Papain-catalyzed

G. Lowe, Y. Yuthavong, Kinetic Specificity in Papain-Catalyzed Hydrolyses , Biochem. J. 1971, 124, 107-115. 

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 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. 

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 9.28 HPLC profile of papain-catalyzed B AEE hydrolysis product. Conditions column, juBondapak CN eluent, 0.05 M ammonium acetate-methanol (85 15) flow rate, 2.0 mL/min detector, 254 nm at ambient temperature (From Chen et al., 1982.)...

Figure 13. A possible mechanism of action for papain catalyzed hydrolysis...

Carrieri, A., Altomare, C, Barreca, M.L., Contento, A., Carotti, A. and Hansch, C. (1994). Papain Catalyzed Hydrolysis of Aryl Esters A Comparison of the Hansch, Docking and CoMFA Methods. II Farmaco, 49,573-585. 

In a papain-catalyzed reaction, at room temperature, proteins can be acylated and enriched in SH groups using A-acetyl-homocysteinethiolactone (Sung et al., 1983) ... 

We have noticed for several systems examined that extrapolations of the Arrhenius plots for intermediate formation to ambient temperatures suggest that the rates of intermediate interconversion will be of the same order of magnitude at the temperatures at which the enzymes normally operate (12). The convergence of Arrhenius plots is shown for the papain-catalyzed hydrolysis of N-carbobenzoxy-L-lysine p-nitroanilide in Figure 3 (28). 

Uemura, T., Fujimori, M., Lee, H.-H., Ikeda, S., and Aso, K. (1990) Polyethylene glycol-modified papain catalyzed oligopeptide synthesis from the esters of L-aspartic and L-glutamic acids in benzene. Agric. Biol. Chem., 54... 

Gln-Gln-Phe-Gly9-OMe was formed by chymotrypsin-catalyzed transpeptidation in the presence of H-Phe-GIy-OMe. In a papain-catalyzed acyl transfer reaction and subsequent tryptic cleavage, the resulting dodecapeptide ester was converted into substance P. These results indicate that peptides can be readily produced by a combination of recombinant DNA technology and peptidase-catalyzed conversion with the advantage of possible incorporation of groups other than coded amino acids into the recombinant product. 

D Cantacuzene, C Guerreiro. Optimization of the papain catalyzed esterification of amino acids by alcohol and diols. Tetrahedron 45(3) 741-748, 1989. 

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.

General procedure for papain-catalyzed oligo( y-ethyl-L-glutamate) synthesis... 

Transpeptidation, transamidation, a reaction involving the transfer of one or more amino acids from one peptide chain to another. This term was first coined by Fruton, in 1950, by analogy with transglycosidation for the papain-catalyzed displacement reaction between Bz-Gly-NH2 and aniline forming Bz-Gly-NHPh. Of special importance in relation to protease-catalyzed transpeptidation reactions in a preparative scale is the one-step tryptic conversion of porcine insulin into human insulin, despite the controversial interpretation of the mechanism involved. A bacterial transpeptidase, serim-type u-Ala-u-Ala carhoxypeptidase (EC... 

The problem of the enzymatic synthesis of peptide bonds assumed a new aspect in around 1937, when Heinz Fraenkel-Conrat [30], then in the laboratory of M. Bergmann, demonstrated the papain-catalyzed formation at pH 5 of sparingly soluble benzoylglycine anilide from benzoylglycine amide or from benzoylglycine (hippuric acid) and aniline as well as the condensation of benzoylleucine with leucine anilide yielding the nearly insoluble dipeptide benzoyldileucine anilide (Fig. 6). 

Mammalian liver and muscle frucose bisphosphate aldolases are also very susceptible to limited proteolysis (5,53-55). Cathepsin B, cathepsin L, and papain catalyze the limited proteolysis of rabbit muscle and rat liver aldolases 50,51). In fact, decrease of aldolase activity in liver is observed during starvation 109) and after administration of lysosome-tropic agents 103). Leupeptin caused an increase in osmotic sensitivity of lysosomes and an increase in the activities of free lysosomal proteinases, such as cathepsin A and cathepsin D, and a moderate increase of cathepsin B and L, and resulted in a decrease in aldolase activity. The molecular properties of aldolase isolated from the livers of control rats and leupeptin-treated rats indicated that the decrease of aldolase activity is attributable to hydrolysis of a peptide linkage(s) near the carboxyterminal of the enzyme. However, care is necessary in determining whether proteolytic modification of enzymes... 

Monti JC, Jost R. Papain-catalyzed synthesys of methionine enriched soy plastein. Average chain length of the plastein peptides. J Agric Food Chem 1979 27 1281-1285. 

Papain catalyzed the polymerization of L-methionine methyl ester hydrochloride to give water-insoluble oligomer with DP = 8-10 (59-61). The resulting water-insoluble oligomer was converted to water-soluble sulfoxide and sulfone derivatives by treatment of DMSO or hydrogen peroxide. Esters of phenylalanine. 

FIG. 4 Amphiphilic structure formed by papain-catalyzed incorporation of L-leucine n-dodecyl ester into sucdnilated ctsi-casein. The position of hydrophihc amino acid residues on the protein molecule are indicated by vertical bars. (From Ref. 34.)... 

Figure 3. Papain-catalyzed formation of pectin amide...

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