Enzyme immobilization techniques

Choice of Method. Numerous enzyme immobilization techniques have been described in the Hterature comprehensive books on this and related subjects, including industrial appHcations, are available (33—36). The more general techniques and some selection criteria are included herein. 

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. 

Methods based on the inhibitory effect of the analyte and the use of an enzyme thermistor have primarily been applied to environmental samples and typically involve measuring the inhibitory effect of a pollutant on an enzyme or on the metabolism of appropriate cells [162]. The inhibiting effect of urease was used to develop methods for the determination of heavy metals such as Hg(II), Cu(II) and Ag(I) by use of the enzyme immobilized on CPG. For this purpose, the response obtained for a 0.5-mL standard pulse of urea in phosphate buffer at a flow-rate of 1 mL/min was recorded, after which 0.5 mL of sample was injected. A new 0.5-mL pulse of urea was injected 30 s after the sample pulse (accurate timing was essential) and the response compared with that of the non-inhibited peak. After a sample was run, the initial response could be restored by washing the column with 0.1-0.3 M Nal plus 50 mM EDTA for 3 min. Under these conditions, 50% inhibition (half the initial response) was obtained for a 0.5-mL pulse of 0.04-0.05 mM Hg(II) or Ag(I), or 0.3 mM Cu(II). In some cases, the enzyme was inhibited irreversibly. In this situation, a reversible enzyme immobilization technique... 

Stability, duration, sensitivity, interference, and availability of substrates to contact enzymes are the criteria for the success of an enzyme sensor. These criteria depend on sources of enzymes, immobilization techniques, and transducers used. Food matrices are much more complicated than the clinical samples, hence, these criteria become extremely important for the application of the enzyme sensor in food analysis. An extensive list of the response time, detection limits, and stability of biosensors was summarized by Wagner (59). 

Alanine and aspartic acid are produced commercially utilizing enzymes. In the case of alanine, the process of decarboxylation of aspartic acid by the aspartate decarboxylase from Pseudomonas dacunhae is commercialized. The annual world production of alanine is about 200 tons. Aspartic acid is produced commercially by condensing fumarate and ammonia using aspartase from Escherichia coli. This process has been made more convenient with an enzyme immobilization technique. Aspartic acid is used primarily as a raw material with phenylalanine to produce aspartame, a noncaloric sweetener. Production and sales of aspartame have increased rapidly since its introduction in 1981. Tyrosine, valine, leucine, isoleucine, serine, threonine, arginine, glutamine, proline, histidine, cit-rulline, L-dopa, homoserine, ornithine, cysteine, tryptophan, and phenylalanine also can be produced by enzymatic methods. 

Enzymatic reactions are commonly observed or practiced in various kinds of food and biotechnology products. With the goals of reducing operating costs and improving product quality, a number of enzyme immobilization techniques have been developed in recent decades [Woodward, 1985]. The availability of robust membranes, particularly porous inorganic membranes, has improved the enzyme immobilization technology. One type of membrane bioieactors immobilizes enzyme in the membrane pores by dead-end filtration of the enzyme solution. 

Miyahara, Y. Matsu, F. Moriizumi, T. Matsuoka, H. Karube, I. Suzuki, S. Micro enzyme sensors using semiconductor and enzyme-immobilization techniques. Anal. Chem. Symp. Ser. 1983, 17, 501-506. 

Another enzyme immobilization technique uses sol-gel film. Recently, amperometric glucose [74], lactate [75], phenolics [76] and hydrogen peroxide... 

Fig. 5. Correlation between heat response and reaction rate of cephalosporin C transformation by immobilized D-amino acid oxidase of Trigonopsis variabilis. Enzyme immobilization techniques entrapment in polyacrylamide gel ( ), cells cross-linked with glutaraldehyde ( ), cells entrapped in polyacrylamide gel (a) [28]...

The number of radiochemically pure L-amino acids that have been synthesized with a short-lived positron-emitting radionuclide is limited at the present time. However, with the further development of rapid organic synthetic procedures and enzyme immobilization techniques it is expected that this number will increase. Such labeled amino acids will be useful for (i) basic research in determining the metabolic fate of a given amino acid in vivo and (ii) imaging of physiological processes in human organs in various disease states. 

Enzyme immobilization allows a wider use of enzymes in fine chemistfy because it facilitates catalyst reuse and downstream processing of the product and, sometimes, it improves enzyme stability. In spite that enzyme immobilization techniques have been used widely during the last 30 years, very few information can be found about aldolases immobilization. 

Several methods for enzyme immobilization can be found in literature. In our laboratory, we have developed a new enzyme immobilization technique making possible response time of the biosensor much inferior to any of the response times so far reported for a penicillin sensor the combined pH electrode to be coated with the enzyme was left... 

Different FIA procedures have been developed for biotechnology applications, especially for oidine glucose analysis. Some methods use continuous enzymatic reagent consumption, and some use enzyme immobilization techniques. The former are more versatile, easier to assemble, and more robust. The latter are simpler and more economical, but due to the nature of enzyme kinetics, it is difficidt to measure substrate concentrations above Igl without the use of dilution techniques, which may affect the final result. 

Table 1.2 Comparison of different enzyme immobilization techniques...

Enzyme immobilization techniques can be roughly divided in three main categories related to the presence, or absence of carrier, and a hybrid third one (Figure 10.1). In the carrier-type immobilization, the enzyme can be linked to the surface carrier, or entrapped... 

Many enzyme immobilization techniques developed in connection with the preparation of heterogeneous biocatalysts have been applied for the construction of enzyme electrodes as well as for other types of biosensors. The immobilization of enzymes or other biocomponents on different membranes is most frequently realized by crosslinking agents, first of all by glutaraldehyde, and with the addition of bovine serum albumin [169] or other proteins, with l,8-diamino-4-aminomethyloc-tane, [170], etc. 

The fourth and probably most popular enzyme immobilization technique is the entrapment technique. In this case, monomer or low molecular weight water soluble polymers are crosslinked in the presence of the enzyme to entrapment the enzyme into the polymer matrix. This has been done with a variety of redox polymer (osmium and ferrocene-based), as well as sol-gels and other hydrogels. This technique effectively covalently links the enzyme to the electrode surface and minimizes leaching and most of these polymers are hydrogels with facile transport of substrate/product in and out of the film. However, frequently this crosslinking affects specific activity of the enzyme. 

Tran-Minh, C. and Pandey, PC. (1990) Insecticide determination with enzyme electrodes using different enzyme immobilization techniques. Biosens. Bioelectron., 5, 461. 

Enzyme immobilization techniques have been revealed as a powerful tool to enhance most enzyme properties such as sta-bihty, activity, specificity and selectivity, and reduction of inhibition. Several methods had been used including cross-linked enzyme aggregates (CLEAs) (Wang et al. 2011), entrapment, and support-based immobilization (Brady and Jordaan 2009). 

The obtainment of fine powders of PCL, poly(co-laurolactam), and their copolymers has attracted relevant attention in recent years for possible commercial utilizations in several fields. Cosmetic formulations, coating and graphic art applications, protein or enzyme immobilization techniques, rotational molding and sintering processes, and filtration devices in food and beverage industry are the major industrial fields where powdered polyamides are currently applied. Their use as stationary phase in chromatography has also been envisaged and introduced for some specific systems. 

Conditions for achieving efficient DET via enzyme immobilization are dictated partly by materials architecture. Enzyme immobilization techniques may include nonspecific adsorption, covalent linkage, entrapment in conductive polymeric films, association with metal colloids, and encapsulation within porous matrices (see Chapter 11). The simplest method is nonspecific adsorption, but control is limited various noncovalent interactions will yield different orientations of the redox center with respect to the electrode interface and, as a result, inefficient DET. 

The degree of enzyme purity will ultimately affect fuel cell performance, particularly when enzyme preparations are used to form immobilized films on electrode surfaces in DET reactions. Contaminating proteins that do not provide electron transfer effectively foul the electrode. When enzyme immobilization techniques are specific to the enzyme, then enzyme purity may not be as much as an issue, but rarely the immobilization technique is absolutely specific to the cathodic or anodic enzyme. For example, an attractive immobilization strategy is to link a particular enzyme to an electrode via its cofactor (e.g., flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), etc.) [59]. The cofactor is linked to the electrode material first and then the apoenzyme is allowed to naturally bind to the cofactor all other proteins in the enzyme preparation that cannot bind the cofactor remain unbound and can be removed. Enzymes used in fuel cells are not so unique, and proteins in the immobilizing preparation may use the same cofactor but not the same fuel during fuel cell analysis or operation. 

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