Enzyme-Modified Protein

Limited digestion of the globular soy proteins (Harosoy variety) with the enzyme rennin affords a modified protein preparation which retains a high molecular weight. The enzyme-modified protein is precipitated and washed with alcohol and subsequently heat-modified. A flavorless product results which is easily dispersed in water and shows excellent functional characteristics. 

The effect of heat denaturation on the native mixed soy 7S and 11S globulins and on the enzyme-modified soy 7S and 11S proteins is shown by the UV difference spectra (Figure 4). The strong negative peak at 232-233 nm indicates rupture of the secondary structure which has occurred in both the native and enzyme-modified protein. The enzyme-modified protein may retain more order and be somewhat more stable to heat denaturation than the native protein. Loss of secondary structure is also apparent in the circular dichroism spectra (Figure 5) with the change in character of the curve below 220 nm. The spectrum resembles that reported by Koshiyama and Fukushima (11) for 7S protein in 0.25% SDS/0.01M tris buffer. 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

Enzyme modified w. SC-PEG (5000) No. ofmPEGs linked per protein molecule % of native activity (Substrate )... 

Enzymes modified with N -carbonyldiimidazole (CDI) include horseradish peroxidase 761 /1-lactamase after nitration and reduction,[771 lysozyme, and urease.[781 Ref. [77] describes how the tyrosine side chain of a protein was nitrated, reduced with dithionite to an amino group, and then treated with CDI or A/-(2,2,2-trifluoro-ethoxycarbonyl)imidazole to give the benzoxazolinonyl alanine moiety ... 

Co-for-Zn substitution in alcohol dehydrogenase from Saccharomyces cerevisiae revealed a 100-fold increase in activity and a higher resistance of the modified protein to the inhibitory action of other divalent transition metals,1208 making the Co-modified enzyme suitable for biotechnological applications. 

Hemoglobin is another heme-containing protein, which has been shown to be active towards PAH, oxidation in presence of peroxide [420], This protein was also modified via PEG and methyl esterification to obtain a more hydrophobic protein with altered activity and substrate specificity. The modified protein had four times the catalytic efficiency than that of the unmodified protein for pyrene oxidation. Several PAHs were also oxidized including acenaphthene, anthracene, azulene, benzo(a)pyrene, fluoranthene, fluorene, and phenanthrene however, no reaction was observed with chrysene and biphenyl. Modification of hemoglobin with p-nitrophenol and p-aminophenol has also been reported [425], The modification was reported to enhance the substrate affinity up to 30 times. Additionally, the solvent concentration at which the enzyme showed maximum activity was also higher. Both the effects were attributed to the increase in hydrophobicity of the active site. 

While partial substitution of egg with emulsifiers is possible there is no viable complete replacement. The most promising egg replacers are enzyme-modified soy proteins. 

Soya Proteins. Early attempts to make albumen substitutes from soya protein also ran into problems. A bean flavour tended to appear in the finished product. A solution to these problems has been found. Whipping agents based on enzyme modified soy proteins are now available. The advantage of enzymatic modification is that by appropriate choice of enzymes the protein can be modified in a very controlled way. Chemical treatment would be far less specific. In making these materials the manufacturer has control of the substrate and the enzyme, allowing the final product to be almost made to order. The substrates used are oil-free soy flakes or flour or soy protein concentrate or isolate. The enzymes to use are chosen from a combination of pepsin, papain, ficin, trypsin or bacterial proteases. The substrate will be treated with one or more enzymes under carefully controlled conditions. The finished product is then spray dried. 

A second approach that can be adopted to overcome the intrinsic requirement for cysteine at the N-terminus of C-terminal fragment utilizes the enzyme subtiligase, a double mutant of subtilisin, which is able to join two unprotected peptides. Thioester-modified proteins were shown to present good substrates of subtiligase [65]. However, although this approach could be potentially useful for general isotope labeling, the efficiency of this process remains to be proven. 

The reader should consult earlier reviews [70, 94, 95] and other chapters in this volume for a detailed discussion of UbL biology and biochemistry. There are two important points for the current discussion. First, the conjugation cascades of UbLs differ from that of ubiquitin chiefly in terms of complexity - there is one conjugating enzyme per UbL, and many fewer E3s. Second, because modifier proteins (including ubiquitin) do not interact strongly with their dedicated E2s (Section 5.6.1), it is believed that El enzymes play the major role in matching E2s with the correct modifier protein (see Ref [96]). 

Post-translational modifications, such as phosphorylation, complex glycosylation, and lipidation, typically occur in eukaryotic organisms. Therefore, their expression in prokaryotic systems like Escherichia coli is difficult. However, it should be noted that via clever engineering and coexpression of specific enzymes, access can be granted to specific lipidated proteins via expression in bacteria, for example, via the expression of A -myristoyltransferase in E. coli Eukaryotic systems that can be used for the expression of post-translationally modified proteins are yeast and Dictyostelium discoidum. Furthermore, lipidated proteins, such as the Rah proteins, can be obtained via purification from tissue sources or from membrane fractions of insect cells that had been infected with baculovirus bearing a Rah gene. ... 

HPMA copolymers are water-soluble biocompatible polymers, widely used in anticancer drug delivery (reviewed in Reference [22]). HPMA copolymers containing reactive groups at side-chain termini were previously used for the modification of trypsin [23], chymotrypsin [23,24], and acetylcholinesterase [25]. The modification dramatically increased the acetylcholinesterase survival in the blood stream of mice and the thermostability of modified enzymes when compared to the native proteins. However, the modification involved multipoint attachment of the copolymers to the substrates, which may cause crosslinking. To modify proteins or biomedical surfaces by one point attachment, semitelechelic polymers should be used. 

Enzymes are proteins that act as biological catalysts. They not only bind molecules, but also provide a special environment in which the molecules are chemically modified. Enzymes cannot promote a reaction that is not energetically favourable, but by binding the reagents in the necessary orientation and in close proximity, they signi-... 

Many bacterial enzymes and proteins, which are modified by the introduction of F-Phe or F-Tryp, have been obtained. Mammalian proteins containing F-Pro or other fluorinated amino acids have also been obtained, either in a direct manner in vivo) or, more efficiently, by the expression of the gene in a bacteria. Thus, trifluoro-methionine has been incorporated by E. coli in the lysozyme of a bacteriophage. Because this enzyme contains three methionines, it has been used to study the interactions of this protein with its ligands by F NMR. ... 

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