Cell Cross-link

Fig. 5. Correlation between heat response and reaction rate of cephalosporin C transformation by immobilized D-amino acid oxidase of Trigonopsis variabilis. Enzyme immobilization techniques entrapment in polyacrylamide gel ( ), cells cross-linked with glutaraldehyde ( ), cells entrapped in polyacrylamide gel (a) [28]...

Lee, K., Hong, H., Larson, R., Mooney, D.J. Hydrogel formation via cell cross-linking. Advanced Materials 15,1828-1832 (2003)... 

Many of these end products of lipid peroxidation are themselves cytotoxic and mutagenic, and can go on to cause further damage to the cell, cross-linking proteins and causing DNA lesions by reaction with amino acid residues and DNA bases. 

Two types of immobilization are used for immobilizing glucose isomerase. The intracellular enzyme is either immobilized within the bacterial cells to produce a whole-ceU product, or the enzyme is released from the cells, recovered, and immobilized onto an inert carrier. An example of the whole-ceU process is one in which cells are dismpted by homogenization, cross-linked with glutaraldehyde, flocculated using a cationic flocculent, and extmded (42). 

In a second example, a cell—gelatin mixture is cross-linked with glutaraldehyde (43). When soluble enzyme is used for binding, the enzyme is first released from the cell, then recovered and concentrated. Examples of this type of immobilization include binding enzyme to a DEAE-ceUulose—titanium dioxide—polystyrene carrier (44) or absorbing enzyme onto alumina followed by cross-linking with glutaraldehyde (45,46). 

Peptidoplycans (14,16) are the primary component of bacterial cell walls. They consist of a heteropolysaccharide called murein cross-linked with short peptide chains. 

The key to hexavalent chromium s mutagenicity and possible carcinogenicity is the abiHty of this oxidation state to penetrate the cell membrane. The Cr(VI) Species promotes DNA strand breaks and initiates DNA—DNA and DNA-protein cross-links both in cell cultures and in vivo (105,112,128—130). The mechanism of this genotoxic interaction may be the intercellular reduction of Cr(VI) in close proximity to the nuclear membrane. When in vitro reductions of hexavalent chromium are performed by glutathione, the formation of Cr(V) and glutathione thiyl radicals are observed, and these are beHeved to be responsible for the formation of the DNA cross-links (112). 

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. 

En2yme techniques are primarily developed for commercial reasons, and so information about immobilisation and process conditions is usually Limited. A commercially available immobilised penicillin V acylase is made by glutaraldehyde cross-linking of a cell homogenate. It can be used ia batch stirred tank or recycled packed-bed reactors with typical operating parameters as iadicated ia Table 2 (38). Further development may lead to the creation of acylases and processes that can also be used for attaching side chains by ensymatic synthesis. 

Cell wall Peptidoglycan a rigid framework of polysaccharide cross-linked by short peptide chains. Some bacteria possess a lipopolysaccharide- and protein-rich outer membrane. Mechanical support, shape, and protection against swelling in hypotonic media. The cell wall is a porous nonselective barrier that allows most small molecules to pass. 

FIGURE 9.22 (a) The cross-link in Gram-positive cell walls is a pentaglycine bridge. 

Several drugs in current medical use are mechanism-based enzyme inactivators. Eor example, the antibiotic penicillin exerts its effects by covalently reacting with an essential serine residue in the active site of glycoprotein peptidase, an enzyme that acts to cross-link the peptidoglycan chains during synthesis of bacterial cell walls (Eigure 14.17). Once cell wall synthesis is blocked, the bacterial cells are very susceptible to rupture by osmotic lysis, and bacterial growth is halted. 

This thiol-disulfide interconversion is a key part of numerous biological processes. WeTJ see in Chapter 26, for instance, that disulfide formation is involved in defining the structure and three-dimensional conformations of proteins, where disulfide "bridges" often form cross-links between q steine amino acid units in the protein chains. Disulfide formation is also involved in the process by which cells protect themselves from oxidative degradation. A cellular component called glutathione removes potentially harmful oxidants and is itself oxidized to glutathione disulfide in the process. Reduction back to the thiol requires the coenzyme flavin adenine dinucleotide (reduced), abbreviated FADH2. 

Figure 6.8 Pendllins are similar to the bacterial peptidogiycan terminal alanylalanine moiety. Because of this similarity, the enzyme transpeptidase recognizes 8-lactam antibiotics as substrate. As a result of this the 8-lactam is incorporated in the peptide chain thereby making peptide-peptide cross-linking impossible. The occurrence of this phenomenon stops the construction of the bacterial cell wall.

Polyazetidine prepolymer may be cross-linked in aqueous solution by reaction with amine, thiol, hydroxyl, carboxylic add or other polyazetidine groups. Cross-linking occurs upon water removal, heating or by changing to a basic pH. The immobilised cell/polymer composition may be prepared in the form of membranes, fibres, tubes or beads. 

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