Lactalbumin Phosphate

Lactalbumin phosphate is produced commercially from whey protein, which constitutes about 20% of the total milk protein. After removal of the casein from skim milk, long-chain polyphosphate salts are added to the whey to precipitate calcium lactalbumin phosphate at pH = 6.0. This product is filtered and dried below bO C so as not to denature any of the proteins present which include some 

I I Phosphorylated residues FIGURE 10.22 Bovine asl-casein—amino acid sequence. 

FIGURE 10.23 Bovine as2 casein—amino acid sequence. Phosphorylation may also occur at Ser 31 andThr 

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Biopolymer phosphates - casein, lactalbumin phosphate, starch phosphates, lecithin... 

Lactalbumin phosphate (molecular mixture) Starch phosphate (molecular mixture)... 

Lactalbumin phosphate can replace sodium caseinate in coffee whiteners and non-butterfat dairy products. A typical formulation for a coffee whitener is... 

Improvements in self-raising flour, biscuits and cake mixes by the addition of about 2% lactalbumin phosphate are claimed in patent literature. Instant cold water-soluble forms of gelatine can be obtained by reaction with lactalbumin phosphate [90,91]. 

Nitrogen sources include proteins, such as casein, zein, lactalbumin protein hydrolyzates such proteoses, peptones, peptides, and commercially available materials, such as N-Z Amine which is understood to be a casein hydrolyzate also corn steep liquor, soybean meal, gluten, cottonseed meal, fish meal, meat extracts, stick liquor, liver cake, yeast extracts and distillers solubles amino acids, urea, ammonium and nitrate salts. Such inorganic elements as sodium, potassium, calcium and magnesium and chlorides, sulfates, phosphates and combinations of these anions and cations in the form of mineral salts may be advantageously used in the fermentation. 

The proteins of milk fall into several classes of polypeptide chains. These have been delineated most completely in bovine milk, and a system of nonmenclature has been developed for them (Chapter 3 Eigel et al. 1984). One group, called caseins, consists of four kinds of polypeptides asr, as2-. and 3-, and k- with some genetic variants, post translational modifications, and products of proteolysis. Almost all of the caseins are associated with calcium and phosphate in micelles 20-300 fim in diameter (see Chapter 9). The other milk proteins, called whey proteins, are a diverse group including /3-lactoglobulin, a-lactalbumin, blood serum albumin, and immunoglobulins (Chapter 3). Almost all... 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

Ribonuclease A, insulin, a-lactalbumin, myoglobin Methacrylic monolith with tertiary amino functions 30% Acetonitrile in 60 mM aqueous sodium phosphate, pH 2.5 390 mm x 50 pm i.d. 290 mm effective length... 

There are a number of media available which are not based on a detailed investigation of growth requirements, but rather include crude mixtures of nutrients added to promote cell growth. These include lactalbumin hydrolysate (Appendix 1 Table 9) or yeast extract (Appendix 4) to provide an inexpensive source of amino acids or vitamins. Thus Melnick s monkey kidney media A and B (Melnick, 1955) contain lactalbumin hydrolysate and calf serum in Hanks and Earle s BSS, respectively. Chick embryo extract and tryptose phosphate broth (Appendix 1, Tables 11 and 12) are also used occasionally and their use is referred to where appropriate throughout the book. Mitsuhashi and Maramorosch mosquito cell medium contains lactalbumin hydrolysate, yeast extract and foetal calf serum in a specially developed saline (Mitsuhashi and Maramorosch, 1964 Singh, 1967). 

Chromatographic separation of lactalbumin from human milk whey on calcium phosphate columns. Nature 181, 996 (1958). 

Protein Composition of Milk. Skim milk is a colloidal suspension of extreme complexity. The particulate phase, the casein micelles, consists primarily of a mixture of asi, as2, / , and x-caseins combined with calcium ions and an amorphous calcium-phosphate-citrate complex. The soluble phase contains lactose, a fraction of the caseins and calcium, and, in raw milk, the whey proteins, which are predominantly /3-lacto-globulin and a-lactalbumin. When milk is centrifuged at high speed (in our experiments, 30 min at 110,000 X gravity), the casein micelles sediment. This permits one to separate the two physical phases of skim milk and to measure changes in composition of the phases resulting from... 

Figure 7 Effect of salt concentration on migration velocity of DMSO, bovine carbonic anhydrase (BCA), a-lactalbumin (a-LAC), soybean trypsin inhibitor (STI), and ovalbumin (OVA). Conditions column 50 pm i.d. x 340/260 mm, packed with strong anion exchanger mobile phase, 50, 100, and 200 mM NaCI in 5 mM phosphate buffer, pH 7.0 applied voltage, -1 5 kV injection, 6 s at-8 kV UV detection, 200 nm. (Reprinted with permission from Ref. 48, copyright 2000, American Chemical Society.)...

In the presence of a-lactalbumin, however, only glucose and cellobiose (Glc)3l-4Glc) [37] are the preferred acceptors. Neither o-mannose, D-allose, D-galactose, o-ribose, nor D-xylose are substrates. Glucuronic acid and a-glucose-1-phosphate (monosaccharides with a negative charge) are also not accepted. 

Severe heat treatments, especially >80°C, adversely affect the rennet coagulation of milk. Although changes in calcium phosphate equilibria are contributory factors, complexation of /3-lactoglobulin and/or ot-lactalbumin with x-casein via intermolecular disulfide bond formation is the principal factor responsible (see Fox, 1988). Most authors agree that the primary and, especially, the seconday (nonenzymatic) phase of rennet coagulation are adversely affected by severe heat treatments, as is the strength of the... 

FIGURE 5.17 Effect of ACN concentration in the eluent on the separation of four proteins. Column, 39 cm (effective length 29 cm) x 50 p.m i.d., fused-silica capillary with porous methacrylic monolith having tertiary amino functions mobile phase, ACN (%, v/v) in 60 mM aqueous sodium phosphate, pH 2.5 applied voltage, —25 kV detection, 214 nm sample (1) ribonuclease A, (2) insuhn, (3) a-lactalbumin, and (4) myoglobin. (Reprinted from Zhang, S., et al., J. Chromatogr. A, 887,465-477, 2000. With permission from Elsevier.)... 

FIGURE 47.2 Repeated isocratic HPLC separations of proteins on chip. (Reprinted with permission from Reichmuth, D. S., etal.. Ana/. Chem., 77,2997,2005. Copyright 2005 American Chemical Society.) Conditions lauryl methacrylate monolith mobile phase 24% acetonitrile + 0.16% heptafluorobutyric acid in 5 nunol/L phosphate buffer (pH 2.0) Injections 6,4 nL, 750 ms, pressure 0.2 MPa. Peaks free dye (a), insulin (b), antibiotin (c), a-lactalbumin (d). 

FIGURE 13.14 Dependence of the denaturation enthalpy on the denaturation temperature for (a) lysozyme and (b) a-lactalbumin, both dissolved in 0.05 M phosphate buffer pH 7. (Adapted from Haynes, C.A. and Norde, W., J. Colloid Interface ScL, 169, 313, 1995.)... 

Figure 11.3 ABTS (2,2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid) radicai scavenging activity of a-lactalbumin (a-LA), P-lactoglobulin (p-LG), bovine serum albumin (BSA), and 3-casein at 5 [xM and in 10 mM phosphate buffer at pH 7.4.

Figure 16 Effects of pH and salt type on band spacing in the anion-exchange separation of a protein sample 1, myoglobin 2, transferrin 3, a,-acid glycoprotein 4, a-lactalbumin 5, soybean trypsin inhibitor. Conditions 5 x 0.4-cm WAX column (300A pore) (Shimadzee Shim-pack) 0-100% B in 20min I.OmL/min. (a) A solvent, 0.02 M phosphate buffer B solvent, 0.5 M phosphate buffer (b) A solvent, 0.02 M phosphate buffer B solvent 0.5 M NaCI or Na2S04. (From Ref. 41.)...

Bovine Albumin, 66 kD, Egg Albumin, 45 kD, Glyceraldehyde-3-Phosphate Dehydrogenase, 36 kD, Carbonic Anhydrase, 29 kD, Trypsinogen, 24 kD, Trypsin Inhibitor, 20 kD and a-Lactalbumin,... 

Scheme 5. Solid-phase enzymatic synthesis of a Lewis tetrasaccharide using a linker of 71 atoms between the monosaccharide acceptor and the Sepharose matrix. Reagents i) 1. cacodylate buffer pH 7.5, 5 mM MnCb, a-lactalbumin, GT, UDP Gal (3 eq.), CIAP, 37 °C, 5 days 2. cacodylate buffer pH 7.35, 0.1% triton X-100, ST3, CMP-NeuAc (2.5 eq.), 3 him MnCb, 0.02% NaNj, 35 °C, 5 days 3. cacodylate buffer pH 6.5, 5 mM MnCL, FT, GDP-Fuc (4 eq.), 37 °C, 5 days ii) 1. DTT, 0.1 M phosphate buffer 2. BioGel P-2, 57% from 14.

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