Extraction and Purification

1963) strong alkali isomerizes, enolizes, and hydrolyzes glycopyranoses. Starch, for example, is decomposed after enediol isomerization antecedent to excision of simple organic acids (Schoch, 1964). High temperatures expectedly accelerate the processes. 

Starch may be extracted simply with hot water, but the mildness of this exercise requires separate inactivation of depolymerases (by heat or ethanol) if this biopolymer is to be used for food, or mercuric chloride (0.01 M) if it is to be used other than for food. The crude water extracts are centrifuged, filtered, and spray- or freeze-dried in preparation for storage. Where there is protein, in small samples, most of it can be removed by shaking the crude extract with one-tenth its volume of toluene and discarding the toluene layer. Lipid matter is dissolved by refluxing with aqueous 80% methanol. 

In one outline, cellulose from Valonia ventricosa, an alga, was boiled in excess 1% aqueous NaOH for 6 h, with a change of alkali solution after 3 h the alkali-treated cellulose was washed with distilled water (Blackwell, et al., 1977), then immersed overnight in 0.05-N HC1 at room temperature. The 

Aoki et al. (1971) obtained the crude enzyme as an acetone powder. The procedure will be described here in brief. A fresh sample is blended with cold acetone (—20°C). The homogenate is filtered and dried in a desiccator under reduced pressure at 0 °C overnight. The acetone powder can be stored in a freezer at —20 °C for at least 6 months with little loss of PAL activity. For preparation of the enzyme solution, 1 g of acetone powder is suspended in 40 ml of 0.05 M sodium borate buffer (pH 8.8) at approximately 3 °C for 1 h, and the suspension is cleared by centrifugation at 7000 g for 10 min at 0 °C. The supernatant obtained is used as the crude enzyme solution. The crude enzyme solution also is stable. 

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This seaweed became known as Irish moss. The extraction and purification of the polysaccharide from Irish moss was patented in 1871 (29). This polysaccharide eventually became known as carrageenan it was not produced and marketed until 1937. 

The properties of a botanical gum are determined by its source, the climate, season of harvest, and extraction and purification procedures. Table 6 illustrates one of the important basic properties of all gums, ie, the relationship between concentration and solution viscosity. The considerable viscosity variation observed among gums from different sources determines, in part, their uses. 

J. C. P. Chen and C.-C. Chou, eds.. Cane Sugar Handbook, John Wiley Sons, Inc., New York, 1993 comprehensive reference with emphasis on sucrose extraction and purification. 

MisceUaneous uses include extraction and purification of penicillin, alkaloids, vitamins, and flavors, and as an intermediate in the preparation of dyes and pesticides. Chloroform has also been used as a fumigant and insecticide, in the formulation of cough symps, toothpastes, liniments, and toothache preparations. These latter uses were banned by the FDA in 1976 (38). 

It is recommended that the concentration of sulfuric acid in the initial solution be kept at 2-4 mol per liter for the extraction of tantalum, whereas for the extraction of niobium, the concentration of sulfuric acid must be increased to a minimum of 6 mol per liter [458,481]. In some cases, the presence, in the initial solution, of titanium in the form of fluorotitanic acid ensures the successful and selective extraction and purification of tantalum and niobium with no addition of any other mineral acid [482]. 

Ion exchange resins have been widely used for the extraction and purification of amino acids from the fermentation broth. 

Extraction and purification of luciferin and luciferase (Viviani etal., 2002a) To isolate luciferin, the lanterns of the Australian A. flava were homogenized in hot 0.1 M citrate buffer, pH 5, and the mixture was heated to 95°C for 5 min. The mixture was acidified to pH 2.5-3.0 with HCl, and luciferin was extracted with ethyl acetate. Upon thin-layer chromatography (ethanol-ethyl acetate-water, 5 3 2 or 3 5 2), the active fraction of luciferin was fluorescent in purple (emission Lav 415 nm when excited at 290 nm). To isolate the luciferase, the cold-water extract prepared according to Wood (1993 see above) was chromatographed on a column of Sephacryl S-300. On the same... 

Extraction and purification. Protein P was extracted and purified from E. pacifica and M. norvegica at near 0°C (Shimomura, 1995a). The outline of the procedure used is as follows. 

Extraction and purification (Shimomura et al., 1978). Dr. Yata Haneda and Dr. Shoji Inoue kindly provided us with specimens of O. gracilirostris, which had been picked out one by one from large commercial catches of Sergestes lucens netted in Suruga Bay, Japan. The material was air-dried or freeze-dried before shipment to the U.S. 

Roberts et al. (198 7) and Muller and Campbell (1990) used slightly different methods to extract and purify pholasin. They used lOmM ascorbate, instead of diethyldithiocarbamate, to inhibit luciferase in the process of extraction and purification, which enabled them to obtain the purified preparations of both pholasin and the luciferase. 

Extraction and purification of luciferin (Shimomura etal., 1963d). 

Extraction and purification of Diplocardia luciferase (Bellisario et al., 1972). About 50 specimens of Diplocardia longa (widespread in southern Georgia about 30 cm in length) were electrically stimulated in 250 ml of 0.1 M EDTA at 4°C to exude coelomic fluid. The suspension of coelomic cells obtained was centrifuged at 480 g for 5 min. The pellets from 200 worms were combined and an acetone powder was prepared. The acetone powder obtained (about 10 g) was stable at -80°C for at least one year. 

Extraction and purification of polynoidin (Nicolas et al., 1982). Specimens of Harmothoe lunulata were collected near Roscoff, Brittany, in France, and were anaesthetized with 0.1 mM tricaine in seawater. The scales were carefully removed with tweezers, and washed several times with seawater to remove tricaine. Then the scales were stored in liquid nitrogen until they were used. 

Extraction and purification (Shimomura, 1989). Fruiting bodies are extracted with 30% methanol, and the extract is concentrated under reduced pressure. The residual solution (pH 6.0) is first washed with ethyl acetate, then acidified to pH 2.0 with 1 M HC1 and extracted with ethyl acetate. The extract is evaporated nearly to dryness. The residue is dissolved in 30% methanol and purified by the following 3-step procedure. 

Extraction and purification (Shimomura, 1991b). The luciferin precursors PS-A and PS-B are extracted from the dried fruiting bodies of Panellus stipticus (5 g) with methanol, and the extract is evaporated under reduced pressure to remove most of the methanol. The residue (pH 6.3) is diluted with a small amount of water and extracted with ethyl acetate, and the extract is evaporated to dryness. The precursors in the residue are purified by three steps of silica gel chromatography and one step of HPLC, while monitoring the absorbance of eluate at 220-230 nm. The procedure is summarized below ... 

Extraction and purification of photoprotein (Shimomura, 1986b). The live animals are chilled to 5°C, and the five arms of each specimen are cut off and dropped into cold 3.5% magnesium acetate solution. After 10 min, the arms are drained from the solution, and stored at... 

The substances involved in bioluminescence reactions are usually unstable. Thus, the extraction and purification of bioluminescent substances should be carried out in the shortest possible period of time, usually at a low temperature. It is known through experience that luminescent substances are almost always more stable in the original animal tissues than in extracts when preserved at a low temperature. Therefore, before starting extraction and purification, the stability of the extracts and purified substances should be investigated by carrying out a small-scale pilot experiment. A pilot experiment is also essential in the course of purification to avoid an unexpected loss of the target substance. If a component of the luminescence system is insoluble in common buffer solutions, it must be solubilized to purify it (see C1.3). 

A small amount of sodium dithionite added to an aqueous medium depletes oxygen almost instantly. Due to the acidity of the reagent, a sufficiently strong buffer must be included in the medium. However, the use of dithionite is not recommended in the stages of extraction and purification because the reagent and its decomposition products are highly reactive and can cause undesirable reactions. 

Extraction and purification. The processes of extraction and purification of luciferins and photoproteins are usually the most critical part of the project, and the work often involves various difficulties and complexities specific to the substance under investigation, such as a highly labile nature and extremely low content. Some discussion on this matter is given in Section Cl. 

Structure determination of luciferin. Once a luciferin is obtained in a sufficient purity, the determination of luciferin structure should be attempted most of the important properties of luciferin are usually already obtained during the course of purification as a necessity. The structural study is considerably more straightforward than the extraction and purification, due to the availability of advanced methods, such as high-resolution mass spectrometry and various NMR techniques. If help or collaboration is needed in structure determination, the attractiveness of a luciferin will make it easy to find a good collaborator. However, the purified luciferin is usually an extremely precious material considering the effort spent in preparing it. To avoid accidental loss of the purified material, the chosen collaborator must have solid knowledge and experience in structure determination a criterion to be considered is that the person has successfully done the structure determination of at least one new natural product. 

Vysotski, E. S., Bondar, V. S., and Letunov, V. N. (1989). Extraction and purification of obelin, a calcium-activated photoprotein from the hydroid polyp Obelia longissima. Biokhimiya 54 965-973. 

Ward, W. W., and Seliger, H. H. (1974a). Extraction and purification of calcium-activated photoproteins from ctenophores. Biochemistry 13 1491-1499. 

Bacterial bioluminescence, 30-46 factors required, 31 general scheme, 32 in vivo luminescence, 41 luminescence reaction, 37, 38 Bacterial luciferase, 33-35, 343 assay, 39 cloning, 34 crystal structure, 34 extraction and purification, 34 inactivation, 34, 35 molecular weight, 34 properties, 34 storage, 35 subunits, 34... 

Balanoglossus luciferase, 315 extraction, 315 Balanoglossus luciferin, 316 extraction, 316 Basidiomycetes, 266 Bathophilus, 338 Beneckea, 31, 33, 34, 333 Bentbalbella, 339 Benthosema, 339 2-Benzylimidazopyrazinone, 168 Beroe, 155, 334 Berovin, 155, 346 extraction and purification, 155 spectral properties, 156 BFP, 101... 

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