Proteolysis functional properties

Vaughan, R. A. and Kuhar, M. J. (1996) Dopamine transporter ligand binding domains. Structural and functional properties revealed by limited proteolysis. J. Biol. Chem. 271, 21672-21680. 

The present study was conducted to obtain additional information on changes in soy protein subunits during limited proteolysis. Enzymatic soy protein deamidation that occurred, in addition to limited proteolysis, during germination of soybean seeds was investigated. The effects of proteolysis and deamidation on solubility and emulsifying activity were compared. Phosphorylation of soy protein with a commercially available protein kinase and its effects on subsequent changes in functional properties of the protein were also studied. 

For comparison, the solubility-pH profile of the deamidated protein was added to the plot containing the profiles for the pronase E-treated proteins (Figure 5). The deamidated protein, with 2.6% peptide bond hydrolysis, showed improved minimum solubility, comparable to the protein with 5.7% peptide bond hydrolysis and no deamidation. The shape of solubility-pH profile for the deamidated sample resembled that of the intact protein more than those of the pronase E-treated samples. For the deamidated sample, both the increase in solubility and the slight shift of minimum solubility to the acid side were the result of the increase in negative charges from deamidation. Obviously, deamidation was more capable of maintaining the original protein structure than proteolysis, which is essential for the development of desirable functional properties. 

Kuehler and Stine (43) studied the functional properties of whey protein with respect to emulsifying capacity as affected by treatment with three proteolytic enzymes. Two microbial proteases and pepsin were examined. The emulsion capacity decreased as proteolysis continued, suggesting that there is an optimum mean molecular size of the whey proteins contributing to emulsification. 

The earliest commercial milk protein enzymatic modification dates back to the 1940s, when the first formulas for allergenic infants were made. The aims of this process were to reduce allergenicity as well as to change the functional properties of proteins while preserving their nutritional value for clinical use. Unfortunately the hydrolysates thus obtained were characterized by bitter taste, and for mainly this reason proteolysis, as a technological process, enjoyed very little popularity. 

More details of the plastein reaction and its application to remove pigments such as chlorophyll, or to remove off-flavor components such as the beany taste of soybeans, are shown in Figure 2. The protein of the food system is solubilized and denatured (in order to achieve proteolysis), a protease is added, and the hydrolytic reaction is allowed to proceed. On partial hydrolysis of the protein the pigments and flavor constituents are released from the protein they are removed, the hydrolyzate is concentrated, and resynthesis and/or rearrangement of the amino acid sequence of the polypeptides is catalyzed by the same or a different protease. Resynthesis also can be carried out in the presence of added amino acid esters in order to improve the nutritional/functional properties of the protein. 

Proteolysis in cheese has been studied extensively and reviewed (Fox and Wallace 1997 McSweeney, 2004 Upadhyay et al., 2004). It contributes directly to flavor, via the formation of peptides and free amino acids (FAA), and indirectly via the catabolism of free amino acids to various compounds including amines, acids, thiols (Curtin and McSweeney, 2004). Proteolysis directly affects the level of intact casein, which is a major determinant of the fracture and functional properties, and of cheese texture (de Jong, 1978b Creamer and Olson, 1982 Creamer et al., 1982 Guinee, 2003 Brown et al., 2003). 

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. 

In conjunction with research on protein extraction from yeast, we investigated methods for the maximum recovery of protein possessing good functional properties but low in nucleic acid. Therefore, we examined the feasibility of making the yeast protein resistant to proteolysis during extraction and nucleic acid reduction. Using established extraction procedures (76), we observed... 

Enzyme hydrolysis is occasionally used to modify the functional properties of proteins and yeast autolyzates are used commercially as food flavorants (66,86). Partial proteolysis of... 

Lieske, B. Konrad, G. Physico-chemical and functional properties of whey protein as affected by limited papain proteolysis and selective ultrafiltration. Int. Dairy J. 1996, 6(1), 13-31. 

Solubilization of Protein. Fish protein concentrate has high nutritional quality as determined both from its essential amino acid composition and from animal feeding experiments. Unfortunately, the concentrate is quite insoluble in water because of its denaturation by the solvent extraction method used in processing thus it contributes no functional properties to a food and must be used in bakery products primarily. A potentially useful method of solubilizing the protein is by proteolysis (9-12). As is the case with protein hydrolysates of casein and soybean protein, bitter peptides are formed during the hydrolysis. Papain and ficin produce more of these bitter peptides than does Pronase, for example (12). Pronase was found to produce a more brothy taste (13). A possible method of removing the bitter peptides is to convert the concentrated protein hydrolysate to plastein by further proteolytic enzyme action (14) to remove the bitter peptides. 

Enzymic treatment of less-well-defined protein systems results in an alteration of the functional properties of the proteins. One of the primary functional properties is solubility since a protein generally must be in solution before it can exert many of its other functionalities. Unfortunately, many protein concentrates are either naturally insoluble or rendered insoluble at pH values in the neutral range by processing treatments which denature the proteins. Many protein concentrates tend to be insoluble at acidic pH values around 5(6) and require alkaline conditions for solubilization. Supplementation of an acidic soft drink or citrus juice with protein, for example, would require solubilization of the protein at acidic pH values. Consequently, proteolytic enzymes have been employed to solubilize proteins from various sources and thus alter their solubilities through proteolysis and modify their other functional properties. 

The functional properties of proteins depend also on their structure and interactions with the environment. The functional properties of surfactants depend on their hydrophilic-hydrophobic balance, too. Protein chains modified by proteolysis, amino acid incorporation, and transpeptidation may display different functional properties. As milk proteins possess good surface activities [131], the question of the changes in the functional properties of the enzymatically modified protein products is of especial interest. 

Enzymatic hydrolysis of proteins results in changing the molecular mass of the molecules. However, the effects of protein size on the hydrophobic behavior of amino acids are of great importance [132]. There is a meaningful relationship between hydrophobicity (which may affect the surface of the molecule) and functionality of food proteins [11,133] proposed using the term relative surface hydrophobicity. The proteolysis should be carefully limited for improving the functional properties of food proteins [147]. Mild hydrolysis improves functionality of proteins, while extensive hydrolysis depresses it [139,148],... 

Deamidation of proteins is important for improving functional properties of the product under mild reaction conditions. But enzymatic deamidation of proteins has not had real attention until recently. Kato et al. [155] developed a method for enzymatic deamidation of food proteins by treatment with proteases at pH 10. Salt and disulfide reducing agents have little effect on soy protein deamidation. Heat treatment and proteolysis of soy proteins are the major factors affecting deamidation [156]. 

Solubility. Solubility is an important functional property per se, i.e. in fluid products, and is essential for other functionalities since insoluble proteins can not perform useful functions in foods. The caseins are, by definition, insoluble at their isoelectric points, i.e. in the pH range c. 3.5-5.S the insolubility range becomes wider with increasing temperature. Insolubility in the region of the isoelectric point is clearly advantageous in the production of acid casein and is exploited in the production of two major families of dairy products, i.e. fermented milks and fresh cheeses. However, such insolubility precludes the use of casein in acid liquid foods, e.g. protein-enriched fruit juices or carbonated beverages. Acid-soluble casein can be prepared by limited proteolysis or by interaction with certain forms of pectin. 

Partial proteolysis of soy protein isolate with neutral protease from Aspergillus oryzae altered certain functional properties ( ). Solubility was increased in the enzyme-treated soy isolate at both neutral pH and at the isoelectric point (pH... 

Because the size of polypeptides produced from proteolysis relate directly to their functional properties, the application of DH is the most common approach to monitor the process of proteolysis. However, it should be pointed out that the calculation of PCL from the DH is a theoretical concept only. Although it has been widely used and documented in almost all of the literature, it cannot be used for comparing protein hydrolysates from different proteolytic reaction systems. When a protein substrate is hydrolyzed by different proteases, the differences in the specificity of enzymes may produce protein hydrolysate with significantly different-size distributions, although they have the same DH values. 

Once the polypeptide chain is folded, further events are often required to permit the protein to reach its functional conformation. Covalent processing, such as further limited proteolysis and chemical modifications is an important event in determining functional properties. These processes induce conformational refinements which can be considered as the last events of protein folding. 

---