Immobilization, enzymes

Enzyme immobilization is a widely accepted technology in industries, which enables reuse of the enzyme and is able to address most of the above issues. 

Over the last few decades, intense research in the area of enzyme technology enabled it to facilitate its practical applications. Among them, the novel technological developments in the field of immobilized enzymes offer the possibility of a wider and more economical exploitation of enzymes for industrial appHcations, waste treatment, medicine, and in the development of devices hke biosensor for bioprocess monitoring.  

Immobilized enzymes are defined as enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, which can be used repeatedly and continuously. This definition is applicable to the enzymes as well as aU types of biocatalysts such as cellular organelles, microbial cells, plant cells, and animal cells. In some cases, these biocatalysts are bound to or within insoluble supporting materials (carriers) by chemical or physical binding. In other cases, biocatalysts are free, but confined to limited domains or spaces of supporting materials (entrapment). 

Techniques for the immobilization have been broadly classified into four categories, namely, entrapment, covalent binding, cross-linking, and adsorption and the combination of one or more of these physical techniques together with chemical conjugation techniques. It must be emphasized that in terms of economy of a process, its productivity is important, which is the activity integrated over the operational time. 

Biocatalysts can be immobilized using either the isolated enzymes or the whole cells. Immobilization of whole cells is an easier alternative to immobilization of isolated enzymes due to operational facility. But, at the same time, immobilized cells show lower catalytic activity compared with immobilized enzymes. 

Immobilized enzymes are often more stable than the soluble counterparts and can be recycled. They are also removable from a reaction mixture. PHA synthase fusions with the enzyme of interest were successfully developed and enabled production of recombinant enzyme already attached to a support material (PHA) in one step. The fl-galactosidase LacZ [21] was the first immobilized enzyme followed by alpha-amylase, organophosphohydrolase, and enzymes involved in sialic acid synthesis [76-78]. These demonstrated applicability PHA-immobilized enzymes in food processing, biomass conversion, bioremediation, and fine chemical synthesis. 

Many of the fairly large number of enzyme thermistor biosensors reported so far have been used for the determination of biological substrates or, to a much lesser extent, inorganic substrates. Experimental set-ups similar to that depicted in Fig. 3.22.C were used to determine the substrates listed in Table 3.3, which also gives the primary enzymes and any auxiliary enzymes or reagents employed to improve the determination [158]. 

Analyte Main enzyme Auxiliary enzyme or reagent (effect) 

Alcohols Alcohol oxidase Catalase (increases stability of main enzyme on immobilization) 

Ascorbic acid Ascorbate oxidase Concanavalin A-conjugated Sepharose (facilitates enzyme replacement or renewal) 

Cephalosporins Cephaiosporinase /3-Lactamase (enhances activity of main enzyme) 

The fact that an enzyme can coexist in various oligomeric forms is of major importance for its catalytic expression. Enzyme immobilization is a technique of significant practical utility, especially when used to enhance catalytic potential, resistance to pH and temperature, and continued reusability. Chitosan is known to be an excellent base material for the immobilization of several carbohydrate degrading enzymes, because it exhibits increased thermostability 

Although not associated with the delivery of proteins, multiple emulsions or liquid membranes have been utilized for enzyme immobilization in place of solid-membrane methods or conventional methods (Scheper et al, 1987 May and Li, 1972). Multiple-emulsion systems have proved useful because there is no membrane fouling, they can be utilized in cell-free fermentation broths, and enzyme inhibitors can be excluded. 

Multiple emulsions are unique in that a true liquid phase is maintained separate from an external aqueous phase. This may be especially important for bioactive molecules that cannot be appropriately stabilized in the solid state. In addition, the separation of aqueous phases enables highly specialized environments, conducive to protein activity, to be prepared. The physical instability of conventional systems remains a major factor limiting their wider application. Attempts to improve the physical stability of the aqueous dispersions through interfacial complexation and the use of microemulsions are improving the short-term stability. As an alternative approach, solid-state emulsions attempt to store the multiple emulsion as a solid. Although solid-state emulsions appear to have the potential to be useful protein delivery systems, a substantial experimental data base has yet to be generated. 

Aitken, I. D., 1973, The serological response of the chicken to a protein antigen in multiple emulsion oil adjuvant, Immunology 25 957-966. 

Blackall, P. J., Eaves, L. E., Rogers, D. G., and Firth, G., 1992, An evaluation of inactivated infectious Coryza vaccines containing a double-emulsion adjuvant system. Avian Dis. 36 632-636. 

Brodin, A. F., Kavaliunas, D. R., and Frank, S. G., 1978, Prolonged release fiom multiple 

A list of entrapped enzymes is given in Table 3.2. There are two endo-l,3-P-D-glucanases from marine mollusks Spisula sacchalinensis and Ch. albidus and a-D-galactosidase from marine bacterium Pseudoalteromonas sp. MM 701. They were selected for immobilization for the following reasons  

The enzymes differ significantly in their optimal conditions for enzymatic activity. These include pH, ionic strength and temperature. The variety of conditions enabled us to verify if there could be limits to the approach. 

Endo-1,3-P-D-glucanase Le is a highly labile enzyme. It is especially sensitive to temperature, denaturating at 25 °C. The common approaches were unsuitable for its immobilization. 

5 One-Stage Approach Based on a Silica Precursor with Ethylene Glycol Residues 101 

The main advantage is that the entrapment conditions are dictated by the entrapped enzymes, but not the process. This includes such important denaturing factors as the solution pH, the temperature and the organic solvent released in the course of precursor hydrolysis. The immobilization by THEOS is performed at a pH and temperature that are optimal for encapsulated biomaterial [55,56]. The jellification processes are accomplished by the separation of ethylene glycol that possesses improved biocompatibility in comparison with alcohols. 

The surface of PDMS is hydrophobic which results in poor wettability with aqueous solvents and promotes non-specific protein adsorption. It is also relatively inert to chemical modification [25]. The liquid silicon rubber chosen for fabrication of the reaction plate contained pyrogenic silicic acid as a filler. Aside from its effect on elastomer properties the silicic acid can be expected to provide additional silanol 

The silanized plate was then derivatized with glutardialdehyde solution. In the final step, the pre-activated plate was incubated with enzyme solution, typically containing about 0.1 mg protein/mL A full description of the protocol used has been pubhshed elsewhere [21]. Enzyme immobUization on the GPMR plate took place by an analogous procedure with the exception that a pH of 7.0 was used during the sUanization step to prevent destruction of the wash oat layer of y-aluminum oxide [22]. 

ImmobUization experiments involved recombinant 5-glycosidase CelB from P. Juriosus produced in Escherichia ccAi. A partially purified preparation obtained from the soluble protein fraction of a heat-treated E. coli cell was employed [7]. The specific activity of CelB towards lactose was about 800units/mg protein at 80 °C. We were able to immobUize about 60 pg and up to 100 pg of CelB on the PDMS and CPMR plates, respectively. 

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Enzyme catalysis Enzyme electrode Enzyme immobilization Enzyme immunoassay Enzyme inhibitors... 

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

Immobilized Enzymes. The immobilized enzyme electrode is the most common immobilized biopolymer sensor, consisting of a thin layer of enzyme immobilized on the surface of an electrochemical sensor as shown in Figure 6. The enzyme catalyzes a reaction that converts the target substrate into a product that is detected electrochemicaHy. The advantages of immobilized enzyme electrodes include minimal pretreatment of the sample matrix, small sample volume, and the recovery of the enzyme for repeated use (49). Several reviews and books have been pubHshed on immobilized enzyme electrodes (50—52). 

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. 

Methods of immobilization have already been discussed, and various reactor configurations are possible. An enzyme immobilized on... 

The reactivation of enzymes (after their partial inactivation in an acid medium) upon passing into a medium of pH 8 is also of great importance for oral use (Fig. 25). Enzymes immobilized in crosslinked polyelectrolytes are characterized by a structural memory even after considerable inactivation. Under changed conditions, this leads to a considerable or almost complete reactivation of the enzyme, whereas in the reactivation of a free enzyme in solution under similar conditions the enzymatic activity is restored on a lower level. 

Enzyme immobilization 34-35, 151 2,3-Epoxypropyl methacrylate 161, 162 ESR spectra 141 Excluded volume 136, 139, 152... 

The nonphosphorylated compounds may be readily obtained by chemical or, preferentially, by mild phosphatase hydrolysis of the 1-phosphates. Reactor design17 and enzyme immobilization techniques18 have been evaluated for efficient practical syntheses. Owing to the narrow specificity... 

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

FIGURE 4 Activity of glucose-6-phosphate dehydrogenase as a function of time for ( ) the enzyme immobilized on the polyphosphazene/ alumina support and (o) in the presence of free enzyme and non-activated support. (From Ref. 23.)... 

Kokufuta, E Jinbo, E, A Hydrogel Capable of Facilitating Polymer Diffusion through the Gel Porosity and Its Application in Enzyme Immobilization, Macromolecules 25, 3549, 1992. Kresge, CT Leonowicz, ME Roth, WJ Vartuli, JC Beck, JS, Ordered Mesoporous Molecular Sieves Synthesized by a Liquid-Crystal Template Mechanism, Nature 359, 710, 1992. 

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. 

Enzyme immobilization methods have been extensively reviewed and can be classifled into three categories ... 

Klotzbach T, WattM, Ansari Y, Minteer SD. 2006. Effects of hydrophobic modification of chit-osan and Nafion on transport properties, ion-exchange capacities, and enzyme immobilization. J Membrane Sci 282 276-283. 

Some of these difficulties in using enzymes can be overcome by fixing, or immobilizing, the enzyme in some way. A number of methods for enzyme immobilization have been developed. These can be classified as follows. 

METALLOCENE DERIVATIVES RADIATION CROSSLINKING TCNQ DERIVATIVES ENZYME IMMOBILIZATION... 

Quantitative analytical treatments of the effects of mass transfer and reaction within a porous structure were apparently first carried out by Thiele (20) in the United States, Dam-kohler (21) in Germany, and Zeldovitch (22) in Russia, all working independently and reporting their results between 1937 and 1939. Since these early publications, a number of different research groups have extended and further developed the analysis. Of particular note are the efforts of Wheeler (23-24), Weisz (25-28), Wicke (29-32), and Aris (33-36). In recent years, several individuals have also extended the treatment to include enzymes immobilized in porous media or within permselective membranes. The important consequence of these analyses is the development of a technique that can be used to analyze quantitatively the factors that determine the effectiveness with which the surface area of a porous catalyst is used. For this purpose we define an effectiveness factor rj for a catalyst particle as... 

Hilterhaus, L., Minow, B., Muller, J. et al. (2008) Practical application of different enzymes immobilized on Sepabeads. Bioprocess and Biosystems Engineering, 31, 163-171. 

Fig. 1.11 Applications of LDHs as (A) non-viral vector in gene therapy for transfection of DNA to the cell nucleus, and (B) as matrix for enzymes immobilization in the development of biosensors.

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