Reversible Enzyme Immobilization

As the name suggests, this method is reversible, and the enzymescan be removed from the support easily by simple reactions orreversal of the conditions by which the immobilization was carriedout. The method can be of the following types:

Adsorption

Adsorption (Figure 1a) is the oldest and arguably the simplest ofall techniques. The first industrial process that involved immobilizedenzyme used an aminoacylase adsorbed to a DEAE-Sephadex forcontinuous resolution of amino acids. The first record of largescaleindustrial utilization of immobilized enzyme technology alsoinvolved adsorption of glucose isomerase to DEAE-Cellulose in theproduction of high fructose corn syrup by Clinton Corn Products[11].

Figure 1: Variations in enzyme immobilization technique; (a) adsorption, (b) chelation, (c) disulfide bonding, (d) ionic binding, (e) affinity binding, (f) covalentbinding, (g) crosslinking, (h) entrapment and (i) encapsulation.

Principle: This involves adhering of the enzyme to the surfaceof the carrier via several weak non-covalent interactions such ashydrogen bond, Van Der Waal’s interactions and hydrophobicinteraction [12].

Principle: This involves adhering of the enzyme to the surfaceof the carrier via several weak non-covalent interactions such ashydrogen bond, Van Der Waal’s interactions and hydrophobicinteraction [12].

Advantage: This method is cheap, easy to perform and allows easy recovery of the enzyme from the carrier, thus allowing re-useof both. So, it requires very little activation and no reagents [13]. Theweak interactions involved, hardly cause any distortion of the enzymeretaining maximum enzyme activity [11]. A variety of organic andinorganic materials can be used as support. If the adsorption isbased on hydrophobic interactions, it is stabilized by high ionicconcentrations, thereby permitting the usage of high concentrationsof substrate in the bioreactor [8].

Disadvantage: Significant enzyme loss cannot be avoided in thistechnique as the binding forces are weak, reversible and susceptibleto physical parameters such as pH and temperature [13]. This maylead to the presence of enzyme in the reaction product, which cancontaminate the products and complicate the purification process[11].

Carriers used: Common carriers used in adsorption are activatedcharcoal, alumina, cellulose, Sephadex, agarose, collagen and starch[13]. Researchers have come up with new eco-friendly carriers, suchas coconut fibre with high water retention and cation exchangeproperties, which could significantly reduce costs as well as preventethical issues [14].

Chelation

Chelation or metal binding (Figure 1b) is another common type ofreversible enzyme immobilization mostly used as a chromatographicmethod. Being reasonably expensive and involving safety issues, thismethod is less popular in industries.

Principle: It is based on the ability of charged and polar aminoacids (histidine, lysine, phenylalanine, cysteine and tyrosine) tobind to metal ions via coordinate bonds. The metal ions bound tothe carrier surface have metal ligands weakly bound to them. Uponexposure of the enzyme to the carrier, the weak ligands are replacedby the enzyme molecules [12,15]. The binding can be easily reversedeither by the introduction of a ligand with greater affinity for themetal ion (ethylene diamine tetraacetate, EDTA) or by addition ofexcess of a competing ligand [16].

Advantage: The reversal of the binding provides easy regenerationof the support without any appreciable effect on the yield. The mainadvantage over other reversible enzyme immobilization methodsis that the binding is reasonably strong due to which enzymeleakagecan be maximally restricted. A variety of chelating anionscan be provisionally attached to the matrix followed by metastableattachment of the enzyme to the support [15].

Disadvantage: The reagents involved may not be safe for theproduction of food products and may cause health hazards. Reductionin enzyme activity due to interaction with the metal ions at the activesites of the enzymes is also a major drawback of this process.

Carriers used: The supports used are mostly organic materials,usually cellulose, chitin or silica-based carriers. They are activated asthe enzymes bind to their nucleophilic groups by coordinate bonds.Transition metal salts or hydroxides may also serve as carriers [12].

Disulphide bonding

This technology involves the formation of disulphide bonds between the enzyme and the matrix. Though it is a form of covalentbonding (an irreversible enzyme immobilization method), it isclassified as a reversible technique because of the ease of reversal ofthe binding (Figure 1c).

Irreversible Enzyme Immobilization

Reversible immobilized enzyme technology cannot fulfil the aim of using an immobilized enzyme on a long term basis. Irreversibleimmobilization involves strong chemical bonds and particularlyserves to maintain reasonable stability of the enzymes over a longperiod of time. Most industries use enzymes immobilized by thesemethods, thereby allowing continuous processing of the substrateswithout the need of replacing the enzyme very often. Differenttechniques classified as irreversible enzyme immobilization arediscussed in the following sections.

Covalent Binding

Covalent bonds are highly stable and hence, covalent bindingensures that the enzyme is strongly bound to the support (Figure 1f). It has been used in a number of industries, since 1973, mostprominently in the synthesis of 6-aminopenicillanic acid fromPenicillin G, which utilizes penicillin acylase covalently bound toSephadex G-200 [11].

Advantage: The reaction is highly specific and no contaminants are present on the carrier. If the antibody on the support is highlyspecific for the enzyme, the step of enzyme purification can bebypassed. Enzymes from an impure solution can also specificallyattach to the matrix. Maximum activity of the enzyme is also ensuredif the antibody is targeted at an epitope away from the activity site[21].

Principle: It involves the formation of a covalent bond betweenthe support and the side chains of the amino acids of the enzyme, mostcommonly lysine (ε-amino group), cysteine (thiol group), asparticacid and glutamic acid (carboxylic group), hydroxyl group, imidazolegroup, phenol groups, etc. [11]. These groups are nucleophiles andtend to bind to electrophilic groups of the support. A wide varietyof reactions have been developed, depending on the functionalgroups available on the matrix. For coupling of the enzyme to thesupport, it is often necessary to ‘activate the support’, i.e., modifythe support so as to make it bind to the enzyme more efficiently. Theactivation methods in general can be divided into two main classes,(i) addition of a reactive group to the support polymer to activateit, and (ii) modification of the polymer backbone to produce anactivated group. The activation processes are generally designed togenerate electrophilic groups on the support. This allows the supportto react with the strong nucleophiles on the proteins, allowing stableimmobilization [7].

Advantage: The binding involves covalent interactions andis strong. Hence, leaking of the enzyme into the reaction mixtureis totally prevented. This prevents mixing of the enzyme with theproduct, thereby reducing contamination and the cost of purification[7]. The covalent binding also stabilizes the enzyme in specific proteinorientations, and may promote higher specific activity [14].

Disadvantage: The covalent bond formed between the supportand enzyme may involve the amino acids of the active site of theenzyme, which may lead to significant loss in activity. Since the method is irreversible, the support cannot be recycled, as theenzymatic activity declines. The support along with the boundenzyme has to be discarded [7].

Advantage: The reaction is highly specific and no contaminants are present on the carrier. If the antibody on the support is highlyspecific for the enzyme, the step of enzyme purification can bebypassed. Enzymes from an impure solution can also specificallyattach to the matrix. Maximum activity of the enzyme is also ensuredif the antibody is targeted at an epitope away from the activity site[21].

Carriers used: The supports used are generally stable and easilyavailable and are activated by the appropriate reagents. The commonsupports used are cyanogen bromide (CNBr)-activated Sephadex orCNBr-activated Sepharose. Other common carriers include activatedforms of dextran, cellulose, agarose, etc. [11]. Artificial matricesinclude Polyvinyl chloride, ion exchange resins and porous glass [12].

Crosslinking

It is an irreversible method of enzyme immobilization (Figure1g). It is different from other techniques in the sense that it does notrequire a support for the immobilization. There are two methods ofcross linking in use, (i) Cross Linking Enzyme Aggregate (CLEA),and (ii) Cross Linking Enzyme Crystals (CLEC). Both CLEA andCLEC are modifications of a primitive method, where cross linkingagents such as glutaraldehyde (which react with the amino groupon the protein) were used. The CLEC or CLEA are added to thereaction mixture and can be later removed from the mixture duringproduct purification. Hence, unlike the other systems of enzymeimmobilization, the immobilized enzyme is not bound to any matrix,but is present in the reaction mixture, albeit in an immobilized form.The two methods are described below.

A. Cross Linking Enzyme Crystals (CLEC)

Principle: In this method, glutaraldehyde is used to crystallizethe enzyme. Hence, enzyme crystals are obtained with the help ofthe cross-linking agent. The CLEC, upon addition to the reactionmixture, catalyzes the reaction with reasonably high efficiency [12].

Advantage: The CLEC are very stable and are not easilydenatured by heat or organic solvents. They are moderately resistantto proteolysis. They have a manageable size and stability in operatingthe bioreactor and can be recycled [12, 2]. They also give enhancedenantioselectivity to particular forms of their substrate [23].

Disadvantage: Highly purified enzyme produced through astandard protocol of crystallization is required for the preparationof CLEC. These requirements involve a lot of time and expenses.Diffusion of substrate and product is limited with increase in size ofthe aggregate [24].

B. Cross Linking Enzyme Aggregates (CLEA)

Principle: CLEA is an improved version of CLEC productionand aims at nullifying the disadvantages of CLEC. While CLECrequires the formation of crystals, CLEA could work in aqueoussolutions. Addition of salts, organic solvents or non-ionic polymersresults in the formation of enzyme aggregates which retain theircatalytic properties. These aggregates are called Cross Linked EnzymeAggregates (CLEA) [12] (Figure 2).

Figure 2: The mechanism of action of CLEA via formation of enzyme crystal aggregates.

Advantage: It is cheaper, easier to perform and has a wide rangeof applications. For multi-enzyme catalysis, it is possible to synthesizeCLEA having more than one enzyme in the aggregate (called combi-CLEA) [23].

Disadvantage: The size of the derived aggregates is small andoften similar to the size of the substrate or product; this may causesevere inconvenience during product purification [25]. Diffusion ofsubstrate and product is limited with increase in size of the aggregate[24]. The CLEAs are often found to be fragile and tend to exhibit lowstability in stirred tank fermenters or packed bed reactors [23].

Crosslinking agents used: For most enzymes, the crosslinkingagent used is glutaraldehyde, which is cheap, stable and easilyavailable. However, glutaraldehyde partially or totally inactivatessome enzymes. For such biomolecules, alternative cross-linking agentssuch as dextran polysaccharide, bis-isocyanate, bis-diazobenzidine,diazonium salts and functionally inert proteins, such as bovine serumalbumin (BSA) should be preferred [12,22].

Entrapment

Entrapment is another prominent technique of irreversibleenzyme immobilization, where the enzyme is immobilized byentrapping it within a support matrix or within fibres (Figure 1h).

Principle: Enzymes, being large macromolecules, tend to be largerthan the substrates or products. Thus, the enzyme is immobilizedwithin a matrix of appropriate pore size to allow only the substratesand products of a diameter smaller than the matrix pore size to diffusein and out of the mesh respectively [13]. The enzyme size-to-poresize of support is a deciding factor in selecting the support. Smallerthe pores, lesser the enzyme entrapped, while larger the pores, morethe leaking of the enzyme. Hence, accurate pore size selection of thesupport is crucial [12].

Advantage: The method is fast, cheap and easily carried out under mild or physiological conditions. As the enzyme remains confinedwithin a matrix, it is protected from contamination by microbes,proteases or other enzymes [13].

Disadvantage: The meshwork of the matrix cannot support a hugevolume of enzyme molecules and can lead to enzyme inactivation.Hence, the process can be costly at times. The rate of diffusion of thesubstrate and product dictate the reaction rate. This is because unlessthe substrate molecules diffuse into the mesh, the reaction will not beinitiated and according to Le Chatelier’s principle, the reaction ratedoes not reach a peak unless the products sieve out [12,13].

Matrix used: Common polymers used for enzyme entrapmentinclude alginate, carrageenan, collagen, polyacrylamide, gelatin,silicon rubber and polyurethane [26].

Encapsulation

Encapsulation can be regarded as a special type of entrapmentwhere the enzyme is immobilized by entrapping it in a sphericalsemi-permeable membrane (Figure 1i).

Principle: The basic underlying principle is the larger size of theenzyme as compared to substrates or products. When the enzymecan be entrapped or occluded within a semi-permeable membrane,it would allow the small substrate molecules to diffuse in and theproduct molecules to diffuse out. However, the enzymes being muchlarger, cannot diffuse through the membrane and remains withinit. Hence, the enzyme is restricted within the membrane, althoughit is free floating inside the capsule [12]. The permeability of themembrane is controlled according to the enzyme being immobilized.Encapsulation is achieved by one of the two methods, (i) Coacervation (allowing the polymer to separate out enzyme microdroplets ina water immiscible solvent), and (ii) Interfacial polymerization(when a hydrophobic monomer is added to an aqueous solution ofenzyme and another monomer which has been dispersed in a waterimmiscible solvent. This promotes polymerization at the interface ofthe two droplets and hence around the enzyme).

Advantage: Encapsulation within a membrane maintains theenzyme structure in its native form and prevents leakage of theenzyme, protecting it from the harsh conditions of the medium.Multi-enzyme encapsulations can also be created by trapping morethan one enzyme within a membrane [27].

Disadvantage: As mentioned earlier with entrapment method,the diffusion of substrate and product across the membrane controlsthe reaction rate. The pore size needs to be maintained accuratelyto prohibit enzyme leakage (if the pore is too large) or poor loadingof the enzyme (if pores are very minute). This technique is nonrecommendablefor reactions involving substrate and enzymemolecules of similar diameters as an optimum pore size cannot beselected in such cases [12].

Biopolymers used: A variety of biopolymers like alginate,maltodextrin, cellulose, chitosan, etc. are available which can beused for the purpose of encapsulation of an enzyme. These polymersform a single layered capsule around the enzyme. Double-layermicrocapsules, built of two distinct types of polymers, are alsopopular. The most common ones are composed of chitosan, Poly-Llysine,Polyvinyl acetate, gelatin and boric acid [6].

Whole cell immobilization as an alternative strategy toenzyme immobilization

Cells may be described as living biocatalysts. Every cell containsdifferent enzymes, which can be used to catalyze a large variety ofreactions. Some of these reactions yield products that are required intheir life processes, such as energy production and anabolism. Whenreactions are catalyzed by cells in a bioreactor, the cells are generallynot immobilized, and remain floating in the medium. Hence, theyoften have to be filtered out and discarded during purification ofproducts. In nature, microorganisms are often found growing ona substrate. Biofilms are surface-attached microbial communitiesconsisting of multiple layers of cells embedded in hydrated matrices.This allows bacteria to grow attached to the substrate, and especiallyin niches where the surrounding medium is not static or stable, e.g.,on rocks under flowing rivers, on the walls of pipes, teeth, etc. [28].It is often seen that bacteria bound to a surface grow and proliferatebetter. Hence, this principle was put to use in immobilizing cells, andthen use them for human activity. In this technology, the cells remainbound to a surface, while the nutrients (essential for cells to survive),substrates and products diffuse in and out of the cells (owing to thesemi-permeable nature of the cell membrane) [28]. Whole cells maybe immobilized either in a viable or non-viable form [29].

Principle: The discussion on immobilized cells to be used asbiocatalysts constitute two major points as mentioned below.

(i) The cells need to be properly attached to a suitable support.Different techniques can be adapted for the same and a few techniques include immobilizing the cells under a porous support like polymericgels, attaching them to a micro carrier surface or entrapping thembehind membranes [28]. The last approach is less favoured, as itcreates a constraint of space, and the rate of the reaction is limited bythe diffusion parameters (the nutrients, substrates and products haveto diffuse through two membranes - the external membrane as well asthe membrane of the cells). The strategy used commonly is attachingthem to a carrier; several motifs on the cell membrane or wall ofthe cell being immobilized are utilized. Targeting the characteristicepitopes on the membrane of commercially useful microorganismscan allow efficient binding of the cells to the carrier [29].

(ii) Easy diffusion of the substrates and products within the cellsis essential for economical production at the industrial scale. The cellsmay be permeabilized to act as better intracellular source of enzymesand this may be achieved by physico-chemical techniques. Physicaltechniques include freezing and thawing. Chemical techniquesutilize organic solvents (toluene, chloroform, ethanol, butanol) andmild detergents (N-Cetyl-N, N, N-trimethyl ammonium bromide,i.e., CTAB and digitonin) to increase the membrane porosity [29].However, to avoid unwanted cell lysis, low dosage of the chemicalsis advocated. Permeabilization is only necessary for enzymes thatwork in the cytoplasm. For enzymes active in the periplasmic spacesuch as catalase (in yeasts), urease, Penicillin G acylase (in bacteria),the cells can be utilized without permeabilization. Hence, noveltechniques of using intracellular enzymes include their transport intothe periplasmic space, via genetic engineering. Anchoring proteinsattach the enzymes, either to the exterior of the cell membrane or theinterior of the cell wall. The anchoring proteins with a characteristicLPXTG domain aid enzymes to remain in the periplasmic spacevia attachment with the cell wall [29]. The recombinant Escherichiacoli expressing organophosphorous hydrolase (OPH) gene has beencreated in this way [30].

Advantage: The conditions of the cytosol or resident organellesare the optimum for respective enzymes. Viable cells continue todivide and naturally replenish the enzymes inside a bioreactor.Continuous fermentation processes tend to suffer from the problemof reduction in cell density due to washing away of cells. This can beovercome by using immobilized cells for the process. The cell densityof the culture can be pre-determined, as the number of cells to beimmobilized can be controlled [29]. The major advantage is that thisprocess is more economical than enzyme immobilization, because itis far more expensive to get a purified enzyme as compared to a cellculture.

Disadvantage: Cell immobilization is far less expensive ascompared to enzyme immobilization. Hence, it may seem reasonableto utilize cell immobilization over enzyme immobilization. However,this method involves certain disadvantages which have been discussedas follows:

(i) The rate of catalysis is highly limited by the rates of diffusion ofsubstrate and product across the membrane, as the enzymes remainentrapped within the cells.

(ii) For aerobic cells, proper oxygenation is vital for survival andgrowth. The dissolved oxygen needs to be maintained at a high level, as the cells lie embedded on a solid surface (usually inside the media).

(iii) The matrices may tear due to excessive proliferation of thecells [31].

(iv) Immobilization can only be used for cells that release outthe products to the exterior. Since the cells are immobilized, it is notpossible to extract the cells and lyse them for releasing the products.

(v) A cell is a reservoir of a myriad of enzymes. So, there arechances of cross-reaction of the substrate with unwanted enzymes, orthe product can be converted to some unwanted forms.

Enzyme Modification vs. Enzyme Immobilization

Genetic modulations and experiments have created variationsof enzymes which better catalyse reactions than their normalcounterparts. Such modifications, though seem costly during initialimplementations, are actually cost-effective in the long run, as theyproduce higher yields than their normal counterparts. Enzymemodification is to be used primarily when the native form of theenzyme has either very low activity and is extremely labile or readilydegraded by the conditions in which the reaction is to be carried out.The enzyme immobilization tries to prevent its mobility, while stillretaining its catalytic activity, while enzyme modification focuses onmodifying its activity, sensitivity to various chemicals and alteringthe specificity. This technique seems to maximise the industrialefficiency of an enzyme and tries to retain its catalytic efficiency to themaximum. It fixes usable enzymes to a support, so that they are notlost during purification [6].

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Applications of immobilized enzymes

1. Immobilized enzymes as biosensors

A biosensor is an analytical device that responds to an analytein an appropriate sample and interpretates its concentration as anelectrical signal, via a suitable combination of a biological recognitionsystem and an electrochemical transducer [32]. It contains an optical,electrical, chemical or mechanical as well as a biological component,usually an enzyme, antibody or nucleotide. Biosensors should beable to detect even traces of biological activity and must be able todistinguish between two different living entities as well. The idea ofusing immobilized enzymes is highly sensible, as enzymes show highsensitivity, rapid detection, reasonable costs, etc. As already discussed,enzyme immobilization also prevents enzyme-loss, maintains thestability of the enzyme structure, enhances shelf life and providessolid disposable detection kits that facilitates immediate and onfielddetection. These techniques are applicable in the preparationof medicines. Their high specificity might be used in detection ofvery minute quantities of pathogens and toxins in food, water oreven within human body [4]. The most commonly used exampleof an immobilized enzyme, being used as a biosensor is that of theglucose oxidase-based electrodes, that are used for monitoring thelevel of glucose in a system [5]. The Enzyme Linked ImmunosorbentAssay (ELISA) is another popular diagnostic tool, used as a biosensorfor especially detecting viral infections. Another popular kind ofbiosensor being put to use nowadays involves the principle of enzymeinhibition. It uses an analyte such as organophosphorous pesticides and derivatives of insecticides or heavy metals. These toxic analytesare chosen on the basis that they would inhibit normal functionof enzymes and that the percentage of inhibited enzyme would beproportional to the quantity of the toxic analyte. Hence, it gives anestimate of toxin quantity [4]. Biosensors based on immobilizedenzymes are even used in astrobiology, for detecting the presence orabsence of life in other planets. Proteins characteristic of existenceof life may be detected by the biosensors. This is based on drillingthe ground to a depth of about 1.5-2 m to search for an amino acidsignature of life that got extinct about 3 billion years ago on Mars. Amicrofabricated capillary electrophoresis device, called Life MarkerChip (LMC), that utilizes immobilized enzyme for the determinationof amino acid chirality was developed and incorporated into theUREY instrument selected as part of the Pasteur payload for the ESA(European Space Agency) ExoMars mission [28].

2. Production of antibiotics

Classical methods of antibiotic production do not involve cellcultures. Attempts are being taken to overproduce such secondarymetabolites by keeping the cells under stress. As the immobilized cellsare confined to a small localized region, it is easier to induce stressfulconditions required for the production of secondary metabolites.Immobilized β-acylase shows better efficiency in the hydrolysis ofPenicillin G. Enzymatic production of Cephalexin using immobilizedPenicillin G Acylase also shows promise [4].

3. Biodiesel production

Ethanol and biodiesel have shown the ability to replacepetroleum and fossil fuels. On the basis of extent of energy retained,these hydrocarbons play pivotal roles as biofuel. The alternativehydrocarbons are required, as the traditional fuel is graduallydepleting. Biologically derived fuels are eco-friendly, causing lesserenvironmental hazards and are economically feasible [33]. Biodieselcan be produced from vegetable oils, animal oils, microalgal oils andwaste products of vegetable oil refinery through trans-esterificationof triglycerides and do not emit oxides of sulphur, halogens or othertoxic gases upon burning [34]. Hence, emphasis on the synthesisof biodiesel is very high. Classical methods use chemicals such asH₂SO₄ or NaOH and require a large input of energy and water. Useof immobilized lipase for the production of biodiesel is graduallygaining importance. The method is easy, economic and environmentfriendly,allowing easy recovery of glycerol, shows excellent catalyticactivity and is stable in non-aqueous media [5].

4. Bioremediation

Industries release harmful chemicals as effluents into waterbodies or into the soil, leading to widespread water pollution. Apartfrom toxic heavy metals like arsenic and mercury, industrial effluentsoften contain aromatic azo-dyes. Upon anaerobic transformation,these produce colourless aromatic amines that are carcinogenic andpose a great threat to human health. Use of immobilized enzymes forthe purpose provides some promise. Peroxidases from bitter gourdimmobilized on a support have been effective in decolorizing reactivetextile dyes. Immobilized laccases and polyphenyl oxidases proveeffective in detoxifying phenolic compounds in water bodies [4]. The immobilized laccase enzyme has the ability to degrade anthracinoiddye, Lancet blue and Ponceau red [13]. The Single EnzymeNanoparticle (SEN) is particularly used to chelate heavy metalsfrom polluted areas. In SEN, the enzyme is coated by a nanometrethick material. The SEN retains its activity even in extremely harshconditions [13]. High Efficiency Nano-Catalyst ImmobilizationTechnology (HENCI) is a promising new technology based on nanoscaledcatalysts, which can be used to degrade rare yet dangerouspollutants. Owing to their size and efficiency, they might be veryuseful in removing such pollutants to a very minute level. However,they are reasonably expensive and efficient supports to immobilizethem in large quantities are yet to be found [28]. Soils contaminatedwith pesticides may be treated with immobilized enzymes capable ofdegrading many organophosphorous compounds [28].

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Therapy

Immobilized enzymes find immense application in the fields oftherapeutics and pharmaceutical industry.

Treatment of kidney disorders

Kidney disorders often have several painful consequences,including gout. Gout is characterized by inflammation of joints dueto the accumulation of uric acid. It may be because of decreasedsecretion of urate by the tubules due to excess anions, or due toincreased tubular reabsorption. Present day treatment of goutinvolves treatment with an anti-inflammatory agent, followed byuricosuric agents to enhance renal excretion of urate. A novel methodinvolves the use of uricase immobilized to mesoporous silica. Theenzyme-entrapped mesoporous silica can be used as a transdermalpatch. The uricase converts uric acid to allantoin, which is about 100times more water soluble than urate [35].

Treatment of liver disorders

Hepatic failure is very dangerous, and if untreated may resultin death. Liver performs a huge array of vital metabolic functionsin our system, including detoxification of several toxins. When theliver fails to perform its tasks, the toxins accumulate and may leadto hepatic coma. The enzymes responsible for hepatic detoxification,e.g., alcohol dehydrogenase have been purified from rabbit liver,immobilized to a hemocompatible form of agarose matrix and testedto show fair stability and activity for a prolonged period of time. Thiscould be used for treatment of hepatic failure or other liver disorders[36].

Treatment of thromboembolic disorders

Thromboembolic disorders refer to the fatal syndrome in adultswhere blood clot formed at one location in a blood vessel is carriedby the blood to another location, where it blocks a blood vessel.Immobilized streptokinase may be used for the treatment of suchdisorders. They provide prolonged fibrinolytic activity and hence canbe a valuable asset in the treatment of anticipated thromboembolicdisorders [28].

Treatment of inflammatory manifestations

Superoxide dismutase (SOD) and catalase (CAT) have been immobilized in biodegradable microspheres to obtain efficientdelivery systems. The enzymes were found to remain stable throughoutin the immobilized state. SOD was also seen to be released slowlyand uniformly over a prolonged period of time, around two monthsin in vitro assays. Hence, these could be used for the treatment ofinflammatory manifestations, as in rheumatoid arthritis and variousintra-articular joint diseases [28].

Treatment of inborn errors of metabolism and cancertherapy

Inherent errors of metabolism are caused when an organism lacksthe ability to produce or utilize a particular substrate or component.This is mostly caused due to defective enzyme(s) in the metabolicpathway. This can be treated by encapsulating the enzyme (for whichthe patient is deficient) with erythrocytes (of the patient or someonewith the same blood group). The erythrocytes would then serve tocarry the enzyme throughout the system without generating animmune response. Cancer therapy and the treatment for various otherdiseases, using nanoparticles as carriers are also being researchedupon. These could work at delivering the drugs to their exact targetswith great accuracy [13].

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Detergent synthesis

Synthetic detergents can be produced using immobilized enzymesfor better removal of dirt and stain. Enzymes such as proteases,amylases, cellulases, etc. are immobilized using the granulationsof the detergent. These enzymes are relatively stable under harshconditions of washing and show good activity [5]. Lipases are used toremove stains caused by fats and oils. Proteases remove stains causedby proteins, such as those caused by blood, egg and sweat. Amylasesremove stains caused by carbohydrates such as chocolate and gravies.Cellulase is used to improve the softness and colour brightness ofcotton clothes [13].

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Food Industry

Immobilized enzymes play a key role in the food industry andhave a wide range of applications. We have briefly discussed theapplications in the following sections:

Brewing industry

Use of immobilized yeast cells allows the increase in the cellconcentration in a definite space of the fermenter. It allows retainingthe cells and simplifies the purification step.

Dairy industry

Immobilized enzymes are often put to use in the dairy industryto obtain the starting components and allow various reactions, suchas cheese and yoghurt production and prevention of growth ofpsychotrophic bacteria in milk. Immobilization can also alter boththe lactose and citrate metabolism and promote further nutritionalenrichment of dairy products [5]. Immobilized lipases have allowedthe incorporation of linolenic acid (an anti-carcinogen) into dairyproducts [37].

Meat industry

Using immobilized cells could aid in meat fermentation and retain a continuous cell line. It creates a starter culture that is cheaperand provides a continuous cell line for the process [5].

Alcohol industry

Immobilized cells are used for the production of alcohol. Alcoholproducingbacterial cells are immobilized and the fermentationis carried out in a bioreactor. It is observed that immobilized cellsexhibit a higher rate of product formation as compared to the cellsthat are not immobilized [5].

Organic acid production

Organic acids such as citric acid, malic acid, propionic acid,butyric acid, gibberellic acid and succinic acid may be synthesizedusing immobilized cells. Citric acid is generally synthesized throughAspergillus niger. Increased fungal growth disrupts uniform oxygendistribution and is antagonistic to high product yields. Immobilizedfungal cells show reduced proliferation and hence lower utilization ofoxygen, thereby prolonging organic acid production [5].

Syrup production

Enzymes like β-galactosidase are immobilized to a substrateand are used generally for lactose fermentation [13]. The first largeindustrial process to utilise immobilized enzyme was the productionof high fructose corn syrup by Clinton Corn Products and usedglucose isomerase adsorbed to DEAE-Cellulose as discussed earlier[11].

Textile industry

Common immobilized enzymes such as cellulose, amylase andpectinase derived from microbes have a variety of roles in textileindustry. Immobilization makes the enzyme withstand the harshconditions of the cloth processing. The processes such as scouring,treatment of wools, bio-polishing and denim finishing are achievedusing immobilized enzymes [13].

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Enzyme Immobilization and Nanotechnology

With the development of nanostructures such as nanofibres,carbon nanotubes and gold nanoparticles, Nanotechnology seems tobe offering better and more efficient support. They provide a largesurface area for binding of the enzyme, resulting in a net increasein both enzyme loading and enzymatic activity per unit volume.The enzymes are taken up by a process called nano-entrapment,which leads to discrete nanoparticles. Owing to their porosity andhigh surface area, nanostructures could more often be consideredfor usage as carriers. While enzymes bound to the nanostructuresby adsorption show a little leaking of the enzyme, those bound bycovalent linkage exhibit almost zero leaking [5].

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Further technology developments

Efficient simultaneous production of D-hydantoinase (Dhase)and D-decarbamoylase (Dcase) has been recently reported by wholecell immobilization of the recombinant E. coli strain LY13-05.Fermentation for 12 h with 30 g L^-1 DL-p-hydroxyphenyl hydantoin(DL-HPH) as the substrate for the immobilized strain caused thebroth cell density to reach 1.9 g L^-1. The yield of D-HPG was 29 g L^-1, that of D-p-hydroxyphenylglycine (D-HPG)/HPH was 97% andthe specific productivity was 1.3 g (g.h)^-1 with the productivity as2.4 g (L.h)^-1. The advantages observed in this process was the highsubstrate conversion efficiency during the production of D-HPG andthe specific yield rate, which were better than the yield of D-HPGfrom other reported strains. Conversion efficiency and yield was alsoreported to be higher for the recombinant strain in comparison tothat of the free cells. Moreover, the immobilized strains had betterthermal stability, higher repetition application time and longerstorage duration, which will supposedly have wider implications forindustrial productions [38].

Immobilized enzymes have always posed as a major cost factor inthe utilization of heterogeneous catalysts on an industrial scale. Theformation of foreign peptides associated with the oil body in plantseeds has been strategized. Such production can be manipulated bygenetically modifying rapeseeds at relatively low cost. Floatationcentrifugation can be used to easily separate out the oil bodies. Thecatalytic activity of the reporter β-glucuronidase expressed on theoil body surface is comparable to those of free β-glucuronidaseenzyme [39]. The United States Environmental Protection Agencyhas been engineering stable immobilized enzymes for the hydrolysisand transesterification of triglycerides. The main improvementsundertaken in this project were (i) catalyst improvement by increasingthe specific surface area of the immobilized matrix within a range ofmacropore size; (ii) characterization of the used materials in orderto provide feedback to the catalyst improvement studies regardingpore size distributions, specific surface areas and enzyme distributionwithin the alcogel; (iii) studies related to the bioreactor to improvereactivity, reusability and stability of the used immobilized enzymes(http://cfpub.epa.gov/ncer_ abstracts/index.cfm/fuseaction/ display.highlight/abstract/6727).

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Multi-Enzyme Immobilization

Cells are complex machineries and require many enzymes toperform the life functions. Multiple enzymes work in a cascade toperform a set of complex reactions within all living cells. However,most attempts to utilize more than one enzyme in a bioreactoroften lead to failure due to the sensitivity of the enzymes as wellas cross reactions. Enzyme immobilization promises to tackle theproblem and deliver multi-enzyme immobilized systems, mostlyby the technique of encapsulation [8]. The enzymes may retaintheir activity and have their active sites close to each other. Thiscan allow a mechanism similar to substrate channelling without thediffusion of the intermediates (Figure 3). However, multi-enzymeimmobilized systems face several challenges. The enzymes shouldhave enough spatial separation to prevent steric hindrance betweenthe enzyme subunits that would distort their structure, but shouldbe close enough to ensure maximal activity. The support should besuch that it allows maximal loading of the enzymes, as well as ensuresmaximum enzyme activity. A biosynthetic approach co-localizingthe two enzymes on nanocarriers to mimic multi-enzyme complexsystems that occur in nature can potentially improve the product yieldsignificantly. Different polymeric nanocarriers are being investigatedto act as supports for multi-enzyme systems [40].

Figure 3: Schematic representation of the operation of a multi-enzyme immobilization system; product formation occurs through shifting of the intermediates from one enzyme active site to another, analogous to substratechannelling.

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Conclusion

The specificity of the enzymes as well as their ability to catalyzereactions in mild conditions makes them very precious. Immobilizedenzymes often show remarkable stability, high levels of activityand greatly reduce the cost of product processing [8]. However,immobilization does cause a slight loss of activity in most cases. Thecosts of enzyme purification and immobilization are often high andresult in increasing the cost of the overall reaction [7,24]. Thesedisadvantages need to be addressed to improve the overall reactionand make it more feasible. There is plenty of scope for furtherresearch to develop better methods of immobilization as well as bettercarriers. Nanotechnology is one of the more promising fields thatattempts to provide an efficient substrate. Identification of effectiveimmobilization techniques on appropriate supports could answerseveral pressing questions that challenge industrial developmentand human welfare, viz., bringing down the costs of food products,coming up with more efficient disease detection kits, strategies toclean up contaminated water bodies and even finding a way to cure/control cancer. The possibilities, opportunities and prospects in thefield of enzyme immobilization are therefore endless.

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Acknowledgements

Financial assistance from Science and Engineering ResearchBoard (SERB), Department of Science and Technology (DST),Government of India through the research grant (SR/FT/LS-65/2010)and from Council of Scientific and Industrial Research (CSIR),Government of India, through the research grant [38(1387)/14/EMRII]to Dr. Aryadeep Roychoudhury is gratefully acknowledged.

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