Plastein reaction

The plastein reaction enables peptide fragments of a hydrolysate to join enzymatically through peptide bonds, forming a larger polypeptide of 

The reaction rate is affected by, among other things, the nature of the amino acid residues. Hydrophobic amino acid residues are preferably linked together (Fig. 1.51). Incorporation of amino acid esters into protein is affected by the alkyl chain length of the ester. Short-chain alkyl esters have a low rate of incorporation, while the long-chain alkyl esters have a higher rate of incorporation. This is especially important for the incorporation of amino acids with a short side chain, such as alanine (cf. Table 1.40). 

Enrichment of a protein with selected amino acids can be achieved with the corresponding amino 

The plastein reaction also makes it possible to improve the solubility of a protein, for example, by increasing the content of glutamic acid (Fig. 1.55). A soya protein with 25% glutamic acid yields a plastein with 42% glutamic acid. 

1) Zein hydrolyzate 2) Trp-plastein 3) Thr-plastein 4) Lys-plastein 5) Ac-Lys-plastein 6) Control without addition of amino acid ethyl esters. 

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Pish silage prepared by autolysis of rainbow trout viscera waste was investigated as a substrate for the plastein reaction using pepsin (pH 5.0), papain (pH 6—7), and chymotrypsin (pH 8.0) at 37°C for 24 h (152). Precipitation with ethanol was the preferred recovery method. Concentration of the protein hydrolysate by open-pan evaporation at 60°C gave equivalent yields and color of the final plastein to those of the freeze-dried hydrolysate. 

Currently available proteins are all deficient to greater or lesser extent in one or more of the essential amino acids. The recently advanced plastein reaction (229) has made it possible to use protein itself as substrate and to attach amino acid esters to the protein with high efficiency. By this method, soy bean protein (which is deficient in methionine) has been improved to the extent of having covalently attached L-methionine at 11%. 

The plastein reaction usually involves two steps hydrolysis of protein and resynthesis of peptide links. Yamashita et al. (20) found similar BV, digestibility, and weight gain for denatured soy meal and soy plastein. This same group (49,50) had described a one-step process by which amino acids may 5 enzymatically incorporated in intact protein to improve protein quality. With soy protein they applied a racemic mixture of D,L-methionine ethyl ester and were able to enzymatically incorporate L-methionine. As Schwimmer (51) has pointed out, one expects the methionine so incorporated to be highly available due to its location at the end of the polypeptide chains. 

The protease-catalyzed synthesis of peptide bonds is known as the plastein reaction ( ). Plastein itself is defined as the product formed by this reaction which is insoluble in trichloroacetic acid solutions 6). The plastein reaction has been most extensively investigated by researchers in Japan (, ... 

Z 2)- These scientists have reviewed various aspects of plastein and the plastein reaction and its importance to protein functionality and nutriture (6, JO). 

The conditions necessary Tor the plastein reaction have been reviewed by Fujimaki et al. ( ), and compared to those necessary for proteolysis by Arai et al. ( ). The substrate for the synthetic reaction must consist of low molecular weight peptides, preferably in the tetramer to hexamer range. These are usually produced from proteins by protease action. A number of proteolytic enzymes and protein substrates have been investigated for producing plastein reaction substrates. The most often used proteases are pepsin JJ), and papain (12,13), but others... 

Perhaps the most controversial aspect of the plastein reaction has been the molecular weight of resulting products. Early work... 

Figure 5. Scheme for removing impurities from protein substrate by hydrolysis, purification, and resynthesis via the plastein reaction (i)... 

Another major application of the plastein reaction is nutritional improvement of proteins by the incorporation of limiting amino acids (8). A plastein containing approximately 7% methionine was produced from soy protein hydrolyzate and L-methionine ethyl ester in the presence of papain. This material was shown to be utilized as a source of methionine in the rat, producing a PER of 3.38 when incorporated into soy protein diets to give a methionine level of 2.74% of protein. 

Fujimaki (77,78) condensed bitter soybean protein hydrolysates in a Plastein-Reaction (79) and obtained non-bitter protein-like products, unfortunately without determination of molecular weights. 

Plastein Formation. Plastein formation is another example of using proteases to modify high-protein food systems to drastically change the properties of that system (II). In the plastein reaction a protease such as papain is used to partially hydrolyze the proteins to about a 10,000-20,000-dalton size at a pH near neutrality. After concentrating the hydrolyzate to 35% (based on protein) and a change in pH, the same protease or a different one is used to catalyze the resynthesis of a few peptide bonds. This may result in a decrease in the solubility of the protein. 

Some potential uses of the plastein reaction are given in Table V. The plastein reaction has been proposed for removing bitter peptides formed through previous hydrolysis of proteins by facilitating the resyn-... 

More details of the plastein reaction and its application to remove pigments such as chlorophyll, or to remove off-flavor components such as the beany taste of soybeans, are shown in Figure 2. The protein of the food system is solubilized and denatured (in order to achieve proteolysis), a protease is added, and the hydrolytic reaction is allowed to proceed. On partial hydrolysis of the protein the pigments and flavor constituents are released from the protein they are removed, the hydrolyzate is concentrated, and resynthesis and/or rearrangement of the amino acid sequence of the polypeptides is catalyzed by the same or a different protease. Resynthesis also can be carried out in the presence of added amino acid esters in order to improve the nutritional/functional properties of the protein. 

The mechanism of the plastein reaction is shown by the equation on page 64. 

Table V. Some Potential Uses of the Plastein Reaction in High-Protein Foods...

Most of the experiments on incorporating amino acid esters into proteins during the plastein reaction have been carried out with papain, indicating that it is one of the best enzymes for this purpose. Other enzymes such as chymotrypsin (40) or carboxypeptidase Y from Sac-charomyces cerevisiae (41) are potent catalysts for peptide synthesis in homogeneous systems using N-acylamino acid esters of peptides as substrates and amino acid derivatives or peptides as nucleophile components. Adding organic co-solvents favored peptide bond synthesis (42,43). 

Following protein hydrolysis, an additional reaction can be carried out to obtain plastein, a water-soluble, heat stable antioxidant. Plastein is a mixture of higher molecular weight protein-like substances formed by the condensation of lower-molecular weight peptides, such as protein hydrolysates, during the plastein reaction (Yamashita et al, 1971). A study conducted by Ono et al. (2004) assessed the production of plastein from squid hepatopancreas and indicated its high potential as a commercial antioxidant due to its thermostability, tastelessness, and high solubility in water. 

Yamashita, M., Arai, S., Tsai, S. J., and Fujimaki, M. 1971. Plastein reaction as a method for enhancing the sulfur-containing amino acid level of soybean protein. J. Agric. Food Chem., 19,1151. 

Endopeptidases. Our expanding understanding of the relationship between structure and functionality of food proteins presents the opportunity for designing functionality into proteins by selective, specific proteolytic modification. Control of reaction and prevention of autolysis offered by immobilization are essential to establish the conditions for a highly selective modification. Hydrolysis at specific positions in the primary structure of proteins could be coupled with resynthesis of peptide bonds by selection of conditions, for example, as in the plastein reaction. By careful choice of enzymes and conditions according to the characteristics of the substrate proteins, it may be possible to design new structures from known food proteins. 

Hydrolytic Alkaline Acid Enzymatic (plastein reaction)... 

Partial proteolysis has been used by several researchers to improve functional properties, i.e. foaming, solubility of proteins (7,8,9). The significant problems associated with enzyme hydrolysis of proteins are excessive hydrolysis occurring under batch conditions, the generation of bitter flavors during hydrolysis and the cost of enzymes. Extensive information on factors affecting proteolysis of proteins and the problem of bitterness has been reviewed by Fujimaki et al. (7) in conjunction with studies of the plastein reaction. 

Enrichment of proteins in specific a.a. can be achieved in the plastein reaction, i.e., protease-catalyzed transpeptidation in concentrated solutions of a.a. ethyl esters and protein hydrolyzates (Figure 7.8). Incubation of a protein hydrolyzate, concentrated to 30-40%, with ethyl esters of Lys, Met, or Trp, e.g., with an appropriate endopeptidase at pH 4-7 at about 37°C, leads after a few days to accumulation of peptides of 2-3 kDa enriched in the respective a.a. residues. Plasteins free of Phe residues can also be obtained for phenylketonuric patients. The rate of incorporation of a.a. into the plastein increases with the hydrophobicity of the a.a. Thus selective removal of hydrophobic a.a. from the hydrolyzate and decrease of its bitter taste are possible. 

The nutritional quality of a protein can be increased by the plastein reaction (46). Following partial hydrolysis of a protein by pepsin, the ethyl ester of a limiting amino acid such as methionine or cystine, or a partial hydrolysate of another protein which is limiting in another amino acid residue, can be added to the hydrolysate and covalently linked through plastein formation. 

Fish protein concentrate, although a good protein source nutritionally, is very insoluble this limits its use in foods. The protein can be solubilized by proteolytic enzymes, but the hydrolysate is bitter (12). It would be useful to investigate the plastein reaction for removal of the bitter taste as well as for changing the solubility properties of the digest (14). The plastein reaction is described more fully in Chapter 6 of this monograph. 

Mechanism of Peptide Bond Resynthesis. Peptide bond resynthesis can occur by either transpeptidation or condensation. It is likely that in plastein reactions both reactions occur to variable extents depending on the experimental conditions and the nature of the peptides available. Below, we examine some of the features of each of these reactions. 

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