Enzymes solid-supported

Immobilized cell Technique used for the fixation of enzymes or cells onto a solid support. 

Porous glass (PG) modified with covalently adsorbed poly(p-nitrophenyl acrylate), as described in Sect. 4.1, turned out to be a highly suitable carrier for immobilization of various biospecific ligands and enzymes. When the residual active ester groups of the carrier were blocked by ethanolamine, the immobilized ligands when bound to the solid support via hydrophilic and flexible poly(2-hydroxyethyl acrylamide). The effective biospecific binding provided by the ligands... 

Since 1978, several papers have examined the potential of using immobilised cells in fuel production. Microbial cells are used advantageously for industrial purposes, such as Escherichia coli for the continuous production of L-aspartic acid from ammonium fur-marate.5,6 Enzymes from microorganisms are classified as extracellular and intracellular. If whole microbial cells can be immobilised directly, procedures for extraction and purification can be omitted and the loss of intracellular enzyme activity can be kept to a minimum. Whole cells are used as a solid catalyst when they are immobilised onto a solid support. 

The field of synthetic enzyme models encompasses attempts to prepare enzymelike functional macromolecules by chemical synthesis [30]. One particularly relevant approach to such enzyme mimics concerns dendrimers, which are treelike synthetic macromolecules with a globular shape similar to a folded protein, and useful in a range of applications including catalysis [31]. Peptide dendrimers, which, like proteins, are composed of amino acids, are particularly well suited as mimics for proteins and enzymes [32]. These dendrimers can be prepared using combinatorial chemistry methods on solid support [33], similar to those used in the context of catalyst and ligand discovery programs in chemistry [34]. Peptide dendrimers used multivalency effects at the dendrimer surface to trigger cooperativity between amino acids, as has been observed in various esterase enzyme models [35]. 

Immobilization by chemical bonding gives strong, irreversible attachments to a solid support. The bonds are normally covalent but they can be electrostatic. Typical supports are functionalized glass and ceramic beads and fibers. Enzymes are sometimes cross-linked to form a gel. Occasionally, enz5anes can be flocculated while retaining catalytic activity. 

For most applications, enzymes are purified after isolation from various types of organisms and microorganisms. Unfortunately, for process application, they are then usually quite unstable and highly sensitive to reaction conditions, which results in their short operational hfetimes. Moreover, while used in chemical transformations performed in water, most enzymes operate under homogeneous catalysis conditions and, as a rule, cannot be recovered in the active form from reaction mixtures for reuse. A common approach to overcome these limitations is based on immobilization of enzymes on solid supports. As a result of such an operation, heterogeneous biocatalysts, both for the aqueous and nonaqueous procedures, are obtained. 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

Babiak P, Reymond JL (2005) A high-throughput, low volume enzyme assay on solid support. Anal Chem 77 373-377... 

The concept of zeolite action was tested in a particular reaction where the enzyme is exposed from the beginning to an acidic environment the esterification of geraniol with acetic acid catalyzed by Candida antarctica lipase B immobilized on zeolite NaA [219]. Lipases have been used for the hydrolysis of triglycerides and due to their ambivalent hydrophobic/hydrophilic properties they are effective biocatalysts for the hydrolysis of hydrophobic substrates [220]. When water-soluble lipases are used in organic media they have to be immobilized on solid supports in order to exhibit significant catalytic activity. 

Butler, J.E. (2000a) Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. Methods 22, 4-23. 

Butler, J.E. (2000b) Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. In Methods in Molecular Medicine Molecular Diagnosis of Infectious Diseases. (J. Decker, and Reischl, eds.), Vol. 94, pp. 333-372. Humana Press, Inc., Totowa, NJ. 

In conventional synthetic transformations, enzymes are normally used in aqueous or organic solvent at moderate temperatures to preserve the activity of enzymes. Consequently, some of these reactions require longer reaction times. In view of the newer developments wherein enzymes can be immobilized on solid supports [183], they are amenable to relatively higher temperature reaction with adequate pH control. The application of MW irradiation has been explored with two enzyme systems namely Pseudomonas lipase dispersed in Hyflo Super Cell and commercially available SP 435 Novozym (Candida antarctica lipase grafted on an acrylic resin). 

Enzymes are usually used in aqueous or organic media and the temperature is limited to 40 °C to preserve enzyme activity as a consequence the reactions need very long times. With enzymes immobilized on solid supports [80] it is possible to operate at higher temperatures. 

Compared with enzymes fewer reports are available on immobilization of antibody (Ab) in sol-gels and their applications in immunosensing. Immobilization of Abs on a solid support was first reported in 1967 [128] and the technology has widespread application in affinity chromatography and other areas. However, the major problem associated with covalent immobilization of antibody on solid surface is partial loss of biological activity due to the random orientation of the asymmetric macromolecules,... 

Immobilized enzymes are attached to a solid support by adsorption or chemical binding or mechanical entrapment in the pores of a gel structure but retain their catalytic power. Their merit is ease of separation from the finished reaction product. 

Targeted libraries have been most effective when based upon the display of diverse functionality about a minimal mechanism-based pharmacophore targeting an enzyme family. Early successes with this approach were first achieved with proteases.1221 Our own efforts to design libraries which target enzyme families require that the minimal pharmacophore serves as the site for attachment to solid support.1231 The pharmacophore is the only invariant part of the inhibitor structure, which... 

Roth and Breaker1331 used in vitro selection to evolve DNA molecules of the type illustrated in Scheme 4, which self-cleave in the presence of histidine. (This is in principle mimicking a disabled mutant enzyme, of the sort mentioned in the introduction.1101) In this case a pool of 2 x 1013 modified DNAs was attached at the 5 -end, tightly but reversibly, to a solid support The polynucleotide was made up of a randomised sequence of 40 deoxynucleotides (N40 in Scheme 4) flanked by two regions of basepairing complementarity to the sequences on either side of a single upstream RNA linkage (rA). 

Glycal assembly on a solid support eliminates the repetitive purifications usually associated with oligosaccharide synthesis. As a method, it has a certain generality as it does not require any specific enzymes or complex starting materials. Both natural and nonnatural sugars may be used in the constructions. 

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