Lactose purification

Bissett, D.L. Anderson, R.L. Lactose and D-galactose metabolism in Staphylococcus aureus. Ill Purification and properties of D-tagatose-6-phosphate kinase. J. Biol. Chem., 255, 8745-8749 (1980)... 

A general, recent trend has been to apply the principles of ion exchange to the purification of whey or lactose solutions. Anionic and cationic exchange resins are used to remove impurities from the solution, which can then be condensed and crystallized or spray-dried directly. Ahlgren (1977) and Delaney (1976) have reviewed developments... 

Guy, E. J. 1979. Purification of syrups from hydrolyzed lactose in sweet whey permeate. J. Dairy Sci. 62, 384-391. 

I The second purification procedure we examine illus- Vri trates an unusual approach to the purification of a membrane-bound protein. The lactose carrier protein of E. coli is normally tightly bound to the plasma membrane. This protein is involved in the active transport of the dissaccharide lactose across the cytoplasmic membrane. When lactose carrier protein is present, the intracellular concentration of lactose can achieve levels 1,000-fold higher than those found in the external medium. Ron Kaback devised a simple yet elegant procedure for the purification of this protein. 

Purification of the membrane-bound lactose carrier protein is a very different problem from the purification of the soluble OMP synthase. Both the approach to purification and the assays for the protein during purification are quite novel. The assay involves reconstituting a transport system with membranes that are free of lactose carrier protein, then adding the partially purified carrier protein and radioactively labeled lactose. The activity in this assay system is proportional to the transport of radioactive lactose across the membrane in the cell-free reconstituted system. 

Outline of purification procedure for lactose carrier protein from E. coli. 

The purification of two proteins, UMP synthase from mammalian tumor cells, and lactose carrier protein from E. coli bacteria is described in detail to illustrate how different fractionation methods can be combined most effectively. 

Methods for Purification and Characterization of Proteins, the primary goal is to acquaint the reader with the techniques used for protein purification. The first part of chapter 6 presents methods for protein fractionation. In the second part of this chapter, purification procedures for two proteins, UMP synthase and lactose carrier protein, are presented so that the student can see how different purification steps are combined for maximum effectiveness. 

In cases where it has proved impractical to assay for activity at each step, protein purification has been aided by tagging a fraction of the molecules with a photoaffinity reagent (e.g. the lactose carrier of Escherichia coli Newman et al., 1981 and the /J-adrenergic receptor of frog erythrocytes Shorr et al., 1982). 

The specific activity of the enzyme was determined in 0.15M lactose solution (0.02M phosphate buffer, pH 7-0). The activity of the enzyme was expressed in terms of units of activity per mg of enzyme. A unit of activity was defined as a p mole of glucose produced per minute. The soluble lactase activity following affinity chromatography purification was 37-1 units/mg. This represented a 4 fold increase in catalytic potency over the specific activity of the crude enzyme preparation (8.9 units/mg). 

Filtration of small (nano) particles from solvent using a filter with extremely small pores (0.001-0.010 micron) finer than ultrafiltration, not as fine as reverse osmosis. Used for the removal of viruses from plasma protein products. See Yaroshchuk, A.E., Dielectric exclusion of ions from membranes, Adv. Colloid Interface Sci. 85,193-230,2000 Rossano, R., D Elia, A., and Riccio, R, One-step separation from lactose recovery and purification of major cheese-whey proteins by hydroxyapatite — a flexible... 

Application of NF in the simultaneous demineralization and concentration of whey, concentration of lactose from the permeate of the UF whey, and the purification of membrane cleaning solutions. 

The purification process for UF retentates is optimized by including a diafiltration (DF) step [3,34]. Compared with conventional processes, UF with DF has the advantage of allowing high product purity and process yield such as the removal of lactose from milk to the desired degree [3,35]. This process commonly includes three basic steps (1) a pre-concentration stage,... 

Lactose is one of the most widely used excipients in tablet manufacture. It is available in a number of different forms, differing in hydration and crystal states. Isolation and purification may involve treatment with sulphur dioxide. However, there are no reports of complications from residues of this powerful oxidizing agent. 

Tautomeric lactose hydrazone 77/78 was obtained from 2,2,2-trifluoroethyl-3-oxobutanoate 76 with hydrazine tautomers 74/74 in a 1 3 ratio using acetonitrile and water to give, after lyophilization, a mixture of pyrazol-3-one 79 and l-lactosylpyrazol-3-one 80 (00CAR169) (Scheme 19). They were identified by HPLC-ESIMS, their stability being limited due to rapid atmospheric oxidation. Further reaction of this mixture with 4-methylbenzenediazonium chloride in aqueous sodium bicarbonate followed by addition of 2,4-pentadione gave, after purification by HPLC, stable 2-/)-D-lactosyl-4-[2-(methylphenyl)diazenyl]pyra-zol-3-one 81 in 15% yield. 

The separation,purification and concentration of a thermosensitive bioactive compound from a lysate has been carried out combining UF, ion exchange and RO with significant cost reduction and productivity increase. Enzyme membrane reactors have been used for triglyceride enzymatic hydrolysis and product separation. Thermophi1ic,thermostable enzyme ultrafiltration membrane have been prepared, and used in high temperature lactose hydrolysis. 

GalU, and o -l,3-GalT were immobilized onto a Ni resin column, and then a solution of Glu-l-P, UTP, and lactose containing 0.01 mol Tris-HCl and 0.01 mol MnCl2 was circulated through the column driven by a pump at room temperature. After the reaction was completed, the solution was pumped out for purification, and the column was ready for the next batch of reaction. 

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