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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. 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. 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.
Methods of Immobilization: 1. Carrier-Binding : the binding of enzymes to water-insoluble carriers. 2. Cross-Linking : intermolecular cross-linking of enzymes by bi-functional or multifunctional reagents. 3. Entrapping : incorporating enzymes into the lattices of a semi-permeable gel or enclosing the enzymes in a semi-permeable polymer membrane
the amount of enzyme bound to the carrier and the activity after immobilization depend on the nature of the carrier. Some of the most commonly used carriers for enzyme immobilization are polysaccharide derivatives such as cellulose. iii. an increase in the ratio of hydrophilic groups and in the concentration of bound enzymes. According to the binding mode of the enzyme. Physical Adsorption Ionic Binding Covalent Binding i. Physical Adsorption Mode . the carrier-binding method can be further sub-classified into: i. In this method. 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. dextran. ii. and polyacrylamide gel.The carrier-binding method is the oldest immobilization technique for enzymes. results in a higher activity of the immobilized enzymes. agarose. as well as the: • • • • Particle size Surface area Molar ratio of hydrophilic to hydrophobic groups Chemical composition In general.
it has the disadvantage that the adsorbed enzyme may leak from the carrier during use due to a weak binding force between the enzyme and the carrier. multiple salt linkages. Ionic Binding Mode The ionic binding method relies on the ionic binding of the enzyme protein to waterinsoluble carriers containing ion-exchange residues. this method can be both simple and cheap.This method for the immobilization of an enzyme is based on the physical adsorption of enzyme protein on the surface of water-insoluble carriers. The processes available for physical adsorption of enzymes are: • Static Procedure • Electro-deposition • Reactor Loading Process • Mixing or Shaking Bath Loading Of the four techniques. and Van der Waal's forces. Hence. is often observed. A major advantage of adsorption as a general method of immobilizing enzymes is that usually no reagents and only a minimum of activation steps are required. the method bears the greatest similarity to the situation found in natural biological membranes and has been used to model such systems. Adsorption tends to be less disruptive to the enzymatic protein than chemical means of attachment because the binding is mainly by hydrogen bonds. If a suitable carrier is found. ionic strength or even the mere presence of substrate. For commercial purposes the preferred method is Reactor Loading . Another disadvantage is non-specific. the rate will probably decrease depending on the surface mobility of enzyme and substrate. further adsorption of other proteins or other substances as the immobilized enzyme is used. However. if the substance adsorbed is a substrate for the enzyme. This may alter the properties of the immobilized enzyme or. and the conditions are much milder than those needed for the covalent binding method. the most frequently used in the lab is Mixing-Bath Loading. Hence. the ionic binding method causes little changes in the conformation and the active site of . ii. In this respect. pH. The earliest example of enzyme immobilization using this method is the adsorption of beta-D-fructofuranosidase onto aluminum hydroxide. Because of the weak bonds involved. the method causes little or no conformational change of the enzyme or destruction of its active center. desorption of the protein resulting from changes in temperature. Polysaccharides and synthetic polymers having ion-exchange centers are usually used as carriers. The binding of an enzyme to the carrier is easily carried out.
The main difference between ionic binding and physical adsorption is that the enzymes to carrier linkages are much stronger for ionic binding although weaker than in covalent binding. Covalent attachment to a support matrix must involve only functional groups of the enzyme that are not essential for catalytic action. Covalent Binding Mode The most intensely studied of the immobilization techniques is the formation of covalent bonds between the enzyme and the support matrix. The conditions for immobilization by covalent binding are much more complicated and less mild than in the cases of physical adsorption and ionic binding. peptide and alkylation methods according to the mode of linkage. Leakage of enzymes from the carrier may occur in substrate solutions of high ionic strength or upon variation of pH. Therefore. even in the presence of substrate or solution of high ionic strength. However. Therefore. covalent binding may alter the conformational structure and active center of the enzyme. This is because the binding forces between enzyme proteins and carriers are weaker than those in covalent binding.the enzyme. iii. resulting in major loss of activity and/or changes of the substrate. When trying to select the type of reaction by which a given protein should be immobilized. The functional groups that may take part in this binding are listed below: Amino group Carboxyl group Sulfhydryl group Hydroxyl group Imidazole group Phenolicgroup Thiol group Threonine group Indole group This method can be further classified into diazo. the choice is limited by two characteristics: (1) the binding reaction must be performed under conditions that do not cause loss of enzymatic activity. and (2) the active site of the enzyme must be unaffected by the reagents used. Higher activities result from prevention . this method yields immobilized enzymes with high activity in most cases. The covalent binding method is based on the binding of enzymes and waterinsoluble carriers by covalent bonds. the binding force between enzyme and carrier is so strong that no leakage of the enzymes occurs.
The wide variety of binding reactions and insoluble carriers (with functional groups capable of covalent coupling or being activated to give such groups) makes this a generally applicable method of immobilization. 2. As with cross-linking. Hence. There must be ample space between the enzyme and the backbone. This provides alternative reaction sites to those essential for enzymatic activity. This is true even if very little is known about the protein structure or active site of the enzyme to be coupled. covalently linked enzyme-inhibitor complex. A zymogen precursor. A number of protective methods have been devised: • • • • Covalent attachment of the enzyme in the presence of a competitive inhibitor or substrate. Amide bond formation : SUPPORT--CO-NH--ENZYME Alkylation and Arylation: SUPPORT--CH2-NH-ENZYME SUPPORT--CH2-S--ENZYME Schiff's base formation : SUPPORT--CH=N--ENZYME Amidation reaction : SUPPORT--CNH-NH--ENZYME Thiol-Disulfide interchange : SUPPORT--S-S--ENZYME Mercury-Enzyme interchange Gamma-Irradiation induced coupling Carrier binding with bi-functional reagents : SUPPORT-O(CH2)2 N=CH(CH2)3 CH=N-ENZYME The active site of the enzyme must not be hindered. A reversible. Cross-Linking .of inactivation reactions with amino acid residues of the active sites. A chemically modified soluble enzyme whose covalent linkage to the matrix is achieved by newly incorporated residues. immobilized enzyme derivatives that do not leach enzyme into the surrounding solution. It is possible in some cases to increase the number of reactive residues of an enzyme in order to increase the yield of the immobilized enzyme. covalent binding can be brought about by the following: • • • • • • • • • Diazotization : SUPPORT--N=N--ENZYME. covalent bonding should provide stable.
It is done in such a way as to retain protein while allowing penetration of substrate. Marshall (1973). Entrapping Enzymes The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. cross-linking is best used in conjunction with one of the other methods. and so may lead to significant loss of activity. for example. Generally. It is used mostly as a means of stabilizing adsorbed enzymes and also for preventing leakage from polyacrylamide gels. as some of the protein material will inevitably be acting mainly as a support. 3. It can be classified into lattice and micro .Immobilization of enzymes has been achieved by intermolecular cross-linking of the protein. 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. These harsh conditions can change the conformation of active center of the enzyme. very little desorption is likely using this method. Since the enzyme is covalently linked to the support matrix. Cross-linking an enzyme to itself is both expensive and insufficient. The most common reagent used for cross-linking is glutaraldehyde. Cross-linking reactions are carried out under relatively severe conditions. This will result in relatively low enzymatic activity. either to other protein molecules or to functional groups on an insoluble support matrix..
Types: i. Therefore. Lattice-Type entrapment involves entrapping enzymes within the interstitial spaces of a cross-linked water-insoluble polymer. 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. Then the same hydrophilic monomer is added to the organic solvent by stirring. Liquid Drying: In this process. An aqueous mixture of the enzyme and hydrophilic monomer are emulsified in a water-immiscible organic solvent. An aqueous • . ii.capsule types. Microcapsule-Type entrapping involves enclosing the enzymes within semi permeable polymer membranes. careful selection of the most suitable conditions for the immobilization of various enzymes is required. The conditions used in the chemical polymerization reaction are relatively severe and result in the loss of enzyme activity. etc. The result is that the enzyme in the aqueous phase is enclosed in a membrane of polymer. polyvinylalcohol. This method differs from the covalent binding and cross linking in that the enzyme itself does not bind to the gel matrix or membrane. Some synthetic polymers such as polyarylamide. Polymerization of the monomers then occurs at the interface between the aqueous and organic solvent phases in the emulsion. enzymes are enclosed in semi permeable membranes of polymers. This results in a wide applicability. and natural polymer (starch) have been used to immobilize enzymes using this technique. a polymer is dissolved in a water-immiscible organic solvent which has a boiling point lower than that of water.
or by molding into membrane form.Enzymes that have been immobilized by entrapment in fibers to form enzyme fibers. Maleic Anhydride based Copolymers. . Polyacrylates. Most immobilized enzymes are in particle form for ease of handling and ease of application. The molding is done after the enzymes have been enclosed within semi-permeate membranes of polymer by entrapment. • Tubes . Vinyl and Allyl Polymers. and filters.Enzyme tubes are produced using Nylon and polyacrylamide tubes as carriers. Carbon. and surfactants. The organic solvent in then removed by warming in vacuum. and a secondary emulsion is prepared. This is accomplished by adding another organic solvent which is miscible with the first. 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.Enzyme membranes can be prepared by attaching enzymes to membrane-type carriers. Some organic supports include: Polysaccharides. • Phase Separation: One purification method for polymers involves dissolving the polymer in an organic solvent and re-precipitating it. • Particles .solution of enzyme is dispersed in the organic phase to form a first emulsion of water-in-oil type. Proteins. but which does not dissolve the polymer. The solid supports used for enzyme immobilization can be inorganic or organic . • Fibers . Polystyrenes. The first emulsion containing aqueous micro droplets is then dispersed in an aqueous phase containing protective colloidal substances such as gelatin. Polypeptides. and Polyamides. • Membranes . The form of an immobilized enzyme can be classified into four types: particles.The particle form is described in the above section. tubes. membranes. A polymer membrane is thus produced to give enzyme micro capsules.
1995. Packed bed Reactors.Applications of immobilized enzymes: 1) Analytical applications. Voet.immobilized nitrite hydratase is used.immobilized penicillin acylase is used in the production of ampicillin.immobilized invertase in a packed bed reactor. Immobilized Enzyme. Halsted Press. 6) Preparation of acrylamide. Ichiro Chibata. 2nd edition. 3) Production of amino acids. Immobilized Enzymes for Industrial Reactors. Continuous Flow Stirred Tank Reactor. 7) High Fructose Corn Syrup production. 8) Detection of glucose in blood and urine. 9) Enzyme Reactors: Batch Reactors. Biochemistry . 3. . Continuous Flow Membrane Reactor.Immobilized glucose oxidase for monitoring blood glucose. Donald and Judith.immobilized glucose isomerase and hexokinase is used.immobilized amino acylase is used to resolve racemic mixtures of amino acids. References 1. 4) Production of sucrose-Immobilized raffinase used to remove raffinose and stachyose from soybean milk. 2) Production of antibiotics. Academic Press. Batch Membrane Reactor. Ralph Mesing. 5) Manufacture of golden syrup.. Fluidized Bed Reactor. 1995. John Wiley & Sons Inc. 1978 2. Kodansha Ltd.
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