Covalent enzyme immobilization

Scheme 1 Covalent enzyme immobilization Aminopropyl-modified silica reacts with glutaraldehyde and the N-terminus of the enzyme.

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... 

FIGURE 11.6 Covalent enzyme immobilization via reaction of (a) lysine residues with NHS esters or aldehydes, (b) cysteine residues with maleimide groups, and (c) carhoxyl residue of glutamic acid or aspartic add with amine-functionalized materials via NHS-mediated ester chemistry. 

Longo L, Vasapollo G, Guascito MR, Mahtesta C. New insights fiom X-ray photoelectron spectroscopy into the chemistry of covalent enzyme immobilization, with glutamate dehydrogenase (GDH) on silicon dioxide as an example. Anal Bioanal Chem 2006 385 146 152. 

The increased operation time of enzyme-membrane reactors (EMR) and the formation of larger amount of inulin have both been attributed to the ability of nanoparticles to work as turbulence promoters and supports, which supply an increased surface area for covalent enzyme immobilization. 

Rasor and Tischer (1998) have brought out the advantages of enzyme immobilization. Examples of penicillin-G to 6-APA, hydrolysis of cephalospwrin C into 7-ACA, hydrolysis of isosorbide diacetate and hydrolysis of 5-(4-hydroxy phenyl) hydantom are cited. De Vroom (1998) has reported covalent attachment of penicillin acylase (EC 3.51.11) from E.Coli in a gelatine-based carrier to give a water insoluble catalyst assemblase which can be recycled many times, and is suitable for the production of semi-synthetic antibiotics in an aqueous environment. The enzyme can be applied both in a hydrolytic fashion and a synthetic fashion. 6-APA was produced from penicillin-G similarly, 7-ADCA was produced from desa acetoxycephalosporin G, a ring expansion product of penicillin G. 

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 vitro enzymatic polymerizations have the potential for processes that are more regio-selective and stereoselective, proceed under more moderate conditions, and are more benign toward the environment than the traditional chemical processes. However, little of this potential has been realized. A major problem is that the reaction rates are slow compared to non-enzymatic processes. Enzymatic polymerizations are limited to moderate temperatures (often no higher than 50-75°C) because enzymes are denaturated and deactivated at higher temperatures. Also, the effective concentrations of enzymes in many systems are low because the enzymes are not soluble. Research efforts to address these factors include enzyme immobilization to increase enzyme stability and activity, solubilization of enzymes by association with a surfactant or covalent bonding with an appropriate compound, and genetic engineering of enzymes to tailor their catalytic activity to specific applications. 

Immobilization onto a solid support, either by surface attachment or lattice entrapment, is the more widely used approach to overcome enzyme inactivation, particularly interfacial inactivation. The support provides a protective microenvironment which often increases biocatalyst stability, although a decrease in biocata-lytic activity may occur, particularly when immobilization is by covalent bonding. Nevertheless, this approach presents drawbacks, since the complexity (and cost) of the system is increased, and mass transfer resistances and partition effects are enhanced [24]. For those applications where enzyme immobilization is not an option, wrapping up the enzyme with a protective cover has proved promising [21]. 

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 ... 

A difficult problem in utilizing enzymes as catalysts for reactions in a non-cellular environment is their instability. Most enzymes readily denature and become inactive on heating, exposure to air, or in organic solvents. An expensive catalyst that can be used only for one batch is not likely to be economical in an industrial process. Ideally, a catalyst, be it an enzyme or other, should be easily separable from the reaction mixtures and indefinitely reusable. A promising approach to the separation problem is to use the technique of enzyme immobilization. This means that the enzyme is modified by making it insoluble in the reaction medium. If the enzyme is insoluble and still able to manifest its catalytic activity, it can be separated from the reaction medium with minimum loss and reused. Immobilization can be achieved by linking the enzyme covalently to a polymer matrix in the same general manner as is used in solid-phase peptide synthesis (Section 25-7D). 

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-... 

To test the reusability of the biocatalyst, five sequential reaction cycles with CPO immobilized on SBA-16 of different pore sizes were completed [6]. The authors found that immobilization on material with larger pore, 143 A, improved the reusability of the catalyst. Enzyme immobilized by covalent attachment to silica-based materials retained a higher residual activity after five reaction cycles than the physical approach. 

The amino group can be activated with bifunctional reagents. A commonly applied procedure is the introduction of an aldehyde function by the bifunctional reagent glutardialdehyde. The activated carrier can be used directly for the covalent bonding of the enzyme as shown in Fig. 7. Table 1 summarizes various examples of enzyme immobilization. 

Forccd flow mode. Invertase, an enzyme, can be chemically immobilized to the surfaces of ceramic membrane pores by the technique of covalent bonding of silane-glutaraldehyde [Nakajima et al., 1989b]. The substrate (reactant), during the sucrose conversion process, enters the membrane reactor in a crossflow mode. Under suction from the other side of the membrane, the substrate flows into the enzyme-immobilized membrane pores where the bioconversion takes place. Both the product and the unreacted substrate indiscriminately pass through the membrane pores. Thus, no permselective properties are utilized in this case. The primary purpose of the membrane is to provide a well-engineered catalytic path for the reactant, sucrose. 

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