Isoleucine purification

Purification. The objective of crystallization also can be purification of a chemical species. For example, L-isoleucine (an essential amino acid) is separated by crystallization from a fermentation broth that has been filtered and subjected to ion exchange. The recovered crystals contain impurities deleterious to use of the product, and these crystals are, therefore, redissolved and recrystalHzed to enhance purity. 

The experimental crystallizer was a 350-mL jacketed glass unit that was provided with sufficient agitation to keep the contents well mixed. The compositions of the charges to the crystallizer were adjusted by adding the amino acids in predetermined ratios concentrations of the amino acid impurities were maintained in ranges comparable to those found in the recovery and purification of L-isoleucine from industrial fermentation reaction masses. The experiments were divided according to the mode of crystallization ... 

Additions of valine, leucine, and isoleucine were found to relieve the growth inhibitory effects of TP on Bacillus cell cultures, soybean cell cultures, and Arabidopsis seedlings. ALS isolated from a number of sources was found to be sensitive to TP at nM levels. The barley enzyme has been amenable to purification. A purification procedure that gives >60 % recovery and 235-fold purification is described. 

R. Carney, of Zoecon Corporation, Palo Alto, Calif., who is collaborating in this research, developed a synthesis of 96% ee (R)-2-methylbutyric acid from D-isoleucine (Figure 14). Another approach that involved HPLC purification of diastereomeric amides with subsequent cleavage of the amide link gave the R-acid in 94% ee. 

Viscotoxin, a basic peptide of molecular weight ca. 9000 (Samulsson, 1961), moves unretarded in strongly cross-linked gels such as Sephadex G-25 (Fig. 4a) (in phosphate buffer, ionic strength 0.05, pH 6.8. When filtered under similar conditions in weakly cross-linked dextran, viscotoxin behaves quite differently (Fig. 4b). In fact it moves behind isoleucine. The gel of the first kind can be used to remove solutes of lower molecular size, the purification being based on molecular exclusion. Filtration in the second kind of gel may be used not only for separating solutes of different molecular size but also to separate peptides and other substances of similar molecular size when they differ in certain structural features. 

Ikeda, Y. Tanaka, K. (1983) J. Biol. Chem. 258, 9477-9487. Purification and characterization of 2-methyl-branched chain acylcoenzyme A dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria. 

A mixture of 10 mmol amino acid, 0.9 g ammonium thiocyanate, 10 mL acetic anhydride, and 1.3 mL acetic acid was heated at 100°C for 30 min. Then the mixture was poured into 50 mL water, and an oil separated from the water, which subsequently crystallized. Further purification was accomplished by recrystallization of the crude 2-thiohydantoin from ethanol. The 5- ec-butyl-2-thiohydantoin from racemic oL-isoleucine has melting point of 163°C. (no yield was given for this preparation). 

Synthesis. Dn-Isoleucine is synthesized in about 27% over-all yield by the method of Marvel (554). Diethyl sec.-butylmalonate (A) is prepared in 83% yield from diethyl malonate, sodium, absolute ethanol and sec.-butyl bromide (b.p. 91.3°C.) essentially by the method of Romburgb (673). a-Bromo- S-methylvaleric acid (B) is pr ared in about 67% yield by the alkaline hydrolysis of (A), isolation of sec.-butylmalonic acid ((j), and bromination and decarboxylation of (C). DL-Isoleucine (D), prepared by amination of (B), is recrystallized from 30% ethanol. It has been suggested (39, 485) that the product should be repeated recrystallized from 80% ethanol to free it from allo-isoleucine. It has been found in the writers laboratory that recrystallization from 20% ethanol is an effective purification procedure. The described s thesis is essentially that originated by Brasch and Friedman (125) and Ehrlich (237) and employed by Abd halden and Zeisset (39). 

Various amino adds have been detected as impurities in available samples of other amino acids by several investigators. Hegsted and Wardwell (385) found isoleucine as a eontaminant in five of seven samples of OL-leudne examined while Baumgarten et al. (78) observed that a sample of L-leucine contained isoleucine and methionine and that one of L-tryptophan contained t3rrosine. In the wnters laboratory, microbiologioal assay methods have been used extendvely to determine various amino acid impurities in amino acids in process of purification as well as... 

Rudman and Meister IJ ) first showed the presence of a transaminase in cell-free extracts of E. colt that catalyze transamination reactions between glutamate and isoleucine, valine, leucine, norleucine, and norvaline. These monocarboxylic amino acids transaminated with each other as well as with glutamine. Preparations of an E. cdi mutant which did not respond to a-keto- 8-methylvalerate was unable to transaminate isoleucine or valine. The transaminase responsible for activity with the branched-chain amino acids was separated from other transaminases and considerably purified by standard methods of protein purification. It was shown to... 

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