Immobilized enzyme

From Wikipedia, the free encyclopedia

An immobilized enzyme is an enzyme that is attached to an inert, insoluble material such as calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride). This can provide increased resistance to changes in conditions such as pH or temperature. It also allows enzymes to be held in place throughout the reaction, following which they are easily separated from the products and may be used again - a far more efficient process and so is widely used in industry for enzyme catalysedreactions. An alternative to enzyme immobilization is whole cell immobilization.

[edit]Commercial

use

Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include:

Convenience: Minuscule amounts of protein dissolve in the reaction, so workup

can be much easier. Upon completion, reaction mixtures typically contain only solvent and reaction products.

Economical: The immobilized enzyme is easily removed from the reaction

making it easy to recycle the biocatalyst.

Stability: Immobilized enzymes typically have greater thermal and operational

stability than the soluble form of the enzyme.

[edit]Immobilization

of an Enzyme

There are three different ways by which one can immobilise an enzyme, which are the following, listed in order of effectiveness:

Adsorption on glass, alginate beads or matrix: Enzyme is attached to the

outside of an inert material. In general, this method is the slowest among those listed here. As adsorption is not a chemical reaction, the active site of the immobilized enzyme may be blocked by the matrix or bead, greatly reducing the activity of the enzyme.

Entrapment: The enzyme is trapped in insoluble beads or microspheres, such

as calcium alginate beads. However, this insoluble substances hinders the arrival of the substrate, and the exit of products.

Cross-linkage: The enzyme is covalently bonded to a matrix through a chemical

reaction. This method is by far the most effective method among those listed here. As the chemical reaction ensures that the binding site does not cover the enzyme's active site, the activity of the enzyme is only affected by immobility. However, the inflexibility of the covalent bonds precludes the self-healing properties exhibited by chemoadsorbed self-assembled monolayers. Use of a spacer molecule like poly(ethylene glycol) helps reduce the steric hindrance by the substrate in this case.

Enzyme Immobilization
Enzyme engineering is a fast-growing application in the pharmaceutical market. Enzymes are key to new processes because they are environmentally friendly and reduce hazardous waste. Enzymatic reactions can occur under milder conditions, at a faster rate, while being highly specific. Therefore, enzymatic process allows to minimize process steps. Enzymes can be operated in the liquid form or immobilized on various supports. Immobilized enzymes enhance process robustness, allow longer duration of activity of enzymes, and re-use of the same enzymes in multiple cycles. Process design gains flexibility thanks to the different modes of operation: batch or column. Further more, the use of immobilized enzyme eliminate the enzyme separation step from the main process thus simplifying and increasing the overall process yield.

Easy separation from reaction mixture, providing the ability to control reaction times and minimize the enzymes lost in the product. Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme mediated reactions. Ability of enzymes to replace multiple standard chemical steps and provide enatomerically pure products. Fixation Anionic Cationic Examples of immobilized Enzymes Maltose phosphorylase, trehalose phosphorylase Lysozyme (recovery), Cytochrome C, Acylase

Enzyme carrier Amberlite FPA54 Amberlite FPC3500

AmberliteXAD7 HP Adsorption Thermolysin, Penicillin acylase, Lipase, -amylase AmberliteXAD761 Duolite A568 Duolite A7 Adsorption Anionic -amylase, -Galactosidase, Lactase, Papain, Chymotrypsin, Glucoseoxidase, Lipase. Glucose isomerase, Lipase.

Adsorption Trypsin, Aspartase, Aminocylase, RNase, Lactase.

Methods of fixation Three main modes of operation are currently used with enzymes. From simple absorption, ionic to covalent binding. These modes of operation allow a wide range of enzyme and applications to be covered.

Simple adsorption onto a hydrophobic resin. Adsorption onto a resin followed by glutaraldehyde crosslinking. Ionic bonding ( with H+ or OH- form resins). Covalent bonding via -NH2 groups. Via enzyme cofactors such as Fe, Ni, Al etc.

Methods of immobilisation
There are four principal methods available for immobilising enzymes (Figure 3.1): a. b. c. d. adsorption covalent binding entrapment membrane confinement

Figure 3.1. Immobilised enzyme systems. (a) enzyme non-covalently adsorbed to an insoluble particle; (b) enzyme covalently attached to an insoluble particle; (c) enzyme entrapped within an insoluble particle by a cross-linked polymer; (d) enzyme confined within a semipermeable membrane. Carrier matrices for enzyme immobilisation by adsorption and covalent binding must be chosen with care. Of particular relevance to their use in industrial processes is their cost relative to the overall process costs; ideally they should be cheap enough to discard. The manufacture of high-valued products on a small scale may allow the use of relatively expensive supports and immobilisation techniques whereas these would not be economical in the large-scale production of low added-value materials. A substantial saving in costs occurs where the carrier may be regenerated after the useful lifetime of the immobilised enzyme. The surface density of binding sites together with the volumetric surface area sterically available to the enzyme, determine the maximum binding capacity. The actual capacity will be affected by the number of potential coupling sites in the enzyme molecules and the electrostatic charge distribution and surface polarity (i.e. the hydrophobic-hydrophilic balance) on both the enzyme and support. The nature of the support will also have a considerable affect on an enzyme's expressed activity and apparent kinetics. The form, shape, density, porosity, pore size distribution, operational stability and particle size distribution of the supporting matrix will influence the reactor configuration in which the immobilised biocatalyst may be used. The ideal support is cheap, inert, physically strong and stable. It will increase the enzyme specificity (kcat/Km) whilst reducing product inhibition, shift the pH optimum to the desired value for the process, and discourage microbial growth and non-specific adsorption. Some matrices possess other properties which are useful for particular purposes such as ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the biocatalyst by means of magnetic fields), a catalytic surface (e.g. manganese dioxide, which catalytically removes the inactivating hydrogen peroxide produced by most oxidases), or a reductive surface environment (e.g. titania, for enzymes inactivated by oxidation). Clearly most supports possess only some of these features, but a thorough understanding of the properties of immobilised enzymes does allow suitable engineering of the system to approach these optimal qualities. Adsorption of enzymes onto insoluble supports is a very simple method of wide applicability and capable of high enzyme loading (about one gram per gram of matrix). Simply mixing the enzyme with a suitable adsorbent, under appropriate conditions of pH and ionic strength, followed, after a sufficient incubation period, by washing off loosely bound and unbound enzyme will produce the immobilised enzyme in a directly usable form (Figure 3.2). The driving force causing this binding is usually due to a combination of hydrophobic effects and the formation of several salt links per enzyme molecule. The particular choice of adsorbent depends principally upon minimising leakage of the enzyme during use. Although

the physical links between the enzyme molecules and the support are often very strong, they may be reduced by many factors including the introduction of the substrate. Care must be taken that the binding forces are not weakened during use by inappropriate changes in pH or ionic strength. Examples of suitable adsorbents are ion-exchange matrices (Table 3.1), porous carbon, clays, hydrous metal oxides, glasses and polymeric aromatic resins. Ion-exchange matrices, although more expensive than these other supports, may be used economically due to the ease with which they may be regenerated when their bound enzyme has come to the end of its active life; a process which may simply involve washing off the used enzyme with concentrated salt solutions and resuspending the ion exchanger in a solution of active enzyme.

Figure 3.2. Schematic diagram showing the effect of soluble enzyme concentration on the activity of enzyme immobilised by adsorption to a suitable matrix. The amount adsorbed depends on the incubation time, pH, ionic strength, surface area, porosity, and the physical characteristics of both the enzyme and the support.

Table 3.1 Preparation of immobilised invertase by adsorption (Woodward 1985) Support type DEAE-Sephadex CM-Sephadex anion exchanger cation exchanger 0 100 100 75 100 34

% bound at pH 2.5 pH 4.7 pH 7.0

Immobilisation of enzymes by their covalent coupling to insoluble matrices is an extensively researched technique. Only small amounts of enzymes may be immobilised by this method (about 0.02 gram per gram of matrix) although in exceptional cases as much as 0.3 gram per gram of matrix has been reported. The strength of binding is very strong, however, and very little leakage of enzyme from the support occurs. The relative usefulness of various groups, found in enzymes, for covalent link formation depends upon their availability and reactivity (nucleophilicity), in addition to the stability of the covalent link, once formed (Table 3.2). The reactivity of the protein side-chain nucleophiles is determined by their state of protonation (i.e. charged status) and roughly follows the relationship -S- > -SH > -O- > -NH2 > -COO- > -OH >> -NH3+where the charges may be estimated from a knowledge of the pKa values of the ionising groups (Table 1.1) and the pH of the solution. Lysine residues are found to be the most generally useful groups for covalent bonding of enzymes to insoluble supports due to their widespread surface exposure and high reactivity, especially in slightly alkaline solutions. They also appear to be only very rarely involved in the active sites of enzymes. Table 3.2 Relative usefulness of enzyme residues for covalent coupling Residue Aspartate Arginine Cysteine Cystine Glutamate Histidine Lysine Methionine Serine Threonine Tryptophan Tyrosine C terminus N terminus Carbohydrate Others Content + + + + ++ ++ ++ + - ~ ++ - ~ ++ Exposure ++ ++ ++ ++ ++ + ++ ++ ++ Reactivity + ++ + + ++ + + ++ + Stability of couple + + + ++ + + _+ + ++ + - ~ ++ Use + + + ++ + + + -

The most commonly used method for immobilising enzymes on the research scale (i.e. using less than a gram of enzyme) involves Sepharose, activated by cyanogen bromide. This is a simple, mild and often successful method of wide applicability. Sepharose is a commercially available beaded polymer which is highly hydrophilic and generally inert to microbiological attack. Chemically it is an agarose (poly-{ -1,3-D-galactose- -1,4-(3,6-anhydro)-L-galactose}) gel. The hydroxyl groups of this polysaccharide combine with cyanogen bromide to give the reactive cyclic imido-carbonate. This reacts with primary amino groups (i.e. mainly lysine residues) on the enzyme under mildly basic conditions (pH 9 11.5, Figure 3.3a). The high toxicity of cyanogen bromide has led to the commercial, if rather expensive, production of ready-activated Sepharose and the investigation of alternative methods, often involving chloroformates, to produce similar intermediates (Figure 3.3b). Carbodiimides (Figure 3.3c) are very useful bifunctional reagents as they allow the coupling of amines to carboxylic acids. Careful control of the reaction conditions and choice of carbodiimide allow a great degree of selectivity in this reaction. Glutaraldehyde is another bifunctional reagent which may be used to cross-link enzymes or link them to supports (Figure 3.3d). It is particularly useful for producing immobilised enzyme membranes, for use in biosensors, by cross-linking the enzyme plus a noncatalytic diluent protein within a porous sheet (e.g. lens tissue paper or nylon net fabric). The use of trialkoxysilanes allows even such apparently inert materials as glass to be coupled to enzymes (Figure 3.3e). There are numerous other methods available for the covalent attachment of enzymes (e.g. the attachment of tyrosine groups through diazo-linkages, and lysine groups through amide formation with acyl chlorides or anhydrides). (a) cyanogen bromide

[3.1] (b) ethyl chloroformate

[3.2] (c) carbodiimide

[3.3] (d) glutaraldehyde

[3.4] (e) 3-aminopropyltriethoxysilane

[3.5] Figure 3.3. Commonly used methods for the covalent immobilisation of enzymes. (a) Activation of Sepharose by cyanogen bromide. Conditions are chosen to minimise the formation of the inert carbamate. (b) Chloroformates may be used to produce similar intermediates to those produced by cyanogen bromide but without its inherent toxicity. (c) Carbodiimides may be used to attach amino groups on the enzyme to carboxylate groups on the support or carboxylate groups on the enzyme to amino groups on the support. Conditions are chosen to

minimise the formation of the inert substituted urea. (d) Glutaraldehyde is used to cross-link enzymes or link them to supports. It usually consists of an equilibrium mixture of monomer and oligomers. The product of the condensation of enzyme and glutaraldehyde may be stabilised against dissociation by reduction with sodium borohydride. (e) The use of trialkoxysilane to derivatise glass. The reactive glass may be linked to enzymes by a number of methods including the use thiophosgene, as shown. It is clearly important that the immobilised enzyme retains as much catalytic activity as possible after reaction. This can, in part, be ensured by reducing the amount of enzyme bound in non-catalytic conformations (Figure 3.4). Immobilisation of the enzyme in the presence of saturating concentrations of substrate, product or a competitive inhibitor ensures that the active site remains unreacted during the covalent coupling and reduces the occurrence of binding in unproductive conformations. The activity of the immobilised enzyme is then simply restored by washing the immobilised enzyme to remove these molecules.

Figure 3.4. The effect of covalent coupling on the expressed activity of an immobilised enzyme. (a) Immobilised enzyme (E) with its active site unchanged and ready to accept the substrate molecule (S), as shown in (b). (c) Enzyme

bound in a non-productive mode due to the inaccessibility of the active site. (d) Distortion of the active site produces an inactive immobilised enzyme. Nonproductive modes are best prevented by the use of large molecules reversibly bound in or near the active site. Distortion can be prevented by use of molecules which can sit in the active site during the coupling process, or by the use of a freely reversible method for the coupling which encourages binding to the most energetically stable (i.e. native) form of the enzyme. Both (c) and (d) may be reduced by use of 'spacer' groups between the enzyme and support, effectively displacing the enzyme away from the steric influence of the surface. Entrapment of enzymes within gels or fibres is a convenient method for use in processes involving low molecular weight substrates and products. Amounts in excess of 1 g of enzyme per gram of gel or fibre may be entrapped. However, the difficulty which large molecules have in approaching the catalytic sites of entrapped enzymes precludes the use of entrapped enzymes with high molecular weight substrates. The entrapment process may be a purely physical caging or involve covalent binding. As an example of this latter method, the enzymes' surface lysine residues may be derivatised by reaction with acryloyl chloride (CH2=CH-CO-Cl) to give the acryloyl amides. This product may then be copolymerised and cross-linked with acrylamide (CH2=CH-CO-NH2) and bisacrylamide (H2N-CO-CH=CH-CH=CH-CO-NH2) to form a gel. Enzymes may be entrapped in cellulose acetate fibres by, for example, making up an emulsion of the enzyme plus cellulose acetate in methylene chloride, followed by extrusion through a spinneret into a solution of an aqueous precipitant. Entrapment is the method of choice for the immobilisation of microbial, animal and plant cells, where calcium alginate is widely used. Membrane confinement of enzymes may be achieved by a number of quite different methods, all of which depend for their utility on the semipermeable nature of the membrane. This must confine the enzyme whilst allowing free passage for the reaction products and, in most configurations, the substrates. The simplest of these methods is achieved by placing the enzyme on one side of the semipermeable membrane whilst the reactant and product stream is present on the other side. Hollow fibre membrane units are available commercially with large surface areas relative to their contained volumes (> 20 m2 l-1) and permeable only to substances of molecular weight substantially less than the enzymes. Although costly, these are very easy to use for a wide variety of enzymes (including regenerating coenzyme systems, see Chapter 8) without the additional research and development costs associated with other immobilisation methods. Enzymes, encapsulated within small membrane-bound droplets or liposomes (see Chapter 7), may also be used within such reactors. As an example of the former, the enzyme is dissolved in an aqueous solution of 1,6diaminohexane. This is then dispersed in a solution of hexanedioic acid in the immiscible solvent, chloroform. The resultant reaction forms a thin polymeric (Nylon-6,6) shell around the aqueous droplets which traps the enzyme.

Liposomes are concentric spheres of lipid membranes, surrounding the soluble enzyme. They are formed by the addition of phospholipid to enzyme solutions. The micro-capsules and liposomes are washed free of non-confined enzyme and transferred back to aqueous solution before use. Table 3.3 presents a comparison of the more important general characteristics of these methods. Table 3.3 Generalised comparison of different enzyme immobilisation techniques. Characteristics Preparation Cost Binding force Enzyme leakage Applicability Running Problems Matrix effects Large diffusional barriers Microbial protection Adsorption Simple Low Variable Yes Wide High Yes No No Covalent binding Difficult High Strong No Selective Low Yes No No Entrapment Difficult Moderate Weak Yes Wide High Yes Yes Yes Membrane confinement Simple High Strong No Very wide High No Yes Yes

What is an Immobilized Enzyme?

Well, let's review what an enzyme is first. Enzymes are protein molecules which serve to accelerate the chemical reactions of living cells (often by several orders of magnitude). Without enzymes, most biochemical reactions would be too slow to even carry out life processes. Enzymes display great specificity and are not permanently modified by their participation in reactions. Since they are not changed during the reactions, it is cost-effective to use them more than once. However, if the enzymes are in solution with the reactants and/or products it is difficult to separate them. Therefore, if they can be attached to the reactor in some way, they can be used again after the products have been removed. The term "immobilized" means unable to move or stationary. And that is exactly what an immobilized enzyme is: an enzyme that is physically attached to a solid support over which a substrate is passed and converted to product. Right about now you're probably asking yourself, "So, who cares?". Well click here to find out the "Benefits of Immobilization".

It is important to understand the changes in physical and chemical properties which an enzyme would be expected to undergo upon immobilization (sometimes referred to as insolubilization). There are a number of factors that affect the rate of the enzyme's catalytic activities. Changes have been observed in the stability of enzymes and in their kinetic properties due to the micro environment and the product's characteristics.

Methods of Immobilization
When immobilizing an enzyme to a surface, it is most important to choose a method of attachment that will prevent loss of enzyme activity by not changing the chemical nature or reactive groups in the binding site of the enzyme. In other words, attach the enzyme but do as little damage as possible. Considerable knowledge of the active site of the enzyme will prove helpful in achieving this task. It is desired to avoid reaction with the essential binding site group of the enzyme. Alternatively, an active site can be protected during attachment as long as the protective groups can be removed later on without loss of enzyme activity. In some cases, this protective function can be fulfilled by a substrate or a competitive inhibitor of the enzyme. The surface on which the enzyme is immobilized is responsible for retaining the structure in the enzyme through hydrogen bonding or the formation of electron transition complexes. These links will prevent vibration of the enzyme and thus increase thermal stability. The micro environment of surface and enzyme has a charged nature that can cause a shift in the optimum pH of the enzyme of up to 2 pH units. This may be accompanied by a general broadening of the pH region in which the enzyme can work effectively, allowing enzymes that normally do not have similar pH regions to work together.

Carrier-Binding : the binding of enzymes to water-insoluble carriers Cross-Linking : intermolecular cross-linking of enzymes by bi-functional or multi-functional reagents. Entrapping : incorporating enzymes into the lattices of a semi-permeable gel or enclosing the enzymes in a semi-permeable polymer membrane

Carrier-Binding
The carrier-binding method is the oldest immobilization technique for enzymes. In this method, the amount of enzyme bound to the carrier and the activity after immobilization depend on the nature of the carrier. The following picture shows how the enzyme is bound to the carrier:

The selection of the carrier depends on the nature of the enzyme itself, as well as the:

Particle size Surface area Molar ratio of hydrophilic to hydrophobic groups Chemical composition

In general, an increase in the ratio of hydrophilic groups and in the concentration of bound enzymes, results in a higher activity of the immobilized enzymes. Some of the most commonly used carriers for enzyme immobilization are polysaccharide derivatives such as cellulose, dextran, agarose, and polyacrylamide gel. According to the binding mode of the enzyme, the carrier-binding method can be further sub-classified into:

Physical Adsorption Ionic Binding Covalent Binding

Cross-Linking

Immobilization of enzymes has been achieved by intermolecular cross-linking of the protein, either to other protein molecules or to functional groups on an insoluble support matrix.. Cross-linking an enzyme to itself is both expensive and insufficient, as some of the protein material will inevitably be acting mainly as a support. This will result in relatively low enzymatic activity. Generally, cross-linking is best used in conjunction with one of the other methods. It is used mostly as a means of stabilizing adsorbed enzymes and also for preventing leakage from polyacrylamide gels. Since the enzyme is covalently linked to the support matrix, very little desorption is likely using this method. Marshall (1973), for example, reported that carbamy phosphokinase cross-linked to alkyl amine glass with glutaraldehyde lost only 16% of its activity after continuous use in a column at room temperature for fourteen days. The most common reagent used for cross-linking is glutaraldehyde. Cross-linking reactions are carried out under relatively severe conditions. These harsh conditions can change the conformation of active center of the enzyme; and so may lead to significant loss of activity.

Entrapping Enzymes
The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. It is done in such a way as to retain protein while allowing penetration of substrate. It can be classified into lattice and micro capsule types.

This method differs from the covalent binding and cross linking in that the enzyme itself does not bind to the gel matrix or membrane. This results in a wide applicability. The conditions used in the chemical polymerization reaction are relatively severe and result in the loss of enzyme activity. Therefore, careful selection of the most suitable conditions for the immobilization of various enzymes is required. Lattice-Type entrapment involves entrapping enzymes within the interstitial spaces of a cross-linked water-insoluble polymer. Some synthetic polymers such as polyarylamide, polyvinylalcohol, etc...and natural polymer (starch) have been used to immobilize enzymes using this technique. Microcapsule-Type entrapping involves enclosing the enzymes within semi permeable polymer membranes. The preparation of enzyme micro capsules requires extremely well-controlled conditions and the procedures for micro capsulation of enzymes can be classified as:

Interfacial Polymerization Method: In this procedure, enzymes are enclosed in semi permeable membranes of polymers. An aqueous mixture of the enzyme and hydrophilic monomer are emulsified in a water-immiscible organic solvent. Then the same hydrophilic monomer is added to the organic solvent by stirring. Polymerization of the monomers then occurs at the interface between the aqueous and organic solvent phases in the emulsion. The result is that the enzyme in the aqueous phase is enclosed in a membrane of polymer. Liquid Drying: In this process, a polymer is dissolved in a water-immiscible organic solvent which has a boiling point lower than that of water. An aqueous solution of enzyme is dispersed in the organic phase to form a first emulsion of water-in-oil type. The first emulsion containing aqueous micro droplets is then dispersed in an aqueous phase containing protective colloidal substances such as gelatin, and surfactants, and a secondary emulsion is prepared. The organic solvent in then removed by warming in vacuum. A polymer membrane is thus produced to give enzyme micro capsules. Phase Separation: One purification method for polymers involves dissolving the polymer in an organic solvent and re-precipitating it. This is accomplished by adding another organic solvent which is miscible with the first, but which does not dissolve the polymer.

The form an of immobilized enzyme can be classified into four types: particles, membranes, tubes, and filters. Most immobilized enzymes are in particle form for ease of handling and ease of application.

Particles - The particle form is described in the above section. Membranes - Enzyme membranes can be prepared by attaching enzymes to membrane-type carriers, or by molding into membrane form. The molding is

done after the enzymes have been enclosed within semi-permeate membranes of polymer by entrapment. Tubes - Enzyme tubes are produced using Nylon and polyacrylamide tubes as carriers. The polymer tube is first treated in a series of chemical reactions and the enzyme is bound by diazo coupling to give a tube in a final step. Fibers - Enzymes that have been immobilized by entrapment in fibers to form enzyme fibers.

The solid supports used for enzyme immobilization can be inorganic or organic . Some organic supports include: Polysaccharides, Proteins, Carbon, Polystyrenes, Polyacrylates, Maleic Anhydride based Copolymers, Polypeptides, Vinyl and Allyl Polymers, and Polyamides.

Types of Reactors
In an enzyme reactor, the highest specific enzyme activity is desirable. It is considered an added bonus if the support that is used also aides in separation. One approach is to use a molecular sieve as the support and pulse the reactor bed with the alternating passage of substrate solution and water. The result is that bands of unused substrate and product progress down the column. It so happens that the enzymes for which this technique would be useful are also those which in some cases benefit in having the enzyme immobilized on a porous support. For an industrial reactor, it is preferable to use supports that are non-biodegradable such as glass, silica, Celite, Bentonite, alumina, or titanium oxide, if possible. Even the linkages between enzyme and support can be non-biodegradable, as they are in the case of titanium. In some of these supports the physical nature of the surface becomes a major problem. Thus, some supports that form excellent packed beds fail to do so when coated with enzyme. Particles which ideally self-suspend in a fluid bed may form aggregates during use which will require more power to pump through substrate. Many problems were encountered using porous glass supports until someone realized that the glass itself could dissolve. This problem has been eliminated by treatment of the glass surface with zirconium. Many types of reactors have been proposed including the following:

Batch reactors may include: o Stirred Tank for Soluble Enzymes o Stirred Tank for Immobilized Enzymes o Stirred Tank with Immobilized Enzyme Basket Paddles o Stirred Tank with Immobilized Enzyme Basket Baffles o Total Recycle Packed Bed Reactor o Total Recycle Fluidized Bed Reactor

Continuous reactors may include: o Stirred Tank Reactor with Filtration Recovery o Stirred Tank Reactor with Settling Tank Recovery o Stirred Tank Reactor with Immobilized Enzyme Basket Paddles o Stirred Tank Reactor with Ultra filtration Recovery o Packed Bed Reactor (same link as above) o Packed bed with recycle o Flat Bed Reactor o Filter Bed Reactor o Fluidized Bed Reactor, Same but better design (expanded top section)
o

Membrane Reactor using hollow fibers

Advantages of Immobilised Enzymes

Easier purification of the product as the separation of the enzyme beads is not a problem. Easy to recover and recycle the enzymes more economical process. The enzymes remain functional for much longer as it is a gentler process.

Benefits of Immobilizing an Enzyme

There are a number of advantages to attaching enzymes to a solid support and a few of the major reasons are listed below:

Multiple or repetitive use of a single batch of enzymes The ability to stop the reaction rapidly by removing the enzyme from the reaction solution (or vice versa) Enzymes are usually stabilized by bounding Product is not contaminated with the enzyme (especially useful in the food and pharmaceutical industries) Analytical purposes - long 1/2-life, predictable decay rates, elimination of reagent preparation, etc.

ADVANTAGES: 1.ENZYMES ARE NOT PRESENT IN THE PRODUCTS, SO NO NEED OF PURIFICATION. 2. ENZYMES ARE IMMEDIATELY AVAILABLE FOR REUSE. SO A CONTINUOUS PROCESS CAN BE CARRIED OUT. 3. IMMOBILIZED ENZYMES ARE MORE STABLE. DISADVANTAGES: 1. IMMOBILIZATION REQUIRED ADDITIONAL TIME, MORE EQUIPMENT AND MATERIALS. 2. THESE ENZYMES ARE LESS ACTIVE HAS THEY DON'T MIX FREELY WITH SUBSTRATE. 3. ANY CONTAMINATION WILL EFFECT ALL THE WHOLE SYSTEM.