Protein products aqueous extraction processing

Soybean concentrate production involves the removal of soluble carbohydrates, peptides, phytates, ash, and substances contributing undesirable flavors from defatted flakes after solvent extraction of the oil. Typical concentrate production processes include moist heat treatment to insolubilize proteins, followed by aqueous extraction of soluble constituents aqueous alcohol extraction and dilute aqueous acid extraction at pH 4.5. 

Also free amino acids and peptides have been extracted from enamel. The material is very similar to that of dentine. Peptides from bone and teeth extract have an apparent molecular size of 750 to 5000 daltons163. Also peptides have been found in aqueous extracts of egg shells from birds and reptiles164 It has been assumed that free peptides and glycopeptides may have effects on the nucleation of the mineral phase185). On the other hand, those products could be split off from matrix proteins during crystallization processes. Perhaps there may be an association between free peptides and mucopolysaccharides or glycopeptides. It cannot be excluded that they are artefacts due to isolation procedures164). 

Reverse micelles are self-organized aggregates of amphiphilic molecules that provide a hydrophilic nano-scale droplet in apolar solvents. This polar core accommodates some hydrophilic biomolecules stabilized by a surfactant shell layer. Furthermore, reverse micellar solutions can extract proteins from aqueous bulk solutions through a water-oil interface. Such a liquid-liquid extraction technique is easy to scale up without a loss in resolution capability, complex equipment design, economic limitations and the impossibility of a continuous mode of operation. Therefore, reverse micellar protein extraction has great potential in facilitating large-scale protein recovery processes from fermentation broths for effective protein production. 

Purification of b-galactosidase from E. coli may be compared Higgins (26) outlines a process of succeeding centrifugations to remove cell debris, nucleic acids precipitate, and protein precipitate (product). Veide (23) outlines a single aqueous extraction with PEG and salt in which b-galactosidase partitions to the PEG-rich phase. Cells, nucleic acids, and a major part of the contaminating proteins partition to the salt-rich phase. 

Besides the extraction of organic pollutes from aqueous solutions, ILs can be also applied to the extraction of target products in biological conversion and separation process. For example, several ILs were reported as extractants to separate antibiotics and proteins from aqueous solutions (Cull et al., 2000 Tzeng et al., 2008). 

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

The two major leafy crops used for the production of recombinant proteins are tobacco and alfalfa, both of which have high leaf biomass yields in part because they can be cropped several times every year. The main limitation of such crops is that the harvested leaves tend to have a restricted shelf life. The recombinant proteins exist in an aqueous environment and are therefore relatively unstable, which can reduce product yields [16]. For proteins that must be extracted and purified, the leaves need to be dried or frozen for transport, or processed immediately after harvest at the production site. This adds considerably to the processing costs. 

Aqueous two-phase cultivation of plant cells was found to be possible when the culture conditions were optimized. The selection of proper polymers and the optimization of their concentrations are needed not only for cell growth, but also for the desirable product partition. Promising examples of the use of aqueous two-phase systems for in situ extraction have been reported recently. However, more detailed and broader studies are necessary to utilize fully the potential of aqueous two-phase systems in plant cell culture. Finally, we believe that the development of fully integrated processes for plant cell cultures will improve the production efficiency of different kinds of secondary metabolites, enzymes, and recombinant proteins. 

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