Plastein formation

Pallavicini et al. (16) utilized a-chymotrypsin immobilized on chitin to catalyze plastein formation from leaf protein hydrolyzates. When analyzed by gel exclusion chromatography, the products were comparable to those produced by soluble enzymes. Modification of Specific Functional Properties... 

Preparation of cheeses and soy derivatives Solubilization of protein concentrates Production of protein hydrolysates Gluten modification in bread doughs Chillproofing of beer Plastein formation Tenderization of meats Quality determination of proteins... 

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. 

Enzyme-catalyzed attachment of amino acids to proteins represents an attractive and interesting way for improving the nutritional value of food proteins. The enzymes that participate in the gastrointestinal digestion of food proteins catalyze exclusively hydrolytic reactions under physiological conditions. However the synthetic activity of proteolytic enzymes was reported first by Danilewski in 1886, and more recently a number of studies have been devoted to plastein formation from con-... 

Legumes Hydrolyzed protein products. Removal of flavor. Plastein formation. 

Of the many functions of proteolytic enzymes listed in Table I, the most extensively used commercially are chillproofing of beer, production of cheese, tenderization of meats, and production of protein hydrolysates. Two of the most active research areas at the moment include use of proteolytic enzymes for plastein formation (see Proteolytic-Induced Aggregation of Proteins, p. 99) and the solubilization of fish protein concentrate. 

Plastein Formation. The ability of some proteolytic enzymes to convert soluble proteins to an insoluble aggregate was noted as early as 1947 ( 42,43) in the formation of plakalbumin from albumin by subtilisin. More recently, Fujimaki and co-workers have investigated this reaction for removal of flavor constituents from soybeans as well as a method of covalently attaching essential amino acids to the protein. 

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. 

Of special interest is the pH, the third factor influencing plastein formation. The optima for most proteases for plastein formation generally lie in a narrow range of pH 4-7 (Table I) measured with soybean globulin hydrolysates. Pepsin, for example, is an acid protease, and one of its characteristics is high degradative activity at low pH. Even at pH 1 it is active and able to degrade proteins. As Yamashita et al. (37) have demonstrated, no appreciable amount of plastein is formed at pH 1-2... 

Several workers have considered plastein formation to be primarily a type of transpeptidation which, as Horowitz and Haurowitz (44) have postulated, can be written schematically as ... 

Not only transpeptidation, but also a certain amount of peptide-peptide condensation, is possibly involved in the plastein reaction. With some of the proteases used for plastein formation, especially a-chymotrypsin (26, 52, 53, 54), the acyl-enzyme intermediate can be formed at pHs 5 from the reversal of the degradative reaction (Equation 1 E-OH + HOOC-CHR-NH E-O-OCCHR-NH- + H20). Once the acyl-enzyme intermediate is formed, the acyl group can be transferred to a nucleophile resulting in peptide bond synthesis. 

Although from an energetic point of view condensation would be a less favorable contributor to the plastein formation than transpeptida-... 

Model Experiment. Any L-amino acid ester (D-amino acid esters do not work Table III), when added to a reaction medium containing a protein hydrolysate, is generally incorporated during the resynthesis reaction under the appropriate reaction conditions. In this way it is feasible to prepare a plastein whose amino acid composition has been altered. Therefore, the plastein reaction, accompanied by new amino acid incorporation, would be expected to be much more valuable than when plastein formation is carried out in the absence of new amino acids. 

Figure 3. Correlation between the initial velocities of the papain-catalyzed aminolysis of ethyl hippurate by amino add esters (abscissa) and the initial velocities (extent after 2 hr) of their incorporation during the plastein formation from an ovalbumin hydrolysate by papain (ordinate). Open circles ethyl esters. Filled drcles n-hexyl esters. Unusual abbreviations Abu = a-aminobutyric acid, Nva = norvaline.

The resulting protein hydrolysates, though free of nonprotein impurities, are accompanied by another problem in most cases—a problem caused by a bitterness resulting from protease action on proteins. As discussed below, the resynthesis reaction leading to plastein formation... 

Enzymatic techniques can be used to endow proteins with surface-active functionality. An enzymatic technique that has shown promise in enhancing surface properties of proteins is a modified version of the classical plastein reaction. The plastein reaction is known to be a protease-catalyzed reverse process in which a peptide-peptide condensation reaction [11,12] proceeds through the peptidyl-enzyme intermediate formation [13]. It is essentially a two-step process enzymatic hydrolysis of a protein and plastein formation from the hydrolysate peptides. A novel one-step process was developed as a modified type of the plastein reaction by Yamashita et al. [14,15], which... 

The classical method of plastein reaction is based on the use of protein hydrolysates from protease reaction as substrates. Therefore, plastein formation requires two quite different unit processes. The first step is protein hydrolysis, with purification of the resulting hydrolysate, and the second is plastein synthesis with the covalent incorporation of an expected amino acid. Yama-... 

Tauber observed plastein formation in peptic digests of several proteins with chymotrypsin as enzyme (trypsin is inactive in the synthesis). Hitherto plastein formation had always been carried out in the neighborhood of pH 4 with chymotrypsin the reaction proceeds rapidly at pH 7.3. The average molecular weights of the synthetic products as determined in the analytical centrifuge are estimated to be in the range 250,000-400,000. As in the case of plasteins formed by pepsin, those formed by chymotrypsin are hydrolyzed in dilute suspension by pepsin, chymotrypsin, and trypsin. 

In plastein synthesis the free energy of formation of the peptide bonds is small, as attested to by the reversal of hydrolysis merely by concentrating certain enzymatic hydrolytic products the synthetic product is insoluble, which tends to drive the reaction toivards synthesis. In these two respects plastein formation resembles reactions of the type benzoyl-L-leucine - - L-leucine anilide to benzoyl-L-leucyUeucine anilide. 

The free energy of formation of the peptide bonds in plastein synthesis must be small this is attested to by the possibility of inducing peptide bond synthesis merely by concentrating an enzymatic hydrolyzate. The synthetic product is practically insoluble and this drives the reaction toward synthesis. It is in these two respiects that plastein formation resembles reactions of the type benzoyl-L-leucine + L-leucine anilide —> benzoyl-L-leucylleucine anilide. 

There is no information at present on the biological significance, if any, of plastein formation. It is an interesting phenomenon, in any event, in that it exemplifies condensation of unsubstituted peptides, entailing only a small free energy change, and catalyzed by proteases. 

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