Papain trypsin hydrolysis

There are four basic mechanistic classes of enzyme which catalyse the hydrolysis of peptide bonds serine proteinases such as trypsin and chymo-trypsin, cysteine proteinases such as papain, acid (aspartic) proteinases such as pepsin, and zinc-containing metalloproteinases such as carboxypeptidase. X-ray crystal structures of representative examples of each class of enzyme are available, and the detailed reaction pathways probably taken by all four classes of enzyme have been subject to analysis in terms of ALPH, These analyses have been for the most part permissive rather than compelling, and are considered in turn below. 

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... 

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). 

Procedure 3 papain, leucine aminopeptidase, prolidase This early procedure for enzymic hydrolysis of proteins was reported by Hill and Schmidt (1962) to be successful for hydrolysis of several proteins. Papain was found to be superior to subtilisin or a combination of trypsin and chymotrypsin for the initial hydrolysis. The method might be improved if aminopeptidase M (discovered after the method was developed) is used in place of the leucine aminopeptidase, but to our knowledge this has not been tested. The problem with diketo-piperazine formation from X-Pro dipeptides in aminopeptidase M hydrolysates of peptides (see above) may make this substitution less desirable than it would seem at first. 

Tappel (99) reported on the hydrolytic activity of various enzymes. They found that the relative hydrolysis of muscle proteins increased in the following order pepsin, Rhozyme A-4, ficin, papain, bromelain, protease 15, Rhozyme P-11, and trypsin. Wang et al. (100) observed that papain was twice as active as ficin towards elastin, a minor component of connective tissue. Ficin and bromelain had equal enzyme activity towards collagen, a major component of connective tissue. Kang and Rice (101) studied the effects of various tenderizing enzymes on water-soluble sarcoplasmic proteins, salt-soluble myofibrillar proteins, and the insoluble stromal proteins. Table VII tabulates the results of some of these studies. 

For the example of hydroxamic acid formation given above, the efficiency of the exchange reaction depends on the relative nucleophilic strength of H2O and NH2OH and on the concentration of the latter. Hydroxylamine is a stronger nucleophilic replacement reagent but is present in relatively low concentration it is therefore favored by the more stable enzyme-acyl substrate bond. We have shown that the stability of the acylated enzyme is characterized by fcs and that for comparable reactions of trypsin and ficin on papain the rate of hydrolysis of the acyl-enzyme compound of the former is ten times as fast as that of the latter. The findings of Durell and Fruton... 

Next to trypsin chymotrypsin is the most preferred proteolytic enzyme in sequencing. Its specificity is less absolute than that of trypsin. Primarily the bonds that follow phenylalanine, tyrosine and tryptophan are cleaved, but measurable hydrolysis takes place next to leucine and methionine residues as well. It is advisable, therefore, to determine in preliminary experiments the conditions (enzyme-substrate ratio, time, temperature) best suited for the formation of a few and well separable fragments. Occasionally also less specific enzymes, such as pepsin, papain or thermolysin find application in structure elucidation. For the hydrolysis of specific bonds new microbial proteases can be isolated. There are known prolidases and also enzymes which hydrolyze solely the bond which follows a pyroglutamyl residue and so on. 

Proteases (trypsin, pepsin, elastase, papain, microbial enzymes, etc.) Hydrolysis of peptide bonds Digestion aids removal of necrotic tissue acne treatment meat tenderizers preparation of special diets and peptone media for microorganisms removal of proteins from carbohydrates and fats prevention of cloudiness in beer laundry additive leather ttuming. 

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)... 

Fig. 4.12. Amino acid sequences and sites of enzymatic cleavage in the hinge regions of human and rabbit-y chains. The symbol H represents the site of an interheavy chain disulfide bond and the symbol L designates the site of a heavy-light chain disulfide bond. The halfcystine at position 220 of the rabbit y-chain forms an intrachain disulfide loop with Cys 131 or 132. The Eu numbering system is used (2). Pap, Pep, and T refer to cleavage sites for papain, pepsin, and trypsin, respectively. The site of cleavage of the rabbit H chain by papain is that for molecules of allotype dll. Hydrolysis by papain of molecules of allotype dl2 is described in the text. Cleavage of the rabbit H chain by trypsin at position 220 occurs only after reduction and aminoethylation (by ethyleneimine) of Cys-220. The symbol CHO represents carbohydrate. References are given in the legend of Fig. 4.9.

A simple case of the general transpeptidation reaction was the trans-amidation resulting in the formation of hippuric anilide from aniline and hippuric amide in the presence of papain. Since this reaction proceeded much faster than the enzymatic synthesis of hippuric anilide from hippuric acid and aniline, it seems reasonable to infer that exchange, in the former reaction, took place between the aniline and ammonia. Waley and Watson subjected L-lysyl-L-tyrosyl-L-lysine and L-lysyl-L-tyrosyl-L-leucine to treatment with chymotrypsin and trypsin at pH 7.8. In the hydrolysis mixture of either of these substrates they were able to identify lysyllysine which could have arisen only by rearrangement of the amino acids in peptide bond. The peptide may have reacted with the lysine liberated by hydrolysis ... 

Claradiastase and trypsin in phosphate buffer have been used for hydrolysis of sample matrix for the determination of supplemental folic acid (27). Jacoby and Henry (26) further modified the method of Hoppner and Lampi (41) for folic acid by HPLC, in which the folic acid added to infant formulas and liquid medical nutritionals is quantitatively extracted with the aid of bacterial protease and papain. One disadvantage of the enzymatic extraction method was the large number of UV-absorbing compounds that are formed during enzymatic hydrolysis. These can interfere in the quantitation of the folate peaks. Thus, addition of a-amylase and protease to enhance extraction has not been common practice for methods involving HPLC. However, Pfeiffer et al. (13) suceessfiilly used triple-enzyme treatment prior to HPLC analysis with a purification method based on affinity chromatography. 

---