CS206971B1 - Method of isolation of the /u-14c maltooligosacharides from the hydrolyzators of u 14c/alpha-glucanes - Google Patents

Description

(54) Insulation method (U14

C) maltooligosaccharides from hydrolyzates (U-C). glucan

It is an object of the invention that the lower maltooligosaccharides - maltose to maltooctose -, after conversion to the corresponding 4-nitrophenylhydrazones, are very efficiently separated by paper chromatography.

The preparative separation of maltooligosaccharides is carried out on an activated carbon column by appropriately varying the gradient of the water-ethanol elution system (R. L. Whistler, D. F. Durso;

J. Am. Chem. Soc. 72, 677 (1950); ibid. 74, 5140 (1952)), respectively. on a Bio-Gel P-2 column eluting with water (M. John, G. Trenel, H. Dellweg; J. Chromatogr. 42, 476 (1969)) or an XE 200 ion exchanger (JF Deaton; Ger. Offen. 2725964, CA. 88, 63494b (1978)). Qualitative evidence or the determination of maltooligoaaccharides is also based on their mutual separation by paper chromatography (J. Stanek, M. Cervny, J. Pacak; Oligosaccharides, p. 328, Nakl. CSAV, Prague 1962; H. Bender; Carbohydr. Res. 65). 85 (1978), K. Umeki, K. Kainuma, J. Chromatogr.

242 (1978)). Cellulose thin layer chromatography (R. Spitschan; J. Chromatogr. 61, 169 (197D), diatomaceous earth (FW Collins, KR Chandorkar; J. Chromatogr. 56. 163 (197D)) provides a quick reference to the composition of a mixture of oligosaccharides of the α-glucan type. , silica gel, D. Nurok, A. Zlatkis, Carbohyd, Res., 65, 265 (1978)) and a mixture of silica gel and diatomaceous earth (CT Mansfield, HG McElroy Jr., Anal. Chem. 586 (197D)). The maltooligosaccharides over the above chromatographic separations are that the maltooligosaccharides in the form of their 4-nitrophenylhydrazones are separated by high-performance paper chromatography.

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SUMMARY OF THE INVENTION The maltooligosaccharides with 4-nitrephenylhydrezine in methanol-aqueous solution are converted into the corresponding 4-nitrophenylhydrazones, which are separated by chromatography on paper and from the isolated hydrazones and by known procedures, for example, by reaction with benzaldehyde to release the corresponding maltooligosaccharides.

The technical implementation of the isolation of maltooligosaccharides is very simple and the individual maltooligosaccharides obtained show a high degree of purity.

Example

Water-soluble (U-c) ε-glucan (10 pCi; glucan isolated from dishes Chlorella sp., Product of the Institute of Exploration, Production and Utilization of Radioisotopes, Prague), optionally diluted with unlabeled glucan (50 mg; glycogen from oats, product JT Baker Chemical Co.) is added to water (2 mL) to which is simultaneously added immobilized α-amylase (ex saliva) on a cellulose carrier (5 mg;

up to 8 U / mg, prepared according to J. Zemek, J. Augustin; Cs. 189472 (1978) 1 and the reaction mixture is allowed to stand at 20-25 ° C for 3-5 days. The immobilized enzyme was filtered off, a 2% methanol solution of 4-nitrophenylhydrazine (4 ml) was added to the filtrate, and the mixture was heated at 60 ° C for 7 hours. The reaction mixture is loaded onto chromatography paper (46 x 56 cm, Whatman No 1) and chromatographed with 1-butanol-ethanol-water (5: 1: 4 v / v) as eluent at 20-25 ° C for 24-30 ° C. hours. The mobility of the individual 4-nitrophenylhydrazones of maltooligosaccharides is shown in Table 1 (at sufficient concentrations of the individual hydrazones, their corresponding bright yellow zones can be observed in the chromatogram).

Table 1

Mobility of 4-nitrophenylhydrazones saccharides in paper chromatography

4-nitrophenylhydrazone R) R ⁺⁺⁾ glucose 1.00 3.43 maltose 0.63 2.00 maltotriose 0.43 1.50 maltotetraose 0.30 1.00 maltopentaose 0.18 0.62 maltohexaose 0.11 0.39 -heptaose 0.06 0.21 maltooktaózy 0,035 0.12 4-nitrophenylhydrazine 1.60 5.49

♦ ^ related to the mobility of 4-nitrophenylhydrazone D-glucose, ♦♦ ^ related to the mobility of D-glucose

From chromatographic paper e individual zones of 4-nitrophenylhydrazones cut out (from the original radioactivity (U- ¹⁴ C) of α-glucan is maltose in the 4-nitrophenylhydrazone zone

206 971-34%, maltotriosis 19-22%, maltotetraosis 10-13% and maltopentaosis 10-12% activity), water (10 ml) and after half an hour at room temperature methanol (1 ml) was added to each fraction. and benzaldehyde (1 mL) and the mixture aa was kept at 60 ° C for 15 hours. Then the solution is filtered, the filtrate is concentrated under reduced pressure (bath temperature up to 40 ° C), the distillation residue is applied to chromatographic paper and chromatographed with 1-butanol-acetol-water at room temperature for 5 to 6 days to give a particularly pure single maltooligosaccharide (ca 50% · natural phenylhydrazone activity).

Maltotriosis as well as other oligosaccharides of the maltose series are important substrates for biochemical and histochemical examinations, both in the field of industrial microbiology (monitoring of fermentation processes), in clinical biochemistry and histochemistry (determination of amylolytic enzymes and chemi- cemia and their location). The use of labeled substrates allows the elaboration of micromethods suitable for the analysis of biological material collected in limited quantities (e.g., to diagnose congenital disorders of glycogen metabolism).

Claims (1)

OBJECT OF THE INVENTION SpSaob of isolating (U-C) maltooligosaccharides from hydrolysates of (U-C) α-glucanes, characterized in that maltooligosaccharides are converted in the methanol-aqueous solution with 4-nitrophenylhydrazine to the corresponding 4-nitrophenylhydrazones with maltooligosaccharide paper, and from the isolated hydrazones and the individual carbohydrates released in a known manner, preferably by reaction with benzaldehyde.