Isoelectric precipitation, protein

Soybean Protein Isolates. Soybean protein isolates, having a protein content of >90 wt%, are the only vegetable proteins that are widely used in imitation dairy products (1). Most isolates are derived from isoelectric precipitation, so that the soybean protein isolates have properties that are similar to those of casein. They are insoluble at thek isoelectric point, have a relatively high proportion of hydrophobic amino acid residues, and are calcium-sensitive. They differ from casein in that they are heat-denaturable and thus heat-labile. The proteins have relatively good nutritional properties and have been increasingly used as a principal source of protein. A main deterrent to use has been the beany flavor associated with the product. Use is expected to increase in part because of lower cost as compared to caseinates. There has been much research to develop improved soybean protein isolates. 

Purification. Enzyme purity, expressed in terms of the percent active enzyme protein of total protein, is primarily achieved by the strain selection and fermentation method. In some cases, however, removal of nonactive protein by purification is necessary. The key purification method is selective precipitation of the product or impurities by addition of salt, eg, sodium sulfate, or solvent, eg, ethanol or acetone by heat denaturation or by isoelectric precipitation, ie, pH adjustments. Methods have been introduced to produce crystalline enzyme preparations (24). 

Petanate, A.M. and Glatz, C.E., 1983. Isoelectric precipitation of soy protein. I. Factors affecting particle size distributions. II. Kinetics of protein aggregate growth and breakage. Biotechnology and Bioengineering, 25, 3049. 

Fig. 1. Relationship between reactor mean velocity gradient and particle size for isoelectric soya protein precipitate. Open symbols represent precipitate diameter for 50% oversize Closed symbols represent precipitate diameter for 90% oversize [51]...

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Soy isolate was prepared by the isoelectric precipitation procedure developed at the Food Protein Research and Development Center, Texas A M University System (23). A commercial soy isolate. Promine F (Central Soya Inc., Fort Wayne, IN), was also used in this study. 

Isoelectric precipitation results in loss of whey protein as well as undesirable non-protein components. Gillberg (35) has shown that the cystine composition of the isolate is lower than for the meal extract because non-precipitated whey protein has a relatively larger proportion of the total sulfur amino acids. This observation is reinforced by Mattil (36), who showed that the range for amino acid composition was different for several commercial concentrates as compared to commercial isolates. He found methionine to be lower for isolates than for concentrates. 

The properties of many dairy products, in fact their very existence, depend on the properties of milk proteins, although the fat, lactose and especially the salts, exert very significant modifying influences. Casein products are almost exclusively milk protein while the production of most cheese varieties is initiated through the specific modification of proteins by proteolytic enzymes or isoelectric precipitation. The high heat treatments to which many milk products are subjected are possible only because of the exceptionally high heat stability of the principal milk proteins, the caseins. 

Although the gelation properties of whey proteins are of great importance in many foods (Mulvihill, 1992) and it is possible to form a weak gel in creams by the formation of a continuous network of fat globules, most important milk gels are those involving casein micelles which can be made to form a gel matrix either by isoelectric precipitation (acid-induced gel) or by the action of a proteolytic enzyme (rennet-induced gel). Both gel types... 

Isoelectric precipitation and acid precipitation are also used to separate antibodies. Isoelectric precipitation (also called euglobulin precipitation) uses the solubility properties of a protein near its isoelectric point.69 When a concentrated protein solution in a low ionic strength buffer is titrated to its isoelectric point, it precipitates very slowly. Table 4 shows a balance of a process for purification of murine monoclonal IgM antibodies.70 The success of precipitation can be followed by the cumulative IgM content in the centrifugate and the sediment. The procedure is very gentle, but very sensitive to environmental conditions. 

Soy beans contain several globular proteins which can be separated from the whey proteins by isoelectric precipitation at pH 4.5-4.9. About 90% of the extracted protein is recovered in this isolate. Of the globular proteins, the 7S and 11S proteins predominate although the actual amount of each varies with soy variety. The isolation and properties of these proteins has been the subject of recent reviews(2 3). Some of the undesirable features of soy proteins are illustrated by the following observations. 

The water-extractability of both the 7S and 11S proteins decreases with aging of the soy meal (4). During isoelectric precipitation 15-30% of the globular fraction is denatured and fails to redissolve in neutral buffer (4, 5). As the pH is lowered to 2-3 the 11S component becomes increasingly sensitive to irreversible denaturation. This acid-sensitive fraction not only limits the solubility of such isoelectric protein isolates, and therefore their functional uses, but this fraction may also be responsible for retention of undesirable flavors (5). 

Our studies on 7S globulin isolated under conditions which avoided the acidic denaturation conditions created by isoelectric precipitation showed about 5% helix, 60% g-structure and 35% random coil. It is quite possible that the isolation conditions have determined the secondary order of the isolated protein. 

For preparative purposes, it is perhaps more convenient to start from pancreas acetone powder (pancreatin). Ninty-five per cent pure bovine procarboxypeptidase A has been obtained by ammonium sulfate fractionation of pancreatin extracts and isoelectric precipitations (47). When the proteins precipitated by 0.39 saturated ammonium sulfate are chromatographed on DEAE-cellulose in a concentration gradient of pH 8.0 phosphate buffer, the two last and most acidic peaks contain procarboxypeptidase A in an electrophoretically homogeneous form (48). The molecular weight of the protein determined by light scattering and sedimentation-diffusion is 94-96,000. Its isoelectric point in univalent buffers of 0.2 ionic strength is below 4.5. 

A large proportion of the soy protein used in the food industry is in the form of protein isolates (>90% protein). Separation of proteins by isoelectric precipitation at the isoelectric pH range 4.2-4.6 is the recognized industrial process. The isoelectric point is the pH value at which the net global charge of the protein is neutral. At this pH value, the solubility of the protein is minimal and can result in complete precipitation. 

Separation of soy protein by EDBM has specific advantages over the conventional isoelectric precipitation used industrially for the production of soybean protein isolates. This technology does not use any added acids or bases during the process to adjust the pH of the protein solution, and the chemical effluents generated during the process could be reused at different stages in the... 

Bramaud, C., Mimar, P., and Daufin, G., Thermal isoelectric precipitation of a-actalbumin from a whey protein concentrate influence of whey-calcium complexation, Biotech-nol. Bioeng., 47, 121, 1995. 

After the initial extraction of the protein fractions with gonadotropic activity from the pituitary, and preliminary separation from other components by ethanol, salt, and isoelectric precipitations, the materials are further resolved by ion exchange and adsorption chromatography, gel filtration, and electrophoresis. 

The precipitation and fractionation of soy protein using CO2 in the pressure range Q.1. 7 MPa was also examined (111,112). Pressurized CO2 allowed better control of the solution pH than the addition of mineral acids and prevented local pH overshoots, which can result in poor quality precipitates. Protein precipitates formed by isoelectric precipitation using CO2 were spherical, whereas those formed using sulfuric acid were irregular (111). The decrease of solution pH to a value close to the isoelectric pH of the proteins was the primary cause of protein precipitation. Volatile acid precipitation is unlike conventional GAS, in which pressures of 5-10 MPa are required to achieve precipitation from an expanded organic solution. 

Hofland GW, de Rijke A, Thiering R, van der Widen LAM, Witkamp G-J. Isoelectric precipitation of soybean protein using carbon dioxide as a volatile acid. J Chromatogr B 2000 743 357-368. 

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