Papain catalyzed attachment

The present study deals first with the papain-catalyzed attachment of lipophilic L-norleucine n-dodecyl ester to a hydrophilic protein. To determine more clearly the mode of reaction, well-defined asi-casein was selected as the protein for this purpose. 

Table IV. Chemical and Functional Properties of Products Prepared from Gelatin0 by Papain-Catalyzed Attachment of L-Leucine ft-Alkyl Esters...

With a series of applications to well-known food products, we tested the suitability of the proteinaceous surfactants prepared from gelatin by the papain-catalyzed attachment of L-leucine n-alkyl esters. Using each of these surfactants we tried to prepare snow jelly, ice cream, mayonnaiselike food, and bread. 

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. 

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-... 

Arai et al. [141] described a particular enzymatic reaction for producing a surface-active protein. A highly hydrophobic amino acid was covalently bound to a hydrophilic protein in an enzyme-catalyzed process for this purpose. The covalent attachment of L-Leu n-alkyl ester to gelatin in the presence of papain as catalyst resulted in a proteinaceous surfactant [141,142] with very good emulsifying properties. 

It should be emphasized that the narrow view that proteolytic enzymes are solely hydrolytic in their action must be broadened to include observations that such enzymes can also catalyze S3mthetic and transfer reactions. Such seems to be the case with many other types of so-called hydrolytic enzymes also. From the aspect of mechanism of action, it may be supposed that the role of the enzyme is to participate in a displacement reaction in which part of the substrate becomes attached to the enzyme. In the case of papain, it has been suggested (see above) that a thiol ester is formed. Such an intermediate can participate in various kinds of reaction and, as emphasized by Fruton and co-workers, may play a biological role other than that of hydrolysis. 

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