19] l-Rhamnulose 1-Phosphate

Abstract

Publisher Summary This chapter describes the preparation, purification, and crystallization of L-Rhamnulose 1-phosphate. L-Rhamnulose 1-phosphate may be prepared by using two mutant strains of Escherichia coli. E. coli K12 HfrP72 is grown overnight with shaking at 37° in Difco nutrient broth to the stationary phase. The mutagen N-methyl-N-nitroso-N'-nitroguanidine is added to a final concentration of 80 μg/ml, and the culture is held without shaking at 37° for 1 hr. L-Rhamnulose 1-phosphate aldolase is detected, after removal of nucleoproteins with MnCl2, with a coupled assay system. Two such mutants isolated used in the preparation described are: Rha-58 (kinaseless) and Rha-54 (aldolaseless). They are maintained on nutrient agar slants. The preparation of L-Rhamnulose is discussed. In the preparation of L-Rhamnulose1-phosphate, cells of the E. coli strain Rha -54 from 10 liters of culture medium are suspended in 75 ml of 20 mM potassium-phosphate buffer, pH 7.0, and disrupted in the French pressure cell. The supernatant fluid is concentrated to about 12 ml at 37° and chromatographed on a Sephadex G-10 column prepared and eluted with distilled water. Fractions containing L-rhamnulose 1-phosphate, detected by paper chromatography in butanone:acetic acid:water (15:5:2 v/v/v) followed by molybdic acid spray, are pooled and concentrated at 37°. L-Rhamnulose 1-phosphate, located by spraying a guide strip, is eluted from the paper with water, concentrated, rechromatographed in the same way, eluted with water, and concentrated at 37° to 10 ml. The preparation of crystalline dicyclohexylammonium salt of L-rhamnulose 1-phosphate is also discussed.