Enzyme hydrolysis of proteins

Simply on the basis of the normal composition of marine organisms, we would expect proteins and peptides to be normal constituents of the dissolved organic carbon in seawater. While free amino acids might be expected as products of enzymic hydrolysis of proteins, the rapid uptake of these compounds by bacteria would lead us to expect that free amino acids would normally constitute a minor part of the dissolved organic pool. This is precisely what we do find the concentration of free amino acids seldom exceeds 150 xg/l in the open ocean. It would be expected that the concentration of combined amino acids would be many times as great. There have been relatively few measurements of proteins and peptides, and most of the measurements were obtained by measuring the free amino acids before and after a hydrolysis step. Representative methods of this type have been described [245-259]. Since these methods are basically free amino acid methods, they will be discussed next in conjunction with those methods. 

M Hauck. Enzymic hydrolysis of proteins for determination of amino-acids and sample preparation. Dtsch Lebensm Rundsch 86 12-15, 1990. 

Functional Properties Related to Enzyme Hydrolysis of Proteins... 

Partial proteolysis has been used by several researchers to improve functional properties, i.e. foaming, solubility of proteins (7,8,9). The significant problems associated with enzyme hydrolysis of proteins are excessive hydrolysis occurring under batch conditions, the generation of bitter flavors during hydrolysis and the cost of enzymes. Extensive information on factors affecting proteolysis of proteins and the problem of bitterness has been reviewed by Fujimaki et al. (7) in conjunction with studies of the plastein reaction. 

A newly described procedure for complete enzymic hydrolysis of proteins and peptides (Brown and Wold 1973) makes use of several proteases which are covalently attached to insoluble supports (Cuatrecasas et al. 1968). The advantages of using these enzymes in the insoluble form are that the enzymes may be easily removed from the products, and there is little or no hydrolysis of the enzymes themselves which reduces the corrections for controls. One disadvantage of the insoluble proteases may be that the size of the insoluble particles introduces steric factors hindering the hydrolysis of large peptides or proteins. 

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. 

These derivatives are not stable to complete acid hydrolysis of proteins, but may be recovered in limited amounts after partial acid hydrolysis. For example, after 90 min of hydrolysis at 100°C in 5.7 N HCl about 30% of the covalently-bound phosphate in phosphoglucomutase was recovered as O-phosphoserine (Milstein 1964), but after 20 hr of hydrolysis at 105°C in 5.7 N HCl, no O-phosphoserine was found (Murray and Milstein 1967). The amount liberated at a certain time will depend on the nature of the residues around the phosphoserine residues in each protein. Hydrolysis of peptides containing O-phosphoserine may often give low yields of serine (e.g. see Nolan et al. 1964). Complete enzymic hydrolysis of proteins or peptides containing O-phosphoserine is the only presently available method for quantitative recovery of this derivative. 

Both groups of investigators observed that cosubstrates are apparently produced during enzymic hydrolysis of proteins. Therefore, with protein substrates the specificity of a proteolytic enzyme is influenced by degradation products in the reaction mixture and may appear very different from the specificity observed with synthetic substrates containing a single susceptible peptide bond. 

The above method, due to Sorensen, is of great importance in following the course of hydrolysis of proteins by enzymes (p. 516). For example, if the protein and its hydrolysis are represented thus ... 

Chymotrypsin (Section 27 10) A digestive enzyme that cat alyzes the hydrolysis of proteins Chymotrypsin selectively catalyzes the cleavage of the peptide bond between the car boxyl group of phenylalanine tyrosine or tryptophan and some other ammo acid... 

Currently, a-amino acids are prepared by several routes such as by the fermentation of glucose, by enzyme action on several substances and by the hydrolysis of proteins. Many methods for synthesising the polymers are known, of which the polymerisation of A -carboxyanhydrides is of particular interest, as it yield-products of high molecular weight (Figure 18.24). 

The most recent advance in treating HIV infections has been to simultaneously attack the virus on a second front using a protease inhibitor. Recall from Section 27.10 that proteases are enzymes that catalyze the hydrolysis of proteins at specific points. When HIV uses a cell s DNA to synthesize its own proteins, the initial product is a long polypeptide that contains several different proteins joined together. To be useful, the individual proteins must be separated from the aggregate by protease-catalyzed hydrolysis of peptide bonds. Protease inhibitors prevent this hydrolysis and, in combination with reverse transcriptase inhibitors, slow the reproduction of HIV. Dramatic reductions in the viral load in HIV-infected patients have been achieved with this approach. 

Mazaleyrat, J.-P. Reboud-Ravaux, M. Wakselman, M. Synthesis and enzymic hydrolysis of cyclic peptides containing an anthranilic acid residue. Int. J. Peptide Protein Res. 1987, 30, 622-633. 

Proteolysis The hydrolysis of proteins, usually by enzyme action, into simpler substances. 

Although zinc, cadmium, and mercury are not members of the so-called main-group elements, their behavior is very similar because of their having complete d orbitals that are not normally used in bonding. By having the filled s orbital outside the closed d shell, they resemble the group IIA elements. Zinc is an essential trace element that plays a role in the function of carboxypeptidase A and carbonic anhydrase enzymes. The first of these enzymes is a catalyst for the hydrolysis of proteins, whereas the second is a catalyst for the equilibrium involving carbon dioxide and carbonate,... 

From the realization of the amphoteric nature of proteins and the importance of [H+] in many of their functions, and as increasing numbers of enzymes were identified, it became commonplace to report their optimum pH. Corrections for non-enzymic hydrolysis of the substrates were usually made but, as was critically reviewed by Dixon and Webb (1959), from many of the results it was not possible to distinguish effects of pH on the protein in general from those on the region actually involved in interaction with the substrate. Nevertheless, many... 

T. M. Li and J. F. Burd, Enzymic hydrolysis of intramolecular complexes for monitoring theophylline in homogeneous competitive protein-binding reactions, Biochem. Biophys. Res. Commun. 103, 1157-1165 (1981). 

This enzyme catalyzes the hydrolysis of proteins, including elastin, with preferential cleavage at Ala-Xaa. The following are reviews on the molecular and physical properties of this enzyme [EC 3.4.21.36 (pancreatic) and EC 3.4.21.37 (leukocyte)]. 

A. Digestive enzymes of the gastrointestinal tract begin hydrolysis of protein, fat, and carbohydrates into their component building blocks, namely, amino acids, fatty acids and monoacylglycerols, and simple sugars (such as glucose). 

This unnatural acid is used as a chiral intermediate for the synthesis of a number of products. Chemical asymmetric synthesis was very difficult and so the stereoselective synthetic properties of enzymes were exploited to carry out a selective reduction reaction. The stereoselective hydrolysis of protein amino acid esters had already been commercialised by Tanabe in Japan using immobilised aminoacylase, and selective reduction reactions using whole yeast cells are already used in a number of processes, such as the selective reduction of the anti-cancer drag Coriolin. 

For routine, enzymic hydrolysis of sialic acids on a preparative or an analytical scale in our laboratory, the substrates are incubated with bacterial sialidases in 50 mM acetate buffer at pH 5.5 and 37° for times ranging between a few minutes and several hours, depending on the substrate.107 After incubation, the sialic acids liberated are separated by dialysis, ultrafiltration, or protein precipitation at 0-2°, and purified as will be described in the following Section. 

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