Papain catalyzed aminolysis

Recently a simplified process was developed for incorporating l-methionine directly into soy proteins during the papain-catalyzed hydrolysis (21). The covalent attachment of the amino acid requires a very high concentration of protein and occurs through the formation of an acyl-enzyme intermediate and its subsequent aminolysis by the methionine ester added in the medium. From a practical point of view, the main advantage of enzymatic incorporation of amino acids into food proteins, in comparison with chemical methods, probably lies in the fact that racemic amino acid esters such as D,L-methionine ethyl ester can be used since just the L-form of the racemate is used by the stereospecific proteases. On the other hand, papain-catalyzed polymerization of L-methio-nine, which may occur at low protein concentration (39), will result in a loss of methionine because of the formation of insoluble polyamino acid chains greater than 7 units long. 

Figure 1. A simplified representation of the possible process for the papain-catalyzed hydrolysis and aminolysis E, enzyme (papain) S, substrate (protein) ES, Michaelis complex ES, peptidyl enzyme N, nucleophile (amino acid ester) P and P2, products formed from S by hydrolysis ...

The present study is an attempt to maximize the functionality of food proteins through the smallest possible modification. This object can be attained partly by applying a papain-catalyzed unusual reaction that proceeds efficiently under intentionally abnormal conditions. These conditions were characterized by at least two requirements extremely high substrate concentration and alkaline pH (see Table III). The reaction taking place is defined primarily as a type of aminolysis of a protein (sub-... 

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.

All of the data from Table II indicate that, under the unconventional conditions intentionally set in the present study, papain can catalyze the aminolysis of the ES intermediate that probably occurs from succinylated asi-casein (substrate) and papain (enzyme) by L-norleucine n-dodecyl ester (nucleophile), with formation of a surface-active 20,000-dalton product to which this lipophilic nucleophile is attached covalently as illustrated in Figure 1. The observed amphiphilic function of the 20,000-dalton product is probably a result of the formation of a localized hydro-... 

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