CS267706B1 - Method for isolating D-ribose - Google Patents

Abstract

The solution concerns the isolation of D-ribose from mixtures with D-glucose. The essence of the solution consists in separating the mixture of both aldoses by chromatography of an aqueous solution in a column of cation exchange resin based on spherical sulfonated styrene-divinylbenzene copolymers, containing not more than 8% by weight, preferably 4 to 5% by weight of divinylbenzene, with a grain size of not more than 0.5 mm, preferably 0.1 to 0.3 mm, saturated with alkali metal ions, especially sodium, or alkaline earth metal ions, especially calcium, strontium and barium. The solution has application in the fermentation industry in obtaining an intermediate product of riboflavin synthesis.

Description

The invention relates to a process for isolating D-ribose from mixtures with D-glucose, obtained, for example, by fermenting D-glucose-containing nutrient media, microorganisms which produce D-ribose.

D-ribose is part of some of the basic components of living matter. It is contained in nucleic acids. Its reduced derivative, ribitol, is part of riboflavin, which acts as an active center for flavoprotein enzymes in electron transport systems (in the respiratory chains),

D-ribose is of industrial importance especially for the synthesis of riboflavin - vitamin Bg. The need for this vitamin is constantly increasing, both for the purposes of human medicine and especially as a supplement to feed rations in modern livestock farms.

The classic method of D-ribose production consists in alkaline epimerization of D-arabonic acid, obtained by oxidation of sucrose with oxygen in the presence of KOH, into D-ribonic acid, its isolation, lactonization and reduction with electrochemically prepared amalgam in D-ribose (LC Šnajdman: Proizvodstvo vitamov, Moskva 1973) · Newer processes are based on D-arabinose, which is partially epimerized to D-ribose by the Bílik reaction, ie by the action of molybdate ions at elevated temperatures. Most of the unreacted D-arabinose is separated from the obtained mixture by crystallization (European Pat. No. 20,959).

Several procedures have been described for the preparation of D-arabinose. E.g. oxidative decarboxylation of calcium D-gluconate with hydrogen peroxide in the presence of iron ions (O. Ruff and G. Ollendorf: Ber. 33 (I960), 1898; HG Fletcher and CS Hudson: J.Am. Chem. Soc. 72 (1950), 4546 or in the presence of mixed ions, further by electrochemical means Czech patent certificate No. 225576, oxidation of D-glucose by sodium hypochlorite (RL Whistler and R. Schweiger: J. Am. Chem. Soc. 81 (1959). 5190) and similarly by the action of hypochlorite sodium to sodium D-gluconate, obtained by fermentation from D-glucose '(DOS.pat.Ž 923 267)'.

Fermentation production of D-ribose was made possible only by obtaining artificial mutants of D-ribose-producing bacteria from D-glucose (T. Suzuki, N. Tanaka, K. Mizuhara and S. Kinoshita: J. Gen. Appl. Microbiol. 9 (1963) , 457 and Dutch, U.S. Pat. No. 6,408,606 (1965), T. Azawa and S. Watanabe, Japanese Pat. K. Sasajima and M. Yoneda: Agric. Biol. Chem. 35 (1971), 509; K. Sasajima, M. Doi., T. Fukuhara, A. Yokota and Y. N? Kao: Japanese Pat. 782 (1976): K. Mockizuki and T. Shimbo, Japanese Pat. H. Fukuda: J. Ferment. ”Technol. 56 (1978), 91), the available mutants generally provide a mixture of unchanged D-glucose and D-ribose formed by fermentation. D-glucose by the addition of the yeast Saccharomyces cerevisiae, which practically does not ferment D-ribose.

The disadvantages of said method for isolating D-ribose (loss of glucose, contamination of the solution by alcoholic fermentation by-products, in particular glyoerol) are eliminated by the process which is the subject of the present invention. In this process, D-glucose is separated from D-ribose by chromatography on a cation exchange resin column. The fermented broth, containing the remainder of unchanged D-glucose and D-ribose, is filtered and deionized. It is then passed through a column of cation exchange resin of a suitable type, saturated alkali metal ions, corrosive earth metal ions, silver ions, lead ions and the like. The resin is a beaded polystyrenedivinylbenzene copolymer with a DVB content of 3 to 8%, a grain size of 0.05 to 0.5 mm (preferably 0.1 to 0.3 mm). Under suitable separation conditions (resin load, flow rate, concentration of sugar solutions and temperature) complete separation of both monosaccharides can be achieved. From the fractions of the eluate, both sugars can be obtained in crystalline form. Due to the high purity of the solutions of both sugars after separation, it is advantageous to use the solutions thus obtained directly, or after suitable concentration, for further operations, i.e. the solution

CS 267 706 B1

Return D-glucose to the broth preparation and use the D-ribose solution, eg for nucleoside preparation. '

The details of the process according to the invention follow from the following examples, which merely illustrate the process but do not limit it in any way.

Example 1

After two days of growth, 100 ml of soil containing 2% soluble starch, 2% corn extract, 0.3% potassium hydrogen phosphate and 0.1% potassium dihydrogen phosphate was inoculated with Bacillus pumilus V1IFB 1004 mutant, maintained on broth Slant Agar. After a one-day rest period at 32 ° C, the culture was used to inoculate a 1000 ml medium containing 12.5% D-glucose, 2.5% active dried Engedure yeast, 1.5% ammonium sulfate, 0.5% calcium hydrogen phosphate, .5% tricalcium phosphate and 0.1% calcium carbonate. After incubation for 11 days on a longitudinal shaker at 32 ° C, the culture contained 95 mg of total reducing sugars in 1 ml, calculated as D-glucose and 21.5 mg of non-fermentable sugars, calculated as D-robose in ml. Simultaneous analysis by paper chromatography showed the presence of D-glucose and D-ribose, respectively. D-ribose (after fermentation of glucose). The soil was filtered with 3 g of activated carbon and deionized to remove salts. After concentration to a concentration of 35% dry matter, the solution was subjected to chromatographic separation on a column with a diameter of 5 cm and a height of 280 cm. A strongly acidic cation exchanger Wofatit EPS, crosslinked with 4% DVB, grain size 0.10 to 0.30 mm saturated with calcium ions at 50 ° C was used. The loading load was 25 g of dry sugar per 1 resin. The clusters were performed with water * 2 at a flow rate of 0.42 ml per cm of column cross-section per minute, and the eluate fractions were collected in 120 ml increments every 14.5 min. Fractions Nos. 14 to 23 contained a total of 64.7 g of chromatographically uniform dD-glucose, * i. 52% of the amount used in the fermentation. Fractions Nos. 29 to 38 contained a total of 26.6 g of chromatographically uniform D-ribose, i.e. 21% of theory from the glucose used.

Example 2

Fermentation of 1000 ml of soil containing 125 g of D-glucose and nutrient salt was performed with the same strain and in the same manner as in Example 1. After fermentation, the soil was clarified by activated charcoal filtration and deionized on strongly acidic cation exchange columns in H ⁺ -cycle. weakly basic annex in the OH-cycle. After concentrating the solution under reduced pressure to 37% dry matter, a chromatographic separation of D-ribose from unchanged D-glucose was performed on an Ostion KS 0407 cation exchanger column (grain size 0.15 to 2+ 0.30 mm, 4% DVB, Ba-form, dimensions column 5 x 180 cm) eluting with water at _ p.

° C and a flow rate of 0.48 ml per cnT column cross-section per min. The separation of both sugars was quantitative. Eluate fractions 15 to 23 contained 53.9 g of D-glucose in solution and fractions 30 to 39 contained pure D-ribose in an amount of 22.4 g, i.e. 21.5% of theory, based on the starting D-glucose. . Crystallization of D-ribose was performed after concentrating the aqueous solution to a syrup with a concentration of about 95% and after not diluting with anhydrous ethanol. After inoculation with D-ribose, crystallization took place for 48 h at 5 ° C. A second fraction of D-ribose could be obtained from the mother liquor after the 1st fraction of crystals. The total yield of crystalline D-ribose was 20.6 g, i.e. 92% of the amount found analytically in solution. Melting point 86-87 ° C.

Example 3 ·

Fermentation of 1000 ml of broth containing 125 g of D-glucose and nutrient salt was performed with the same strain and in the same manner as in Example 1. At the end of the incubation, the broth was filtered through a filter cartridge with activated charcoal and deionized on strongly acidic cation exchange columns in H ^+. -cycle and weakly basic anion in the OH-cycle.

CS 267 706 Bl

After concentration to a concentration of 40% dry matter, the solution was subjected to chromatographic separation in a column with a diameter of 5 cm and a height of 180 cm. A strongly acidic cation exchanger Zerolit 225 SRG 10, crosslinked with 4% DVB, grain size 0.15 to 0.30 mm saturated with strontium ions at 60 ° C was used. Elution was performed with demineralized water at a rate of 0.4 ml. 1 —2 min .cm column cross-section. The eluate was collected in fractions of 120 m. Combining fractions 14 to 22, a solution was obtained which contained 58.1 g of pure D-glucose, i.

46.5% of the amount used in the fermentation. Fractions 29 to 38 contain 23.2 g of pure D-ribose, i.e. 22% of theory, based on the starting D-glucose.

Claims (1)

Process for the isolation of D-ribose from mixtures with D-glucose, characterized in that the deionized solution containing D-glucose and D-ribose is subjected to chromatographic separation on a column of ketion exchange resin based on spherical sulfonated styrene-divinylbenzene copolymers containing at most 8% by weight. , preferably 4 to 5% by weight, of divinylbenzene, with a grain size of at most 0.5 mm, preferably 0.1 to 0.3 mm, saturated with alkali metal ions, in particular sodium, or alkaline earth metal ions, in particular calcium, strontium and bar.