Immobilization, enzyme stabilization

Ugarova, N.N., Rozhkova, G.D. and Berezin, TV. (1978) A physico chemical analysis of soluble and immobilized enzyme stabilization. J. Solid-Phase Biochem., 3, 119-131. 

This field summarizes general information on stability, e.g., increased stability of immobilized enzymes, stabilization by SH-reagents, detergents, glycerol or albumins etc. 

Keywords Multi-analyte biosensor Microelectrode-array Enzyme immobilization Enzyme stability Differential scanning calorimetry (DSC) Chip-calorimeter... 

Immobilized enzyme stability was assayed by using 0.4 g of the immobilized CALB on fiber or 0.01 g of Novozyme 435 in successive batches of methyl butyrate hydrolysis. The operational conditions were the same as described for the assay of hydrolytic activity. At the end of each batch, the immobilized lipase was removed from the reaction medium, washed with phosphate buffer to remove any remaining substrate or product, dried under vacuum (10 min), and assayed again. The residual activity of the biocatalyst was calculated in terms of percentage of activity (U) of the immobilized enzyme measured after each cycle compared with the activity of the immobilized enzyme before the first cycle. 

It is important to realize that biocompatibility issues are not only relevant in respect of the well being of the host, but also in respect of the requirements of the sensor itself. More specifically, the required chemical interactions between the sensor and the body must not be interfere with by interactions—either chemical or physical— between the membrane material and contacting/adhering cells. For example, an encapsulating membrane of an electrochemically based sensor must maintain appropriate mass transport conditions for the analyte and electrolyte species, and must exclude species that could interfere with the electrochemistry, or denature or inhibit the activity of immobilized enzymes. Stability of mass transport conditions is especially critic, since any change in the permeability of the membrane or the surrounding tissues can affect the sensor calibration. Satisfactory stability can not be achieved without a biocompatible encapsulation material. 

Active immobilized enzyme stabilizer and activator of co-immobilized D-glucose oxidase... 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

The success of the enzyme electrode depends, in part, on the immobilization of the enzyme layer. The objective is to provide intimate contact between the enzyme and the sensing surface while maintaining (and even improving) the enzyme stability. Several physical and chemical schemes can thus be used to immobilize the enzyme onto the electrode. The simplest approach is to entrap a solution of the... 

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). 

Solutions of surfactant-stabilized nanogels share both the advantage of gels (drastic reduction of molecular diffusion and of internal dynamics of solubilizates entrapped in the micellar aggregates) and of nonviscous liquids (nanogel-containing reversed micelles diffuse and are dispersed in a macroscopicaUy nonviscous medium). Effects on the lifetime of excited species and on the catalytic activity and stability of immobilized enzymes can be expected. 

All soil metabolic proce.sses are driven by enzymes. The main sources of enzymes in soil are roots, animals, and microorganisms the last are considered to be the most important (49). Once enzymes are produced and excreted from microbial cells or from root cells, they face harsh conditions most may be rapidly decomposed by organisms (50), part may be adsorbed onto soil organomineral colloids and possibly protected against microbial degradation (51), and a minor portion may stand active in soil solution (52). The fraction of extracellular enzyme activity of soil, which is not denaturated and/or inactivated through interactions with soil fabric (51), is called naturally stabilized or immobilized. Moreover, it has been hypothesized that immobilized enzymes have a peculiar behavior, for they might not require cofactors for their catalysis. 

Enzyme electrodes belong to the family of biosensors. These also include systems with tissue sections or immobilized microorganism suspensions playing an analogous role as immobilized enzyme layers in enzyme electrodes. While the stability of enzyme electrode systems is the most difficult problem connected with their practical application, this is still more true with the bacteria and tissue electrodes. 

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

It should be pointed out that the addition of substances, which could improve the biocompatibility of sol-gel processing and the functional characteristics of the silica matrix, is practiced rather widely. Polyethylene glycol) is one of such additives [110— 113]. Enzyme stabilization was favored by formation of polyelectrolyte complexes with polymers. For example, an increase in the lactate oxidase and glycolate oxidase activity and lifetime took place when they were combined with poly(N-vinylimida-zole) and poly(ethyleneimine), respectively, prior to their immobilization [87,114]. To improve the functional efficiency of entrapped horseradish peroxidase, a graft copolymer of polyvinylimidazole and polyvinylpyridine was added [115,116]. As shown in Refs. [117,118], the denaturation of calcium-binding proteins, cod III parvalbumin and oncomodulin, in the course of sol-gel processing could be decreased by complexation with calcium cations. 

In order to overcome some limitations of the adsorption process due to surface accessibility or diffusional hindering, immobilization of enzymes by direct in situ encapsulation has been investigated. When inorganic supports can be prepared in mild conditions compatible with the enzyme stability, then such processes allow... 

Summarizing the results of many investigations, monosaccharides and such derivatives as D-mannitol and D-glucitol are rather weak acceptors. Disaccharides, including such acceptor products as isomaltose, are much better acceptors, except for certain molecules, for instance leucrose, which is not an acceptor.29,46,47 The decrease of enzyme activity with time has been described in terms of a first-order reaction. The inactivation parameters have been calculated for the immobilized enzyme. The inactivation constants kd were 0.0135 (1/d) when maltose was the acceptor (stabilizing), and 0.029 (1/d) when fructose was the acceptor.38... 

Guire, P. (1976) Stepwise thermophotochemical cross-linking agents for enzyme stabilization and immobilization. Fed. Proc. 35, 1632. 

S. Sasso, R. Pierce, R. Walla, and A. Yacynych, Electropolymerized 1,2-diaminobenzene as a means to prevent interferences and fouling and to stabilize immobilized enzyme in electrochemical biosensors. Anal. Chem. 62, 1111-1117 (1990). 

Many enzymes require the participation of dissociable coenzymes such as NAD+, NADP+ or ATP for their catalytic activities. The use of coenzymes to activate immobilized enzymes on a large scale is hampered by their relatively low stability and high cost. Attempts are therefore being made to stabilize the coenzymes and to find suitable means for their continuous regeneration. The principal approach has been to covalently attach a co-enzyme to a polymeric water-soluble matrix, thus making the co-enzyme, like the enzyme, potentially reusable (9,10). 

It is possible to bind enzymes to an insoluble matrix by a variety of methods and still retain their catalytic activity. The reusable nature of immobilized enzymes can significantly reduce costs and provides a convenient source of enzymes for performing substrate assays. Such preparations often show a greater stability and reduced inhibition effects than do soluble enzymes, although occasionally optimum pH values may be altered slightly. 

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