Enzyme-based cross-linking

Covalent cross-linking of polypeptides (e.g., enzyme-based cross-linking via lysine-rich segments in the elastin precursor tropoelastin)... 

Figure 5 Selection of common cross-linkers used in molecular imprinting protocols. Both ethyleneglycol dimethacrylate (EDMA) and divinylbenzene (DVB) are very common crosslinkers in molecular imprinting. Other acrylate-based cross-linking monomers conunonly used include the branched cross-linker trimethylolpropane trimethacrylate (TRIM)-[24]. Among the water-soluble cross-linkers, there are phenylene-diacrylamide, V,V-methylene diacrylamide [22], and Z w-acryloylpiperazine [92], which have been used in aqueous systems for the imprinting of, e.g., enzymes.

Enzyme responsiveness in injectable hydrogels has also been used to render the material biodegradable such that it can be removed once it is no longer required. In this case, poly(N-isopropylacrylamide-co-acrylic acid) that included peptide-based cross-links was used as the stimuli-responsive polymer. Hydrogelation was triggered thermally, whereas the enzyme-responsive functionality is used to provide proteolytically degradable sites in the hydrogel (Kim and Healy,2003). 

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. 

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. 

As an example the deactivation of immobilised Pen G acylase, which catalyses the reaction of Pen G to 6-Aminopenicillanic acid and Phenylacetic acid, was studied. This enzyme was covalently bound on an ion-exchanger and cross-linked by glutaric aldehyde. To maintain a high reaction velocity, a neutral pH value (removal of Phenylacetic acid) and therefore the supply of NaOH and stirring for distribution of the base are required. 

Figure 5.12 Hydrolysis of methylparathion to p-nitrophenol. The cross-linked phospholipid-based nanocapsules are permeable to reactant and product, while allowing for the retention of enzyme activity. Reproduced with permission from [92].

Fontes tt al. [224,225 addressed the acid—base effects of the zeolites on enzymes in nonaqueous media by looking at how these materials affected the catalytic activity of cross-linked subtilisin microcrystals in supercritical fluids (C02, ethane) and in polar and nonpolar organic solvents (acetonitrile, hexane) at controlled water activity (aw). They were interested in how immobilization of subtilisin on zeolite could affected its ionization state and hence their catalytic performances. Transesterification activity of substilisin supported on NaA zeolite is improved up to 10-fold and 100-fold when performed under low aw values in supercritical-C02 and supercritical-ethane respectively. The increase is also observed when increasing the amount of zeolite due not only to a dehydrating effect but also to a cation exchange process between the surface proton of the enzyme and the sodium ions of the zeolite. The resulting basic form of the enzyme enhances the catalytic activity. In organic solvent the activity was even more enhanced than in sc-hexane, 10-fold and 20-fold for acetonitrile and hexane, respectively, probably due to a difference in the solubility of the acid byproduct. 

An alternative strategy for co-immobilization of mediator and GOx is based on adsorption of enzyme, cross-linked, as was described for the laccase-based biocatalytic cathodes [30, 37 42], to an osmium-based redox polymer film, on carbon electrodes [1-3, 54],... 

Cyclophosphamide (Cytoxan) is the most versatile and useful of the nitrogen mustards. Preclinical testing showed it to have a favorable therapeutic index and to possess the broadest spectrum of antitumor activity of all alkylating agents. As with the other nitrogen mustards, cyclophosphamide administration results in the formation of cross-links within DNA due to a reaction of the two chloroethyl moieties of cyclophosphamide with adjacent nucleotide bases. Cyclophosphamide must be activated metabofically by microsomal enzymes of the cytochrome P450 system before ionization of the chloride atoms and formation of the cyclic ethylenimmonium ion can occur. The metabolites phosphoramide mustard and acrolein are thought to be the ultimate active cytotoxic moiety derived from cyclophosphamide. 

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