-
bilized enzymes continues to improve the efficiency of
reactions utilizing immobilized biocatalysts. These impro-
vements will likely result in a growth in the application of
immobilized enzymes to new fields increasingly realistic.
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