Proteins/enzymes stabilization enzyme modification

Chemical modification of proteins has been extensively studied over the years to identify which amino acids are involved in catalysis. Much less work has been carried out on its influence on enzyme stability. Chemical modification of proteins may yield stabilization, destabilization or no effect at all. Martinek and Berezin (1978) reported the dependence of the thermostability of chymotrypsin on the degree of alkylation of its amino groups up to 30% alkylation the stability rose slightly at 90% substitution stability increased markedly, with a maximum (110-fold) at 95% stability fell to nearly initial values when 100% amino groups were modified. (With these modifications, the optimum pH of the errzyme can change and one must therefore be cautious in comparing two different... 

Other post-translational modifications of proteins (e.g., phosphorylation) are extremely important mechanisms of regulation of enzyme activity. Very tittle work has been done on the effects of such modifications on enzyme stabilization. 

The properties of many dairy products, in fact their very existence, depend on the properties of milk proteins, although the fat, lactose and especially the salts, exert very significant modifying influences. Casein products are almost exclusively milk protein while the production of most cheese varieties is initiated through the specific modification of proteins by proteolytic enzymes or isoelectric precipitation. The high heat treatments to which many milk products are subjected are possible only because of the exceptionally high heat stability of the principal milk proteins, the caseins. 

Other methods of stabilization include chemical or carbohydrate modification of enzymes. Modifications of reactive groups on proteins without insolubilization has been used to enhance stability in solution. Grafting of polysaccharides or synthetic polymers, alkalation, acetylation and amino acid modification have all been reported (5)... 

Modification of the proteins by addition of proteolytic enzymes prevents the precipitation of the peptides upon chilling. Proteases from papaya (papain) were already applied in 1911. It is even more remarkable that the major chill stabilizing enzyme used today is still papain. 

There have been a limited number of studies on the effects of enzymic modification of protein concentrates on functional properties other than solubility. Studies on functional properties, as modified by enzymic treatments, emphasize foam formation and emulsifying characteristics of the hydrolysates. Treatment of chicken egg albumen alters the functional properties of the egg proteins in terms of foam volume and stability and the behavior of the proteins in angel food cakes (25). Various proteolytic enzymes were used to degrade the egg albumen partially. However, proteolytic enzyme inhibitors indigenous to the egg proteins repressed hydrolysis of the egg proteins compared with casein. 

The effect of enzymic modification on the foaming and emulsification properties of fish proteins has been studied in several laboratories (8, 14, 28). Hermansson et al. (8) observed that solvent-extracted FPC modified with an alkaline bacterial protease yielded a whipped foam volume approximately 70% greater than untreated FPC. However, the stability of the foam from the enzyme-modified FPC was about 50% less than that of the untreated FPC. The foam volume of the enzyme-modified FPC was essentially equal to that of egg white, but the foam stability of the FPC hydrolysate was substantially less. 

As an application of an oligonucleotide catalyst, we immobilized DNAzyme carrying peroxidase activity with hemin on gold particles using a thiol modification of the DNAzyme s end to detect peroxide 116). Usually, horseradish peroxidase is immobilized on a solid support. Compared with protein enzymes, the DNAzyme is advantageous because of its thermal stability and convenience of preparation. 

The concept of protein modification by PEGylation is now well established clinically (5,136,137), and is used to increase protein solubility and stability, reduce immunogenicity, prevent rapid renal clearance of small proteins, and prevent receptor-mediated clearance by cells of the reticuloendothelial system. Many HPMA copolymer-antibody, -protein, and-peptide conjugates have also been synthesized (Table 2). In the main, protein incorporation has been used to promote cell-specific drug targeting, but conjugates of biologically active proteins, enzymes and coiled-coil peptide domains have also been reported. 

The chemical modification of enzymes to solubilize them in completely nonaqueous, nonpolar media has been attempted with some success. All methods of chemical modification of enzymes share the same principle the attachment of amphipatic reagents to the polar groups on the exterior of the native protein to stabilize the enzyme against denaturation by hydrophobic organic solvents. Depending on the nature of the interaction between the enzyme and the solubilizing agent, chemical modifications of enzymes can be classified into covalent and noncovalent ones. Various methods of enzyme modification adopted are summarized in Table 4.4. 

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