Covalent enzyme immobilization methods

The methods for covalent enzyme immobilization have been reviewed extensively in the literature [64,65,80,81]. The functional groups of the enzyme involved in the chemical bonding can be the N-lcrminal and e-amino groups... 

Enzyme immobilization methods are classified as chemical or physical. Chemical methods involve the formation of covalent bonds between functional groups on the... 

Affinity ligands are immobilized through covalent binding to the support material. A dense, stable coverage of the support is desired, and most methods for immobilization use two steps support activation and coupling to the affinity ligand (see Section 4.2 regarding enzyme immobilization methods). 

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

In view of the conductive and electrocatalytic features of carbon nanotubes (CNTs), AChE and choline oxidases (COx) have been covalently coimmobilized on multiwall carbon nanotubes (MWNTs) for the preparation of an organophosphorus pesticide (OP) biosensor [40, 41], Another OP biosensor has also been constructed by adsorption of AChE on MWNTs modified thick film [8], More recently AChE has been covalently linked with MWNTs doped glutaraldehyde cross-linked chitosan composite film [11], in which biopolymer chitosan provides biocompatible nature to the enzyme and MWNTs improve the conductive nature of chitosan. Even though these enzyme immobilization techniques have been reported in the last three decades, no method can be commonly used for all the enzymes by retaining their complete activity. 

It was reported that PEGylated lipase entrapped in PVA cryogel could be conveniently used in organic solvent biocatalysis [279], This method for enzyme immobilization is more convenient in comparison to other types of immobilization that take advantage of enzyme covalent linkage to insoluble matrix, since the chemical step which is time consuming and harmful to enzyme activity is avoided. The application of this catalytic system to the hydrolysis of acetoxycoumarins demonstrated the feasibility of proposed method in the hydrolysis products of pharmaceutical interest and to obtain regioselective enrichment of one of the two monodeacetylated derivatives. 

Covalent linking of protein to insoluble polymers is unsuitable as a method of enzyme immobilization... 

In summary, enzyme immobilization is extremely important in the scale-up of many biocatalytic processes. The preferred method for pharmaceutical production involves covalent binding through cross-linking or attachment to a support. Noncovalent attachment is less attractive, but it is heavily utihzed owing to the commercial availabihty of industrial quantities of some enzymes immobilized using this technique. 

In a second example, Storey et al. demonstrated that one could covalently immobilize amyloglucosidase using hydrophilic prepolymers. A 5-mg/ml solution of the enzyme was mixed with an equal volume of prepolymer. The method was judged superior as a support for enzyme immobilization. The percent activity inuno-bili/ed in the polyurethane foams was 25 1.5%. 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

The covalent attachment of enzymes to water-insoluble carriers is usually the preferred immobilization method for sensor manufacturing. Obviously, the selected procedure should avoid the loss of enzymatic activity and keep the accessibility of the binding site to the substrate molecules. Unfortunately, this is usually not the case and due to the severe conditions of many of these procedures, major activity losses and/or changes on the substrate selectivity are produced during immobilization. Some authors have pointed out that the enzyme activity decreases approximately one fifth per formed bond [66]. 

AOD was successfully immobilized on aminopropyl-functionalized glass beads by covalent bonding through glutaraldehyde with an average retention efficiency of 95.14% (see Table 2). The method used for enzyme immobilization showed good reproducibility with a relative SD of 2.85%. 

In recent years the electrochemistry of the enzyme membrane has been a subject of great interest due to its significance in both theories and practical applications to biosensors (i-5). Since the enzyme electrode was first proposed and prepared by Clark et al. (6) and Updike et al. (7), enzyme-based biosensors have become a widely interested research field. Research efforts have been directed toward improved designs of the electrode and the necessary membrane materials required for the proper operation of sensors. Different methods have been developed for immobilizing the enzyme on the electrode surface, such as covalent and adsorptive couplings (8-12) of the enzymes to the electrode surface, entrapment of the enzymes in the carbon paste mixture (13 etc. The entrapment of the enzyme into a conducting polymer has become an attractive method (14-22) because of the conducting nature of the polymer matrix and of the easy preparation procedure of the enzyme electrode. The entrapment of enzymes in the polypyrrole film provides a simple way of enzyme immobilization for the construction of a biosensor. It is known that the PPy-... 

Chemical immobilization methods. These can be accomplished via the formation of an array between the enzyme and surface active groups or via covalently linked transporters. The advantage is that the enzyme cannot escape to solution the disadvantage is that partial or total enzyme deactivation can occur during immobilization owing to the formation of additional chemical links. 

Physical immobilization methods do not involve covalent bond formation with the enzyme, so that the native composition of the enzyme remains unaltered. Physical immobilization methods are subclassified as adsorption, entrapment, and encapsulation methods. Adsorption of proteins to the surface of a carrier is, in principle, reversible, but careful selection of the carrier material and the immobilization conditions can render desorption negligible. Entrapment of enzymes in a cross-linked polymer is accomplished by carrying out the polymerization reaction in the presence of enzyme the enzyme becomes trapped in interstitial spaces in the polymer matrix. Encapsulation of enzymes results in regions of high enzyme concentration being separated from the bulk solvent system by a semipermeable membrane, through which substrate, but not enzyme, may diffuse. Physical immobilization methods are represented in Figure 4.1 (c-e). 

Covalent immobilization methods rely on functional groups on both the enzyme and the support material for the formation of stable covalent bonds. For this reason, the choice of a support is crucial in that it determines the immobilization chemistry... 

Although four activation steps are required prior to enzyme immobilization, this method possesses the advantages of not only providing selective covalent immobilization through tyrosines, but also of avoiding enzyme-enzyme crosslinks that can result in activity losses.4... 

The behavior of immobilized enzymes differs from that of dissolved enzymes because of the effects of the support material, or matrix, as well as conformational changes in the enzyme that result from interactions with the support and covalent modification of amino acid residues. Properties observed to change significantly upon immobilization include specific activity, pH optimum, Km, selectivity, and stability.23 Physical immobilization methods, especially entrapment and encapsulation, yield less dramatic changes in an enzyme s catalytic behavior than chemical immobilization methods or adsorption. The reason is that entrapment and encapsulation result in the enzyme remaining essentially in its native conformation, in a hydrophilic environment, with no covalent modification. 

Chemical immobilization methods may alter the local and net charges of enzymes, through covalent modification of charged residues such as lysine (NH4), aspartate, and glutamate (COO-). Conformational changes in secondary and tertiary protein structure may occur as a result of this covalent modification, or as a result of electrostatic, hydrogen-bonding or hydrophobic interactions with the support material. Finally, activity losses may occur as a result of the chemical transformation of catalytically essential amino acid residues. 

A number of methods are being tested for enzyme immobilization. The method selected depends on the operating details of the enzyme system employed and the nature of the solvent to be used, which is usually water. Enzyme, or inactivated cells, may be encapsulated in a film, or encased in a gel, which is permeable to both the substrate and product, but not to enzyme [77]. Porous glasses or insoluble polymers such as a derivatized cellulose may be used as a support onto which enzyme is adsorbed. Pendant functional groups of a polymer, such as those of the ion-exchange resins, can be used either to ionically bind the enzyme to the resin active sites or to covalently bond the enzyme to the resin [79]. The enzyme may be bonded to a polymer backbone chain using a bifunctional monomer such as glutaraldehyde to react with enzyme sites that do not affect its catalytic activity [80]. 

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