Purifying crude preparations

After initial cell fermentation and product extraction from the producer cells, the crude preparation is subject to multiple chromatographic steps, including ion-exchange, hydrophobic interaction chromatography and gel-filtration chromatography. The purified product is presented in lyophilized form in vials (1 mg active/vial) and excipients include a phosphate buffer, sodium chloride and serum albumin. 

By use of a crude preparation obtained after the cultivation of Aspergillus niger,104 pectinesterase was purified by repeated chromatography on DEAE-cellulose, using gradient elution. The homogeneity of the product was checked by free electrophoresis, sedimentation analysis, and determination of the N-terminal amino acid (phenylalanine). 

When measured at 40° with Lintner s soluble potato starch, the ratio of the dextrinogenic to the saccharogenic activities is approximately 6 to 1 for the amylase of Aspergillus oryzae. This value is given by both crude and purified preparations of the amylase if the measurements are carried out under comparable conditions. This constancy in the ratio of these two activities has led to the conclusion that, like pancreatic amylase, the amylase of Aspergillus oryzae is not accompanied in nature by beta amylase. Reacting mixtures of the amylase of Aspergillus oryzae and starch exhibit alpha mutarotation.72... 

For electrophoresis of the proteinase, saturated solutions of crude and purified preparations were made in 0.1 M Tris-glycine... 

In pharmaceutical and biochemical research, crude preparations of material will often exhibit significant desired biological activity. However, that activity will sometimes be lost when the material is purified. Comment. 

The present method is the only documented synthesis of trimethylamine-isocyanoborane adduct, the first boryl isocyanide reported. The procedure may be general and thus useful for the preparation of other amine-isocyano and amine-pseudohaloborane adducts. Readily available materials are used, and a high yield of crude or purified product is obtained in an estimated 10-hour working period. Schlenk techniques are advocated for this work, but if Schlenk-ware is not available solutions may be transferred quickly in air and subsequently handled under a nitrogen atmosphere. 

Hjelm prepared purified molybdic acid and obtained a pure regulus, which he examined with the microscope. In an unsuccessful attempt to fuse the molybdenum, he raised the temperature of the wind-furnace with fire-air (oxygen) obtained by adding two pounds of crude pyrolusite to the fire (24). 

Crude, rat-epididymal preparations were found to be much more stable than the purified enzyme,60 and, in the presence of Zn2+, there was no loss in activity in 3 hours at 37° at values of pH between 5 and 6. EDTA accelerated the inactivation at acid pH, for both crude and purified preparations. 

Because a-L-arabinofuranosidase is an extracellular enzyme, a crude preparation may be made simply by fractionation of the culture filtrate with ammonium sulfate. The enzyme can be purified from the crude enzyme-preparation by some suitable combination of ion-exchange chromatography, gel filtration, and similar techniques. Three examples of purification procedures, two from micro-organisms and one from a plant, are given here. 

Calculate the approximate percentage lipid composition of the nutmeg (assume the lipid recovery is quantitative). Prepare a table listing the R values obtained for each standard lipid and for the crude and purified unknown. By comparing R values, identify the general class of lipid to which your purified extract belongs. How effective was your purification step If... 

Early studies revealed that the 3, 5 -cyclic phosphate diesterase is present in all mammalian tissues (38, 33, 36), being most active in cerebral cortex (36, 37). It has also been identified in extracts of liver fluke (Fasciola hepatica), the common earthworm (Lumbricus terrestris), and fly larvae (36) and it has been studied in marine organisms (38), the cellular slime mold Dictyostelium discovdeum (39, AO), and in E. coli (Al). The enzyme has been partially purified from beef heart (30), dog heart, (A8) and bovine brain (37, A3). No highly purified preparations have yet been obtained and most studies have been performed with relatively crude preparations. 

N—O-sulfation of minoxidil in the presence of adenosine-3 -phospho-5 -phosphosulfate (PAPS) (equation 24). The enzyme-synthetized product was identical to authentic N—O-sulfate with respect to chromatographic behavior and mass spectral characteristics and was split to minoxidil when treated with sulfatase183. The pH optimum for minoxidil N—O-sulfation was about 8.0. Enzyme activity in crude preparations was maintained for several months during storage at —76°C, while activity of partially purified enzyme was lost under these conditions183. 

Versteeg (8) tested the heat stability of the purified PEs in orange juice, pH 4.0, and found that PEII was the least stable, being completely inactivated at 55°C (131°F) PEI was inactivated at 65°C (149°F), and HM-PE at 85°C (185°F). Heat inactivation curves of a crude preparation of orange PE in orange juice indicated that about 5% of the total PE activity was due to HM-PE, 60% to PEI and 30% to PEII. 

To a solution of 5-methoxymethoxy-7-oxa-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid methyl ester (4.9 g, 22.9 mmol) in 8/l-Me0H/H20 (175 ml, v/v) was added sodium azide (7.44 g, 114.5 mmol) and ammonium chloride (2.69 g, 50.4 mmol) and the mixture was refluxed for 15 h. The reaction was diluted with water (75 ml) to dissolve precipitated salts and the solution was concentrated to remove methanol. The resulting aqueous phase containing a precipitated oily residue was diluted to a volume of 200 ml with water and was extracted with ethyl acetate (3 times 100 ml). The combined organic extracts were washed with saturated NaCI (100 ml), dried (MgS04), filtered and evaporated. The crude was purified on silica gel (1/1-hexane/ethyl acetate) to afford 5-azido-4-hydroxy-3-methoxymethoxy-cyclohex-l-ene-l-carboxylic acid methyl ester (5.09 g, 86%) as a pale yellow oil. Subsequent preparations of 5-azido-4-hydroxy-3-methoxymethoxy-cyclohex-l-ene-l-carboxylic acid methyl ester provided material which was of sufficient purity to use in the next step without further purification. 

Table I. Aminoacyl Transfer to Microsomes with Crude and Purified Transferring Preparations...

The possible role of biotin in the synthesis of carbamyl-P was reinvestigated (27). The addition of biotin to crude rat liver enzyme resulted, in one series of experiments, in increased synthesis, ranging from 21 to 53% over the control level. This effect could not be reproduced with crude or purified preparations. The activity of frog liver enzyme is also unaffected by biotin at any stage of purification. 

Crude or purified preparations of chemokines may bind to polystyrene, therefore all storage and dilution tubes should be of polypropylene. When adding to the dishes, add to no more than three dishes at a time, followed immediately by the agar/medium mixture. 

Dumas) given by the crude and purified products were as follows Calculated 35.9 per cent. Found acid as prepared, 35.4 per cent once recrystallized, 35.9 per cent reprecipitated, 35.7 per cent. 

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