Lysine-based concentrates

An example of an industrial spray-drying system where the combined sorption-spray drying is applied for manufacturing of forage lysine-based concentrates is shown in Figure 12.16. As reported by Tutova and Kuts (1987), introduction of wheat bran directly into the spray cone of dispersed lysine (with the mass ratio of 0.7 to 1.0 kg of bran/kg of dry lysine) markedly improved the product quahty. In particular, the following features were claimed ... 

E. A. L. Biessen, F. Noorman, M. E. van Teijlingen, J. Kuiper, M. Barrett-Bergshoeff, M. K. Bijsterbosch, D. C. Rijken, and T. J. C. Van Berkel, Lysine-based cluster mannosides that inhibit ligand binding to the human mannose receptor at nanomolar concentration, J. Biol. Chem., 271 (1996) 28024—28030. 

Even with the uncertainty in E2 active-site position, the models have suggested that there would be no E3 residues near the E2 active site, in agreement with the observations made in the c-Cbl-E2 structure. This again ruled out the possibility that the SCF E3 provides acid/base catalysis and the possibility that the SCF positions the -amino group of the lysine at the E2 active site [66]. The only plausible mechanism left accounting for the catalysis mediated by the SCF in substrate ubiq-uitination is that the E3 complex helps increase the effective concentration of a portion of the substrate that contains the physiological ubiquitination-site lysine at the E2 active site. This model made the testable prediction that the distance between the destruction motif and the ubiquitinated lysine is a determinant of the ubiquiti-nation efficiency. 

Dissolve the required amount of peptide (1 mole peptide per about 50 moles of lysine residues of the carrier, e.g., 60 pmol of peptide per 5 mg ovalbumin) in PBS and mix with the activated carrier. Stir at RT for 1 h and block with 10 mg/ml of solid NaBH4 at RT for 20 min. Alternatively, reduce (block) the formed azome-thines (Schiff bases) to secondary amines by addition of ascorbic acid (final concentration 5 mM). 

Amino acids with basic side chains Basic amino acids are the opposite of acidic amino acids. Their most important role is to form ionic bonds to negative ions—phosphate and the like. Lysine is a simple example. The side chain contains four CH2 groups and terminates in —NH3 +. Arginine has an even more basic, if somewhat more complicated and larger, side chain. Conversely, the side chain of histidine is not as basic as that of lysine and the concentrations of the unprotonated and protonated forms of histidine are almost equal at biological pH. Strong Lewis acid-Lewis base complexes between the unprotonated form of histidine and metal ions is very common in proteins. Histidine side chains are also involved in moving protons from one atom to another. 

The most frequently used protein assay is based on a method after Bradford (Bradford, 1976), which combines a fast and easily performed procedure with reliable results. However, the Bradford assay has sensitivity limitations and its accuracy depends on comparison of the protein to be analyzed with a standard curve using a protein of known concentration, commonly bovine serum albumin (BSA). Many commercially available protein assays such as those from Pierce or BioRad rely on the Bradford method. The assay is based on the immediate absorbance shift from 465 nm (brownish-green) to 595 nm (blue) that occurs when the dye Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. Coomassie dye-based assays are known for their non-linear response over a wide range of protein concentrations, requiring comparison with a standard. The dye is assumed to bind to protein via an electrostatic attraction of the dye s sulfonic groups, principally to arginine, histidine, and lysine residues. It also binds weakly to the aromatic amino acids, tyrosine, tryptophan, and phenylalanine via van der Waals forces and hydrophobic interactions. 

That summary is based on the reports of a well-conceived and carefully executed research program carried out by Rohan. Mohr et al. (7) extended these studies and was able to draw additional conclusions. First, without exception, free amino acids are much more sensitive to destruction in this system than the peptide-bound amino acids. Second, differences in the stability of amino acids under these conditions are not great —from 25% loss for isoleucine to 68.5% for lysine, over a relatively short period of time. In this system the reducing sugars must be the limiting factor, since the glucose and fructose are completely destroyed or removed. Third, neither cystine nor cysteine are reported to be present, and the only other sulfur-containing amino acid, methionine, is present at a much lower concentration than any other amino acid. Clearly, as we shall see later, cocoa would probably have a considerably different flavor if cysteine or cystine were present in the fermented beans. 

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