Other Enzymatic Modifications

Initially, the separation of sialic acids was mainly carried out by cellulose chromatography at low temperature [6,237]. However, nowadays HPLC fractionations using different column materials, elution protocols and detection techniques have replaced this approach [6,11,268]. The application of HPLC has also introduced a rapid method for tentative assignments of sialic acids in complex mixtures, based on elution times of known standards, being more reliable when more than one HPLC procedure is followed. Moreover, a rapid method for quantification of released sialic acids has become available. Due to the relatively short HPLC runs, also fast transitions between members of the sialic acid family due to migration of substituents, introduction of substituents, cleavage of substituents, or other (enzymatic) modifications can easily be monitored. 

Several other enzymatic modifications of proteins which probably occur after translation, of a polypeptide chain are listed in Table IX. We shall describe each briefly since none of the modifications would appear at this time to be generally useful for modification of proteins in foods. 

All of the 20 amino acids have in common a central carbon atom (Co) to which are attached a hydrogen atom, an amino group (NH2), and a carboxyl group (COOH) (Figure 1.2a). What distinguishes one amino acid from another is the side chain attached to the Ca through its fourth valence. There are 20 different side chains specified by the genetic code others occur, in rare cases, as" the products of enzymatic modifications after translation. 

Most of the other posttranslational modifications involving the N- or C-terminus (Table 1) as well as the side-chain functionalities (Table 2) of the polypeptide chains occur under the control of enzymes that also dictate the regioselectivity of such chemical transformations. This regioselectivity is difficult to attain by synthetic procedures. Sophisticated protection schemes are required when additional chemistry must be performed on preassembled peptides, unless enzymatic methods can be used to supplement the synthetic strategies. As a consequence, the use of suitably modified amino acids as synthons is generally the preferred approach as will be discussed in the following sections. 

Two general classes of pheromone compound have been identified in moths, and these have some broad, although not uniform, associations with certain taxa. The polyene hydrocarbons and epoxides of various chain lengths are pheromones found in some subfamilies of the Geometridae and Noctuidae, and in the Arctiidae and Lymantridae (Millar, 2000). These compounds are probably derived from dietary Unoleic and linolenic acids. The other major class of pheromone compounds includes acetate, alcohols, and aldehydes, which are found in the Tortrici-dae, Pyralidae, Gelechiidae, Sessiidae, and Noctuidae. This class of compounds is derived from the insect s fatty acid synthesis pathway, with enzymatic modifications discussed above. Both classes of pheromone are broadly represented in the Noctuidae but are typically found in different subfamilies (Am et al., 1992,2003). 

It is essential to consider the physico-chemical properties of each WPC and casein product in order to effectively evaluate their emulsification properties. Otherwise, results merely indicate the previous processing conditions rather than the inherent functional properties for these various products. Those processing treatments that promote protein denaturatlon, protein-protein Interaction via disulfide interchange, enzymatic modification and other basic alterations in the physico-chemical properties of the proteins will often result in protein products with unsatisfactory emulsification properties, since they would lack the ability to unfold at the emulsion interface and thus would be unable to function. It is recommended that those factors normally considered for production of protein products to be used in foam formation and foam stabilization be considered also, since both phenomena possess similar physico-chemical and functionality requirements (30,31). 

Dinitriles and diamides also can sometimes be partially hydrolyzed by use of enzymes. As with other enzymatic reactions, small structural modifications can substantially modify the suitability of a given substrate. Scheme 10.3 gives illustrative examples of the highly substrate-dependent hydrolysis of dinitriles and diamides by use of a microorganism. 

Enzymatic Modification of Proteins for Food Use. Proteolytic enzymes are used extensively for modifying proteins in various ways in food products and for waste management (17, 27. These are used in baked and brewed products, cereals, cheese, chocolate/cocoa, egg and egg products, feeds, fish, legumes, meats, milk, protein hydrolysates and wines. But there are many other uses, and potential uses, of enzymes to modify protein. Whitaker (27, 28), and Whitaker and Puigserver (29) have described more than 100 enzymatic modifications of proteins in vivo, challenging scientists to look at the possibilities of modifying the amino acid side chains by proteolytic and other methods. 

Although whey protein concentrates possess excellent nutritional and organoleptic properties, they often exhibit only partial solubility and do not function as well as the caseinates for stabilizing aqueous foams and emulsions (19). A number of compositional and processing factors are involved which alter the ability of whey protein concentrates to function in such food formulations. These include pH, redox potential, Ca concentration, heat denaturation, enzymatic modification, residual polyphosphate or other polyvalent ion precipitating agents, residual milk lipids/phospholipids and chemical emulsifiers (22). 

The cloning of functional genes from natural microbial consortia is dependent on the high quality of the extracted and purified environmental DNA since the enzymatic modifications required during the cloning steps are sensitive to contamination by various biotic and abiotic components that are present in environmental ecosystems. For example, extraction of DNA from soils always results in coextraction of humic substances, which interfere with restriction enzyme digestion and PCR amplification and reduce transformation efficiency and DNA hybridization specificity [19 -22], Therefore, extraction methods have been developed to remove or minimize contamination of the purified DNA by humic or other interfering substances. Several protocols for the isolation of bacterial community DNA from various environmental samples have been reported in recent years. These methods are based either on re-... 

They contain many unusual bases, typically between 7 and 15 per molecule. Some are methylated or dimethylated derivatives of A, U, C, and G formed by enzymatic modification of a precursor tRNA (Section 28.1.8). Methylation prevents the formation of certain base pairs, thereby rendering some of the bases accessible for other interactions. In addition, methylation imparts a hydrophobic character to some regions of tRNAs, which may be important for their interaction with synthetases and ribosomal proteins. Other modifications alter codon recognition, as will be discussed shortly. 

Of the effective compodnds available at the present time, the )3-lactam antibiotics, the aminoglycosides and the polymyxins are all bacteriocidal. The j8-lactam antibiotics, notably carbenicillin, suffer primarily from the penetration factor but enzyme activity can play an important part. The aminoglycosides, on the other hand, have few or no penetration problems but are subject to enzymatic modification. The polymyxins penetrate to the active site and appear to be resistant to enzymatic modification. However, toxicity problems associated with polymyxin therapy prevent the widespread use of these drugs. 

Enzymatic modification of milk fats with lipolytic enzymes has already been mentioned above. Besides this it is possible to manufacture complex cheese flavours today also by fermentation of raw materials of cheese processing with defined microorganisms. Roquefort and other blue cheese flavours fermented by the mould Penicil-lium Roqueforti are currently in commercial production. 

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