Papain protein hydrolysis

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 ...

Specificity of Papain, for Hydrolysis of Peptide Bonds in Proteins and Polypeptides ... 

Papain Neutral Proteinase Carica papaya Meat and Plant Protein Hydrolysis Yields Amino... 

Meat Porcine myofibrillar proteins Hydrolysis with papain Acidic peptides DSGVT, lEAEGE, DAQEKLE, EELDNALN, VPSIDDQEELM Saiga et al. (2003)... 

Venison protein Hydrolysis with papain, pepsin, bypsin, a-chymo trypsin, alcalase, and neutrase Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly (APVPH I) and Asp-Leu-Ser-Asp- Gly-Glu-Gln-Gly-Val-Leu (APVPH 11). Kim et al. (2009)... 

Peanut protein Hydrolysis with papain Peanut protein hydrolysate with the degree of hydrolysis of 25.5% Tang et al. (2012)... 

Pish protein concentrate and soy protein concentrate have been used to prepare a low phenylalanine, high tyrosine peptide for use with phenylketonuria patients (150). The process includes pepsin hydrolysis at pH 1.5 ptonase hydrolysis at pH 6.5 to Hberate aromatic amino acids gel filtration on Sephadex G-15 to remove aromatic amino acids incubation with papain and ethyl esters of L-tyrosine and L-tryptophan, ie, plastein synthesis and ultrafiltration (qv). The plastein has a bland taste and odor and does not contain free amino acids. Yields of 69.3 and 60.9% from PPG and soy protein concentrate, respectively, have been attained. 

Different enzymes have different specificities. Some, such as amylase, are specific for a single substrate, but others operate on a range of substrates. Papain, for instance, a globular protein of 212 amino acids isolated from papaya fruit, catalyzes the hydrolysis of many kinds of peptide bonds. In fact, it s this ability to hydrolyze peptide bonds that makes papain useful as a meat tenderizer and a cleaner for contact lenses. 

For the SAXS studies a CBH II sample was prepared by affinity chromatography from r. reesei QM 9414 to give the enzyme in a homogeneous form 27. In SDS-PAGE the protein had a size of 58 kDa and the isoelectric point was 4.9. Glycosy-lation was estimated as 8 to 18 % 36. The molar absorptivity at 280 nm was 75 000 M xm To obtain the core protein partial proteolytic hydrolysis with papain was per-... 

This enzyme [EC 3.4.22.25] catalyzes the hydrolysis of peptide bonds with a preference for Gly-Xaa in proteins and small molecule substrates. The enzyme, a member of the peptidase family Cl, is isolated from the papaya plant, Carica papaya. It is not inhibited by chicken cys-tatin, unlike most other homologs of papain. 

Pantothenic acid occurs in foods both in the free form and bonded to coenzyme (CoA) or acyl carrier protein (ACP) therefore hydrolysis is needed to extract it totally. Since it is degraded by acid and alkaline hydrolysis, only an enzymatic digestion can be applied. Enzyme hydrolysis with papain, diastase, clarase, takadiastase, intestinal phosphatase, pigeon liver pantetheinase, or combination of them has been used. 

Hermansson et al. (36) used pepsin and papain to solubilize rapeseed protein concentrate. Papain had a lower solubilizing effect than did pepsin. However, the fact that pepsin has an optimum pH for activity at about 1.6, far below the pH range of most foods, made it possible to study the effects of controlled hydrolysis. At pH 7.0, all hydrolysates were more soluble than the original rapeseed protein concentrate. 

Figure 6. Nitrogen solubility (Kjeldahl) of peanut proteins P.H.P. = partial hydrolysis by 5% papain CON. = control (no papain) (2i).

It should be noted that ascorbic acid is more stable at pH 4-5 than at pH 7, at which the folacin vitamers are more stable. Additional protection from oxidation can be achieved by degassing the extraction solution with an inert gas, such as helium. Homogenization is followed immediately by protein precipitation and release of bound folacin vitamers. This can be accomplished by mild acidification, heating, addition of organic compounds such as trichloroacetic acid, and/or enzymatic (e.g., papain) hydrolysis. The specific conditions used for homogenization and protein precipitation are dictated by the food matrix and the expected profile of folacin vitamers. 

Endogenous biotin in foods is predominately protein bound and is relatively stable (180). Consequently it can be extracted under fairly harsh conditions, e.g., autoclaving in 4 M sulfuric acid for 2 hours at 120°C. Enzymatic hydrolysis with papain will also release biotin from proteins (181). Potential sample-cleanup procedures include adsorption on charcoal and/or ion-exchange chromatography (182,183). 

Enzyme hydrolysis, with papain, diastase, clarase, takadiastase, intestinal phosphatase, or combinations thereof is most commonly used to release pantothenate from food proteins (186). A cold perchloric acid extraction was used to release pantothenic acid from tissue samples (187). Food spoilage prior to analysis may lead to inflated pantothenic acid levels (19). 

Papain Hydrolysis of proteins Papaya fruit (Carica Meat tenderizer ... 

Enzymatic gelation of partially heat-denatured whey proteins by trypsin, papain, pronase, pepsin, and a preparation of Streptomyces griseus has been studied (Sato et al., 1995). Only peptic hydrolysate did not form a gel. The strength of the gel depended on the enzyme used and increased with increasing DH. Hydrolysis of whey protein concentrate with a glutamic acid specific protease from Bacillus licheniformis at pH 8 and 8% protein concentration has been shown to produce plastein aggregates (Budtz and Nielsen, 1992). The viscosity of the solution increased dramatically during hydrolysis and reached a maximum at 6% DH. Incubation of sodium caseinate with pepsin or papain resulted in a 55-77% reduction in the apparent viscosity (Hooker et al., 1982). 

Japanese Society of Miso Science and Technology Figure 18. Schematic representation of effect of heat treatment on soybean protein and its hydrolysis patterns by various enzymes. Pattern A, pepsin and other acid proteinoses pattern B, the proteinoses having an optimum near neutrality, such as papain, bacteria neutral proteinase, Aspergillus alkaline proteinase, Aspergillus neutral proteinase and pattern C, trypsin and in vivo nutritional... 

Table VII summarizes the conditions for chymotryptic hydrolysis of the proteins and peptides listed in Table VI. The parameters which would be expected to determine the rate of hydrolysis (apart from the nature of the bonds in the particular substrates) are temperature, pH, time of hydrolysis, and the molar ratio of chymotrypsin to substrate. All these factors often differ considerably for the substrates listed. Hydrolyses have been performed under conditions which vary from 2 to 24 hr, from pH 7.0 to 9.0, from 22° to 40°C, and at enzyme to substrate molar ratios between 1/360 to 1/21. It is not obvious how variations in pH and temperature affect the apparent specificity of chymotrypsin, but at low molar ratios of enzyme to substrate only the most susceptible bonds would be expected to be hydrolyzed. The lowest molar ratio was employed in the studies with ribonuclease. The only bonds of an unusual nature which were split were those formed by serine and histidine in the following sequences, -Thr-Ser. . . Ala-Ala- and -Lys-His. . . Ileu-Ileu-. Many of the unusual splits listed in Table VI were observed in equine or human cytochrome c and in oxidized papain. Each of these substrates was digested for long periods of time and at high ratios of enzyme to substrate under conditions which would be expected to split bonds that are usually resistant to hydrolysis.

Because of its wide specificity, papain will degrade most protein substrates more extensively than trypsin, pepsin, or chymotrypsin and its action is quite comparable to that of subtilisin (Hill and Schmidt, 1962 Nomoto et al., 1960a,b). Many free amino acids are liberated from proteins by papain, but it would not appear to produce as extensive hydrolysis as S. griseus protease (French et al., 1963). 

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