Enzyme surface-modified

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

An alternative strategy to obtain silica immobilised catalysts, pioneered by Panster [23], is via the polycondensation or co-condensation of ligand functionalised alkoxysilanes. This co-condensation, later also referred to as the sol-gel process [24], appeared to be a very mild technique to immobilise catalysts and is also used for enzyme immobilisation. Several novel functional polymeric materials have been reported that enable transition metal complexation. 3-Chloropropyltrialkoxysilanes were converted into functionalised propyltrialkoxysilanes such as diphenylphosphine propyltrialkoxysilane. These compounds can be used to prepare surface modified inorganic materials. Two different routes towards these functional polymers can be envisioned (Figure 3.4). One can first prepare the metal complex and then proceed with the co-condensation reaction (route I), or one can prepare the metal complex after the... 

The pioneering works of Hill and Eddows have opened the way to realize fast and efficient electron transfer of enzymes at the electrode surface. They modified a gold electrode with 4,4 -bipyrydyl, an electron promoter, not a mediator since it does not take part in electron transfer in the potential region of interest, to accomplish rapid electron transfer of cytochrome [1], Their work has triggered intensive investigation of electron transfer of enzymes using modified electrodes [2]. 

As part of a downstream processing sequence, 10 m3 of a process fluid containing 20 kg m 3 of an enzyme is to be concentrated to 200 kg/m3 by means of ultrafiltration. Tests have shown that the enzyme is completely retained by a 10,000 MWCO surface-modified polysulphone membrane with a filtration flux given by ... 

Classical bacterial exotoxins, such as diphtheria toxin, cholera toxin, clostridial neurotoxins, and the anthrax toxins are enzymes that modify their substrates within the cytosol of mammalian cells. To reach the cytosol, these toxins must first bind to different cell-surface receptors and become subsequently internalized by the cells. To this end, many bacterial exotoxins contain two functionally different domains. The binding (B-) domain binds to a cellular receptor and mediates uptake of the enzymatically active (A-) domain into the cytosol, where the A-domain modifies its specific substrate (see Figure 1). Thus, three important properties characterize the mode of action for any AB-type toxin selectivity, specificity, and potency. Because of their selectivity toward certain cell types and their specificity for cellular substrate molecules, most of the individual exotoxins are associated with a distinct disease. Because of their enzymatic nature, placement of very few A-domain molecules in the cytosol will normally cause a cytopathic effect. Therefore, bacterial AB-type exotoxins which include the potent neurotoxins from Clostridium tetani and C. botulinum are the most toxic substances known today. However, the individual AB-type toxins can greatly vary in terms of subunit composition and enzyme activity (see Table 2). 

Figure 1 The mode of action for bacterial AB-type exotoxins. AB-toxins are enzymes that modify specific substrate molecules in the cytosol of eukaryotic cells. Besides the enzyme domain (A-domain), AB-toxins have a binding/translocation domain (B-domain) that specifically interacts with a cell-surface receptor and facilitates internalization of the toxin into cellular transport vesicles, such as endosomes. In many cases, the B-domain mediates translocation of the A-domain into the cytosol by pore formation in cellular membranes. By following receptor-mediated endocytosis, AB-type toxins exploit normal vesicle traffic pathways into cells. One type of toxin escapes from early acidified endosomes (EE) into the cytosol, thus they are referred to as short-trip-toxins . In contrast, the long-trip-toxins take a retrograde route from early endosomes (EE) through late endosomes (LE), trans-Golgi network (TGN), and Golgi apparatus into the endoplasmic reticulum (ER) from where the A-domains translocate into the cytosol to modify specific substrates.

A particular type of biosensor can be developed by putting a membrane in contact with the semi-conducting layer of a field effect transistor. If the membrane incorporates an enzyme adapted to transform a particular analyte (Fig. 19.8), reaction of that enzyme will modify the polarity at the surface of the insulating layer. This will in turn modify the conduction between the source and the collector of the field effect transistor. The current flowing through these two electrodes (source and collector) serves as the signal. 

Various experimental evidence suggests that only 2 or 3 of the 9 tyrosine residues are on the surface of the enzyme (19, 55). Indeed only a part of the tyrosine residues can be easily modified by acetylimidazole at pH 7.5 or by tetranitromethane at pH 8.0 (H. Kasai, K. Takahashi, and T. Ando, unpublished). As enzymes thus modified have catalytic activity, the tyrosine residues that are probably located at the surface of the enzyme do not seem to be essential for activity. Consistent results were also obtained from the modification by fluorodinitrobenzene or by diazo-lH-tetrazole (H. Kasai, K. Takahashi, and T. Ando, unpublished). Especially noteworthy is the derivative, in which one to two tyrosine residues, amino terminal alanine, and one lysine residue were modified with diazo-lH-tetrazole. The derivative was deprived of most of its activity toward RNA but retained about 50% of its activity toward guanosine 2, 3 -cyclic phosphate. This may be explained by some steric hindrance owing to the modification of a tyrosine residue near the active center. 

Sulfite modified enzyme electrode. (2) L-Lactate/L-malate/ sulfite multibiosensor L-lactate dehydrogenase/L-malate dehydrogenase/ sulfite oxidase surface-modified enzyme electrodes/enzymes were deposited on the composite electrodes and covered with a dialysis membrane ... 

One of the key factors in biosensor design is the immobilisation technique used to attach the biorecognition molecule to the transducer surface so as to render it in a stable and functional form. The challenge is to have a stable layer (or layers) of biorecognition molecules that do not desorb from the surface and that retain their activity. Entrapment or encapsulation techniques avoid the chemical changes that usually change the structure of the enzymes and modify their recognition capacity. 

See also - bifunctional mediator, - biofuel cells, -> catalytic current, - catalytic hydrogen evolution, - dye cell, -> enzyme electrodes, -> ferrocene, - glucose sensor, -> indirect and direct electrolysis, and - surface-modified electrodes. 

C5dochrome-c oxidase (COX) is a membrane-bound high-molecular-weight enzyme containing four redox sites (heme a, heme as, Cua and Cub), which is thought to adsorb on gold surfaces modified with a 3-mercaptopropionic acid SAM forming a monolayer (Fcox = 1-5 X 10 mol cm ), in which the... 

Electrode surfaces modified with a multilayered surface architecture prepared by a layer-by-layer repeated deposition of several enzyme mono-layers show a modulated increase of surface-bound protein with a subsequent increase in output current, which is directly correlated with the number of deposited protein layers. The versatility of this approach allows alternate layers of different proteins for the manufacture of electrode surfaces, which are the basis for multianalyte sensing devices with multiple substrate specificities. The surface chemistry used for the manufacture of multilayered electrode surfaces is similar to that previously described for the preparation of affinity sensors, and is based on the stabilization of self-assembled multilayer assemblies by specific affinity interactions, electrostatic attraction, or covalent binding between adjacent monolayers. 

The Lipid I and II building blocks may be further elaborated by many other enzymes that modify the sugars or amino acid chains. Branched peptides are added to the Lipid I and II peptide chains either by enzymes that act in an ATP-dependent fashion similar to the MurC-F ligases [39], or by enzymes that add amino acid residues from aminoacyl tRNA intermediates, such as the S. aureus enzymes FemA, FemB and FemX, which form the pentaglycine bridge (O Fig. 3) [36], and the S. pneumoniae enzymes FemM and FemN, which form an L-Ser-L-Ala or L-Ala-L-Ala dipeptide bridge [34,35]. Lipid II is also the substrate for the sortase enzymes that catalyze the attachment of surface proteins for incorporation into peptidoglycan [40]. 

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