Spacers, enzyme immobilizations

Nouaimi, M., Moschel, K. and Bisswanger, H. (2001). Immobilization of trypsin on polyester fleece via different spacers. Enzyme Microb. Technol 29 567-574. 

Enzyme Immobilization onto Ultrahigh Specific Surfiace Cellulose Fibers via Amphiphilic (PEG) Spacers and Electrolyte (PAA) Grafts... 

Wang, Y. H. and Y. L. Hsieh (2004). Enzyme immobilization to ultra-fine cellulose fibers via amphiphilic polyethylene glycol spacers. Journal of Polymer Science Part A Polymer Chemistry 42(17) 4289-4299. 

The lower relative activities of the enzymes immobilized without spacer, compared to those of the enzymes immobilized with spacers, suggest that the enzymes undergo greater denaturation when immobilized without spacer than with spacers. This may be explained in terms of structural deformation of the immobilized enzyme molecules as illustrated in... 

Figure 4. The covalently immobilized enzymes without spacer must undergo remarkable deformation in the lower surface concentration region, whereas the enzyme molecules immobilized with spacer may be protected from the heavy structural deformation even in the lower surface concentration region owing to the spacer chain. The increase in the relative activity of the enzyme immobilized without spacer with the increasing surface concentration may be ascribed to a reduced interaction with the substrate, because of high enzyme concentration during the immobilization reaction. In addition, the spacer on the carrier surface probably reduces the steric interference with the substrate binding process, especially toward high molecular weight substrates.

Jervis used porous silica coated with chemisorbed polyacrylhydrazide for immobilization of adenosine monophosphate (AMP) [117]. After periodate oxidation of its ribose residue the ligand was coupled to the carrier and used for isolation of lactate dehydrogenase from rabbit muscle. The specific capacity was 2 mg of protein/g adsorbent with a ligand content of 10 pmol/g, whereas recovery of enzymatic activity after elution was 85%. Hipwell et al. [118] found that for effective binding of lactate dehydrogenases on AMP-o-aminoalkyl-Sepharose the spacer arm length required at least 4 methylene links. Apparently, a macromolecule of polyacrylhydrazide acts itself like an extended spacer arm and thus allow AMP to bind the enzyme. 

Figure 17.6 Redox hydrogel approach to immobilizing multiple layers of a redox enzyme on an electrode, (a) Structure of the polymer, (b) Voltammograms for electrocatalytic O2 reduction by a carbon fiber electrode modified with laccase in the redox hydrogel shown in (a) (long tether) or a version with no spacer atoms in the tether between the backbone and the Os center (short tether). Reprinted with permission fi om Soukharev et al., 2004. Copyright (2004) American Chemical Society.

Although affinity chromatography has not been used directly as an analytical method, it may be modified in the future to produce a viable technique. Leucovorin has been used as an effective spacer in obtaining active samples of dihydrofolate reductase.79 If the enzyme could be immobilized without losing its activity, perhaps it could be used to separate folates. 

The immobilization of enzymes on solid supports is the oldest of new biotechnologies. As for most of the chemical applications, the uses in this field require a multi-step modification. As a first layer a simple silane, mostly APTS, is used. In order to minimize sterical hindrance in the ultimate application reaction, a spacer molecule is often introduced between the active surface group and the immobilizing species. This may either be introduced at once, using a long-chain functional silane, or in a second step, after silanization. 

Affinity chromatography combines the analytical and chemical capacities of chemically bonded stationary phases and immobilized enzymes. Technology and methodology of both techniques are joined in the development of affinity stationary phases. Since steric requirements are even more determining than in simple immobilized enzyme systems, spacer molecules have great importance in these modifications. Commonly used spacer arms are summarized in figure 8.3. 

A reactor module incorporating a piece of the dried membrane coiled around a glass rod with a Vexor mesh spacer was constructed. This module was used to test kinetics of the immobilized pectin lyase and to determine whether it could clarify apple juice on a continuous basis. A reaction mixture containing 0.5% pectin was passed through the module, and the amount of product formed was continuously monitored spectro-photometrically at 235 nm. The reactor was stabilized at a constant activity level by passing sufficient reaction mixture through the module to wash away loosely bound enzyme located near the surface of the mem-... 

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