Enzyme immobilization can be defined as the attachment of free or soluble enzymes
to different types of supports resulting in reduction or loss of mobility of
the enzyme. Selection of an immobilization strategy greatly influences the properties
of biocatalyst. The varying levels in activity and diffusion limitations occurring
with immobilization are mainly dependent on the properties of support material
and the immobilization method. Support materials play an important role in the
usefulness of an immobilized enzyme as it should be low-cost and provide adequate
large surface area together with the least diffusion limitation in the transport
of substrate and product for enzymatic reactions (Krajewska,
2004). In order to fully retain the biological activity, enzymes should
be attached onto surfaces without affecting their conformational and functional
Generally, the choice of a suitable immobilization strategy is determined by
the physico-chemical properties of both supporting surface and the enzyme of
interest. The use of free enzymes as compared to their immobilized forms show
some significant drawbacks such as thermal instability, susceptibility to attack
by proteases, activity inhibition, high sensitivity to several denaturing agents
and the difficulty of separating or reusing the free catalyst at the end of
the reaction from the reaction mixture. Many of these restrictions can be resolved
by using enzymes in immobilized forms (Khan et al.,
2006). Enzymes may be immobilized by a variety of methods which may be broadly
classified as physical where weak interactions between support and enzyme exist
and chemical where covalent bonds are formed with the enzyme (Kirk
et al., 2002; Cherry and Fidantsef, 2003;
Sheldon, 2007). To the physical methods belong: containment
of an enzyme within a membrane reactor; adsorption (physical, ionic) on a water-insoluble
matrix; inclusion (or gel entrapment); microencapsulation with a solid membrane;
microencapsulation with a liquid membrane and formation of enzymatic Langmuir-blodgett
The chemical immobilization methods include: covalent attachment to a water-insoluble
matrix; crosslinking with use of a multifunctional, low molecular weight reagent
and co-crosslinking with other neutral substances, e.g., proteins (Cao,
2005; Khan et al., 2005a). Numerous other
methods which are combinations of the ones listed or original and specific of
a given support or enzyme have been devised. However, no single method and support
is best for all enzymes and their applications. This is because of the widely
different chemical characteristics and composition of enzymes, the different
properties of substrates and products and the different uses to which the product
can be applied. Besides, all of the methods present advantages and drawbacks.
Adsorption is simple, cheap and effective but frequently reversible, covalent
attachment and crosslinking are effective and durable but expensive and easily
worsening the enzyme performance and in membrane reactor-confinement, entrapment
and microencapsulations diffusional problems are inherent. Consequently as a
rule the optimal immobilization conditions for a chosen enzyme and its application
are found empirically by a process of trial and error in a way to ensure the
highest possible retention of activity of the enzyme, its operational stability
and durability (Khan et al., 2005b; Krajewska,
New designs of immobilization supporting materials with tailorable pore size are being studied more and more and in parallel, structure and surface characteristics of the target enzymes are worked out. These all studies have enabled more precise control of the immobilization of vast number of enzymes. The synthesis of vast number of immobilized enzymes has been applied in different fields for the benefit of humanity.
Uses of immobilized enzymes: During the initial years of the development of the field of immobilized enzymology, researchers used to find only the advantage of the immobilized enzymes in comparison to their soluble/free counterparts. Advantages of immobilized versus soluble enzymes included comparative studies in pH profile, various denaturing agents organic solvents, temperature, etc. Now recently during the last couple of decades, immobilized enzyme technology has advanced into and ever-expanding and multidisciplinary fields to analyze clinical, industrial and environmental samples. Here, we present recent developments and used of immobilized enzymes in different fields such as in medicine, antibiotic production, drug metabolism, food industry, biodiesel production, bioremediation, etc.
Use of immobilized enzymes as biosensors: Biosensors are electrical,
optical, chemical or mechanical devices with the capability to detect biological
species selectively. They are often modified with biological entities to enhance
their selectivity. Examples of biological recognition molecules include enzymes,
antibodies and oligonucleotides. The ideal biosensor not only has to respond
to low concentrations of analytes but also must have the ability to discriminate
among species according to the recognition molecules that are immobilized on
its surface. Biosensors have wide applications including biomarker detection
for medical diagnostics and pathogen and toxin detection in food and water (Leung
et al., 2007). Analytical technology based on biosensors is an extremely
broad field which impacts on many major industrial sectors such as the pharmaceutical,
healthcare, food and agricultural industries as well as environmental monitoring.
Because of their exceptional performance, capabilities which include high specificity
and sensitivity, rapid response, low cost, relatively compact size and user-friendly
operations, these properties of biosensors make them an important tool for detection
of various chemical and biological components (Amine et
al., 2006). The development of biosensors based on immobilized enzymes
came out to solve several problems such as loss of enzyme, maintainace of enzyme
stability and shelf life of biosensors and additionally to reduce the time of
enzymatic response and offer disposable devices which can be easily used in
stationary or in flow system.
Biosensors based on principle of enzyme inhibition have by now been applied
for a wide range of significant analytes such as Organophosphorus Pesticides
(OP) organochlorine pesticides, derivatives of insecticides, heavy metals and
glycoalkaloids. The choice of enzyme/analyte system is based on the fact that
these toxic analytes inhibit normal enzymatic function. Typically, the percentage
of inhibited enzyme (1%) that results after exposure to inhibitor is quantitatively
related to the inhibitor (i.e., analyte) concentration (Ivanov
et al., 2003a, b).
Malitesta and Guascito (2005) have described the application
of biosensors based on glucose oxidase immobilized by electropolypmerization
for heavy metal determination. Similarly, urease has been entrapped in both
Polyvinyl Chloride (PVC) and cellulose triacetate layers on the surface of pH-sensitive
iridium oxide electrodes and used for the determination of mercury. The immobilization
of polyphenol oxidase during the anodic electropolypmerization of polypyrrole
has been also reported.
The biosensor has been used for the determination of atrazine concentration
in low ppm level. The determination of pesticides with the help of biosensors
have become increasingly important in recent years because of the widespread
use of these compounds (El-Kaoutit et al., 2004).
As in the literature reports, the development and uses of biosensors for the detection of various compounds as pesticides, heavy metals, toxins, etc., is subject of considerable interest, particularly in the area of food and environmental monitoring (Table 1).
USE OF IMMOBILIZED ENZYMES IN MEDICINE
Presently, immobilized proteins/enzymes are used routinely in the medical fields,
for the diagnosis and treatment of various diseases.
|| Survey of immobilized enzymes used as biosensors for the
detection of some special compounds, pesticides and heavy metals
|BSA: Bovine Serum Albumin, GA: Glutaraldehyde, PVA-SbQ: Polyvinyl
Alcohol bearing Styryl pyridium group, PB-SPE: Prussian Blue Screen Printed
Electrode, TCNQ: 7,7,8,8-tetracyanoquinone diaminomethane,
Immobilized proteins as antibodies, enzymes, receptors have revolutionized
the medical fields in terms of time, manpower, accuracy and reliability.
Enzyme-based electrodes represent a major application of immobilized enzymes
in medicine. The high specificity and reactivity of an enzyme towards its substrate
are properties being exploited in biosensor technology. Biosensors used in clinical
applications possess advantages such as reliability, sensitivity, accuracy,
ease of handling and low-cost compared with conventional detection methods.
These characteristics in combination with the unique properties of an enzyme
render an enzyme based biosensor ideal for biomedical applications (DOrazio,
2003). Recently, numerous clinical trials and intensive research efforts
have indicated that continuous metabolic monitoring holds great potential to
provide an early indication of various body disorders and diseases. In view
of this, the development of biosensors for the measurement of metabolites has
become an area of intense scientific and technological studies for various research
groups across the world. These studies are driven by the need to replace existing
diagnostic tools such as glucose test strips, chromatography, mass spectroscopy
and Enzyme Linked Immunosorbent Assays (ELISA) with faster and cost effective
diagnostic devices that have the potential to provide an early signal of metabolic
imbalances and assist in the prevention and cure of various disorders like diabetes
and obesity (Vaddiraju et al., 2010). In recent
years however, intensive research has been undertaken to decentralize such tests
so that they can be performed virtually anywhere and under field conditions.
Hence, the development of portable, rapid and sensitive biosensor technology
with immediate on-the-spot interpretation of results are well suited for this
purpose. The importance of biosensors results from their high specificity and
sensitivity which allow the detection of a broad spectrum of analytes in complex
sample matrices (blood, serum, urine or food) with minimum sample pretreatment
(Malhotra and Chaubey, 2003). Now-a-days, the use of
Surface Plasmon Resonance (SPR) biosensors is increasingly popular in fundamental
biological studies, health science research, drug discovery, clinical diagnosis
and environmental and agricultural monitoring. SPR allows for the qualitative
and quantitative measurements of biomolecular interactions in real-time without
requiring a labeling procedure. Today, the development of SPR is geared toward
the design of compact, low-cost and sensitive biosensors. Nano-technology is
also increasingly used in the design of biologically optimized and optically
enhanced surfaces for SPR (Hoa et al., 2007).
USE OF IMMOBILIZED ENZYMES FOR ANTIBIOTIC PRODUCTION
Competition with well established, fine tuned chemical processes for antibiotics
production is a major challenge for the industrial implementation of the enzyme
synthesis of biologically important antibiotics such as β-lactam.
|| Survey of the immobilized enzymes used for antibiotic production
Enzyme based routes are acknowledged as an environment friendly approach, avoiding
organic chloride solvents and working at room temperature. Among different alternatives,
the kinetically controlled synthesis, using immobilized Penicillin G Acylase
(PGA) in aqueous environment with the simultaneous crystallization of the product
is the most promising one (Giordano et al., 2006).The
β-lactam acylase is traditionally used for the hydrolytic processing of
penicillin G and cephalosporin C. New and mutated acylase can be used for the
hydrolysis of alternative fermentation products as well as for the synthesis
of semisynthetic β-lactam antibiotics.
The yield of hydrolysis and synthesis has been greatly improved by process
design including immobilization of the enzyme and the use of alternative reaction
media. Significant advances have also been made in the resolution of racemic
mixtures by means of stereo-selective acylation/hydrolysis using β-lactam
acylases (Sio and Ouax, 2004).
Enzymatic production of cephalexin using immobilized penicillin G acylase has
also been studied in greater details. Conversion of 7-Amino-3-Deacetoxy-Cephalosporanic
Acid (7-ADCA) to cephalexin by Immobilized Penicillin G Acylase (IMPGA) have
been investigated and it has been observed that under optimized conditions,
IMPGA can attain 85% conversion of 7-ADCA to cephalexin. Furthermore, IMPGA
can be reused for about 10 cycles (Maladkar, 1994).
Production of cefazolin by immobilised cefazolin synthetase from E. coli
as a biocatalyst has been shown possible. The complex of the physico-chemical
studies makes it possible to design a highly efficient technological process
for production of cefazolin (Kurochkina and Nys, 1999).
Table 2 shows some of the latest references for the synthesis
of various antibiotics by different enzymes immobilized on different supports.
USE OF IMMOBILIZED ENZYMES IN FOOD INDUSTRY
Immobilized enzymes are of great value in the processing of food samples and
its analysis. The extent of lactose hydrolysis whey processing, skimmed milk
production, etc. has been greatly enhanced by using respective enzymes as immobilized
forms. The production of high fructose corn syrups has been greatly facilitated
by the use of immobilized glucose isomerase. Similarly in Japan, the fermentation
industry proved its processing efficiency for amino acids through the use of
immobilized amino acid acylase. A relatively new concept is the use of a single
matrix for immobilizing >1 enzymes to enhance food processing. Immobilized
multi-enzyme systems offer many attractive advantages however such a process
also raises some interesting questions about kinetics. Two systems, amino acylase
and glucose isomerase have been demonstrated to be techno-economically feasible.
Immobilization of other enzymes such as glucoamylase, lactase, protease and
flavor modifying enzymes has received some attention recently for food processing
(Carpio et al., 2000). D-tagatose has attracted
a great deal of attention in recent years due to its health benefits and similar
properties to sucrose. D-tagatose can be used as a low-calorie sweetener as
an intermediate for synthesis of other optically active compounds and as an
additive in detergent, cosmetic and pharmaceutical formulations.
|| Survey of the immobilized enzymes used in food industry
|| Survey of the immobilized lipases used biodiesel production
Biotransformation of D-tagatose has been produced using several biocatalyst
sources. Among the biocatalysts, L-arabinose isomerase has been mostly applied
for D-tagatose production because of the industrial feasibility for the use
of D-galactose as a substrate (Oh, 2007).
Calcium alginate beads have been used very efficiently as an effective supports for immobilization of alpha amylase for starch hydrolysis. Studies have also proved immobilization as an important technique for continuous and repeated use of enzymes in industrial application and also rapid separation of the enzyme from the reaction medium, thus improving their economic feasibility.
Compared to the free enzyme, the higher activity of the immobilized enzyme
at higher temperatures and the ability to hydrolyze raw starch such as that
of potato would help overcome problems related to gelatinization of starch during
hydrolysis (Gangadharan et al., 2009). Table
3 shows the processing of various food substrates using respective immobilized
USE OF IMMOBILIZED ENZYMES FOR BIODIESEL PRODUCTION
Biodiesel has gained importance in the recent past for its ability to replace
fossil fuels which are likely to run out within a century. Especially, the environmental
issues concerned with the exhaust gases emission by the usage of fossil fuels
also encourage the usage of biodiesel which has proved to be eco-friendly far
more than fossil fuels. Biodiesel fuel does not produce sulfur oxide, halogens,
carbon monoxide and minimize the soot particulate (Iso et
Biodiesel fuel (fatty acid methyl esters) produced by transesterification of
triglycerides has attracted considerable attention as a renewable, biodegradable
and nontoxic fuels (Antolin et al., 2002; Tiwari
et al., 2007). Recently, lipase-catalyzed transesterification has
become more attractive for biodiesel production since the glycerol can be removed
easily and the purification of fatty acid methyl esters is simple (Dizge
and Keskinler, 2008). Biodiesel can be produced from vegetable oils, animal
fats, microalgal oils and waste products of vegetable oil refinery or animal
rendering and used frying oils.
|| Survey of the immobilized peroxidases, laccases and polyphenol
oxidases used in bioremediation
Immobilized enzymes could be employed in the biodiesel production with the
aim of reducing the production cost by reusing the enzyme (Jegannathan
et al., 2008). Literature about the biodiesel production by immobilized
enzymes shows that most of the researchers have used lipases from different
sources for biodiesel production (Table 4). Different immobilization
supports like ceramics, kaolinites, silica and zeolites have been used for lipase
immobilization (Yagiz et al., 2007) (Table
5). Costs of chemical biodiesel production have still been lower than those
of the enzymatic processes, however if the pollution of the natural environment
is also taken into consideration, these costs are comparable (Canakci
and Gerpen, 2003).
USE OF IMMOBILIZED ENZYMES FOR BIOREMEDIATION
There are >100,000 commercially available dyes with over 7x105
ton of dyestuff produced annually worldwide and used extensively in textile,
dyeing and printing industry (Akhtar et al., 2005a).
It is estimated that about 10-15% of the dyes are lost in industrial effluents.
The discharge of wastewater that contains high concentration of reactive dyes
is a well-known problem.
Anaerobic transformation of azo dyes begins with the reductive fission of the
azo-linkage, resulting in the formation and accumulation of colorless aromatic
amines which can be highly toxic and carcinogenic (Akhtar
et al., 2005b; Khan and Husain, 2007a). Recent
studies indicate that an enzymatic approach has attracted much interest in the
removal of phenolic pollutants from aqueous solutions as an alternative strategy
to the conventional chemical as well as microbial treatments that pose some
serious limitations (Khan and Husain, 2007b).
Conventional physical and chemical methods of dye decolorization/degradation
are actually outdated due to some unresolved problems. Biodegradation appears
a promising technology but unfortunately the analysis of contaminated soil and
water has been shown that these toxic pollutants persist even in the presence
of microorganisms. Often the environment of the microorganisms is not optimal
for rapid degradation. Recent studies indicate that an enzymatic approach has
attracted much interest in the removal of phenolic pollutants from aqueous solution
as an alternative strategy to the conventional chemical as well as microbial
treatments that pose some serious limitations (Chen and
Lin, 2007). Recently, peroxidases from bitter gourd (M. charantia)
immobilized on some cheaper supports have been found highly effective in decolorizing
reactive textile dyes compared to its soluble counterpart as the immobilized
enzyme looses only 50% activity even after 10 cycles of usage (Akhtar
et al., 2005b). Furthermore, laccases from a number of enzymes have
been immobilized on a number of supports for the decolorization/degradation
of various textile and non-textile dyes and phenolic compounds. Table
5 lists some recent research about the use of peroxidase, laccases, polyphenol
oxidases in different immobilized forms for dye decolorization/degradation and
phenolic compounds removal.
Recent advances in the design of immobilization supporting materials with tailorable pore size and surface functionality has enabled more precise control of immobilization of enzymes. New simulations of the surface characteristics of the target enzymes can be used to aid in the design of appropriate support materials. As the structure and mechanism of action of enzymes becomes available more controlled immobilization methods will be generated. The development of cheaper and disposable array biosensors, bioreactors and biochips for the simultaneous detection of clinically important metabolites and rapid screening of diseases has attracted much attention during the recent past. We believe that the use of more and more immobilized enzymes in clinical, biotechnological, pharmacological and other industrial fields has great promise among future technologies.
The researcher would like to express their sincere appreciation to Prof. Hassan Mirghani Mousa for his valuable discussion and proofreading of this study.