SU562202A3 - The method of obtaining derivatives of amino sugars - Google Patents

Description

Solid and liquid, in particular liquid, aqueous nutrient media are used to carry out the proposed spoein, which, along with carbon sources, contain nitrogen sources, salts and antifoams in usual concentrations. Concentrations can vary widely.

The carbon sources are predominantly carbohydrates, in particular starch, maltose, glucose, and mixtures of two (or three) of these substances, as well as complex mixtures, such as malt extract.

As nitrogen sources, complex mixtures commonly accepted in microbiology can be used, such as casein hydrolyzate, yeast extract, peptone, fish meal, soluble fish extracts, water from swollen maize, meat extract, and also mixtures, in addition, amino acids and / or ammonium salt.

When carrying out the proposed process, they are usually operated under aerobic conditions in ventilated cultures grown under constant shaking or in cultures grown in vessels. The genus and concentration of the carbon source in combination with the strain used for fermentation have such an effect on the genus of the target product in relation to the proportion of the radical R that individual units of the homologous series or at least a very narrow region of homologs can be obtained almost selectively. It turned out that in nutrient media containing more than 2% starch, primarily amino-sugar derivatives with 4-7 hexose units are formed and that the SE 50/13 (CBS 614.71) is a particularly suitable strain for this purpose. However, under known conditions, to obtain mixtures of derivatives of amino sugars with 4-7 units of glucose, it is enough to add nutrient media containing, for example 3.5% glucose, 0.1-3%, preferably 0.5-2%, as the source of basic carbon. starch. In addition, it turned out that in nutrient media that do not contain starch, in particular after the addition of maltose to the nutrient medium, in particular with strain SE 50 (CBS 961.70), aminosugar derivatives with 2-3 hexose units are preferably obtained. Especially beneficial for obtaining the proposed derivative of amino sugars with R-glucose were nutrient media, which, as a carbon source, contain only glucose. If the nutrient medium contains an excess of glucose, then longer fermentation also produces longer chain amino sugar derivatives. This can be prevented within certain limits, so that during the fermentation the depletion of nitrogen sources coincides with the moment of depletion of glucose. If the nutrient media does not contain glucose and maltose serves as the carbon source, then amino acid derivatives with 2 units of hexose are predominantly obtained, and pure maltose can be replaced by cheaper mixtures, for example maltzine, a natural malt extract produced by Diamalt AG, Munich, Germany. Depending on the content of maltotriose, the next higher homolog is also formed. Especially beneficial for obtaining lower homologs was SE 50/110, which, in optimal nutrient media, yields approximately twice the yield of lower homologs than SE 50/13.

During fermentations, the composition of the remaining nutrient medium, in particular the concentration of nitrogen sources, and the composition of salts can vary within wide limits.

Nutrient media are sterilized by conventional methods. The pH of the nutrient medium ranges from 5.0 to 8.5, preferably from 6.0 to 7.8. Growth temperatures are 15-45 ° C, preferably 24-32 ° C. To obtain higher homologues with strains SE 50 and SE 50/13, it is more advantageous to work at a higher temperature, for example at 28 ° C, to obtain lower homologs with SE 50 and SE 50/110 at a lower temperature, for example at 24 ° C. The growing period is 1-8 days, preferably 2-6 days. A longer growth period, in particular with an excess of carbohydrates, favors the formation of higher homologues. The end of fermentation is determined by enzymatic breakdown for the presence of a retarding effect and thin layer chromatography.

The lower homologs of the proposed amino sugar derivatives can also be obtained from the higher homologs by cleavage of hexose units. Cleavage occurs with the help of aqueous acids, in particular 1-5 normal mineral acids for 10-180 minutes at temperatures from 50 to 100 ° C, in particular at temperatures from 90 to 100 ° C, or by incubation with enzymes (hydrolases), in particular, 3-amylases or non-inhibited a-amylases or amyloglucosides of microbial origin, for example, a-amylases from B. subtilis or by incubating with microorganisms that grow in nutrient media, which, as the only carbon source, contain high homologs dlagaemyh derivatives of amino sugars in an amount of from 1 to 10%, preferably from 2 to 5% such as, e.g., Aspengillus niger (ATCC 11.394).

Isolation and purification of the proposed amino sugar derivatives is carried out from microbiological culture broths or acidic hydrolysates, or incubation mixtures, in which enzymatic and / or microbiological decomposition of the high amino sugar derivatives is carried out.

Depending on the limits of molecular weight to which the released substances belong, different separation methods are necessary. Higher homologs are recovered by direct precipitation after previous discoloration and concentration of the solutions.

Low molecular weight homologs are preferably prepared by adsorption on active carbon at neutral pH, with subsequent desorption with aqueous alcohols or acetone, preferably 50-80% acetone. The desorption is completely complete at acidic pH values ranging from pH 1.5 to 4, preferably pH 2 to 3.

If the initial solutions are very dark in color, they are discolored prior to adsorption of the proposed substances by means of active carbon at acidic pH values (pH 1-3) or non-specific adsorption resins, for example, Lewapol Ca 9221 (grain size 0.35 mm) from Bayer AG , in the range of pH from 2 to 7, preferably from 2 to 3. Activated carbon only in the acidic region preferably binds dyes, while Lewapol does not adsorb the proposed amino sugar derivatives having an inhibitory effect either in the neutral or in the acidic regions.

To separate the inhibitory derivatives of amino sugars from inactive saccharides and other inactive compounds, the alcaline character of these components is used. Under suitable conditions (pH 1-8, preferably pH 2-4; with a weak ionic strength corresponding to a conductivity of 10 ml of sym, preferably 2 ml of sym), homologous derivatives of amino sugars in the H + form are bound by strongly acidic cation exchangers, for example, Dowex 50 V (from Dow Cneniicals). As it was established by the applicant, the amiposugar derivatives with a retarding effect are especially horizontally bound to catnonites from an acetone solution (50-80% acetone, pH 1-5, preferably pH 2-4), which under these conditions show a much higher capacity for substances. Provided that the solution contains more than 50% acetone, the connection with slightly acidic acids, for example, Amberlite AL-50 (H + -form), is also quite good.

For desorption of the proposed amino-sugar derivatives from cation exchangers, aqueous bases or acids, preferably ammonia or hydrochloric acid, are used in concentrations of 0.01-1 valine / l. Inhibitor-active derivatives of amino sugars are obtained from the eluates after neutralizing the eluates with an appropriate weakly acidic or basic ion exchanger or after removing the base or acid by vacuum distillation in the case of volatile bases of acids after concentration of the solution by lyophilization or precipitation with organic solvents, preferably 10-20 parts by volume of acetone .

In addition, low molecular weight inhibitory derivatives of amino sugars are separated from inert saccharides by chromatography with cell-based ion exchangers, preferably phosphocellulose (Serna, Gepdelberg). As solvents, buffers are used, per person; telpo-phosphate phosphates with weakly nciiiioii strength, preferably 2-100 mM, in particular 5-10 mM, within the RP from 2.5 to 8, given at pH 5-6. A prerequisite for efficient fractionation is to contain as little as possible 1 salts in preparations subjected to

fractionation, while the sprays almost do not interfere.

For the preparation of individual units of the putative homologous series of inhibitor active proprietary amnposaccharides in pure

In addition, pre-purified preparations are chromatographed on a suitable molecular sieve, for example Bio-GeP P-2 (manufactured by Bio-Rad, Munich), and the fraction of the eluate is examined by thin-layer chromatography. Fractions

containing common propionic amino sugars, they are combined, rechromatographed, and after a concentration center, or, as described above, organic solvent is precipitated:,.

In the following examples, hex units are glucose units.

Prpmer 1. In some pr 1merah as the source PRENARAT, obtained following 1M way.

In the case of formulary with 8 liters of nutrient solution, consisting of 5% starch, 1.0% yeast extract, 0.2% K2HPO4, a three-day strain SE 50/13 (CBS 614.71) and a carefully prepared bottle are fed from a katalochka flask Shunp fan is incubated for 3 days at 28 ° C, and a culture liquid is obtained from 105,000 units. NNG

.

6 l of this fer; e1 and liquid after

cooled to 20 ° C. Sub-clutch from zero: oncentrated nNOs (pM 2.5), 30 g of Carboraf H 1 of the 1st active zl are added, the stirrer is from within 10, the center is fugged for 15 min. npi 10000 v / m1 N and transparent light - the yellow residue after neutralizing NHs is concentrated to 500 ml, 500 ml of synergy over 45 milliards with 200 g amberl 1t IRA410 C1, flushed and 4/5 volume parts (about

400 ml) of methanol, so that the main bulk of high-molecular mass products are diluted starch (together with 1 part of residues and 1 of coal). The center is fugied for 5 minutes at about 5000 rpm, 850 ml of residue is placed in drops from 4 liters of dry vap. White hlonevdpy precipitate PA 1-hour, 3 times washing from, 2 times dried in vacuum at. Output 36 g of white powder with lO-lO units. Amnlazy / g.

EXAMPLE 2: For a fermentation product with a composition of preparations of thin-layer chromatography | 1 and 1 ml of a farm, 1 lp mg preparations of anoscopes C (Cf. Schleicher and

Schiell, Dassel, Tour F 1500) and 2 times in the n-butanol / ethanol / water 50: 3: 20 system are developed. For a color image of the sucrase, imbibition of the sucrase is developed and well-dried plate (20 m / 20 x 20 cm) is sprayed with an enzymatic gel and let it harden.

Then, for 5 min at room temperature, the incubator is incubated in a moist chamber and then saturation with substrate gel. After the second layer of the gel has hardened, the plate is numbed into a wet chamber and the incubators are incubated at 40 ° C. Inbiginion coloration (bright spots, red-brown background) appears after 60- 90 minutes. In the event of optimal color development, the process is interrupted and the plate with agar layers on it is dried with hot air.

Preparation of gels.

Enzymatic gel - 1.5 g of agarose (Llndustrie Biologique Francais) is suspended in 100 ml of 0.2 M Na-maleate buffer with a pH value of 6.0 and then dissolved at the boil. The clear agar solution is cooled to 50 ° C and, with stirring, 250 ml of Triton X-100 solution (2 g of Triton X-100 + 8 g of ethanol) and 0.5 ml of dianindine solution (20 mg of dianisidine / 1 ml of acetone) are added. 1 ml of GOD / POD reagent (12.5 mg of glucose oxidase, purity by Beringer, order no. 15423 and 2.5 mg peroxidase, purity I, by Oehringer, order n ° 15302 dissolved in 5 ml of maleate buffer) and 4-6 units of sucrase from small pig intestines. Prior to spraying, the gel must be removed at 50 ° C, since otherwise it will solidify in the nozzles during the spraying process.

Substrate gel: 0.5 g of agarose is suspended in 100 ml of Na-maleate buffer with pH 6, 0 and dissolved at boiling, cooled to 50 ° C, 100 ml of triton are added (2 g of Triton X-100- | -8 g of ethanol) and 1 g of sucrase (Serva N ° 35579) was added. After dissolving the sucrase, the gel is ready for use. For a color image of the inhibition, the amylase of the developed and dried TX plate is sprayed with amylase gel (20 ml / 20X X20 cm plate) and allowed to solidify. After a five minute pre-incubation; at room temperature, the plate coated with a layer of gel is immersed in a 0.5% starch solution (I g of Merck 1252 starch in 200 ml of 0.2 M glycerophosphate buffer, 0.01 СaЬ, pH 6.9, dissolved npc chenny) and kept in it for 2 minutes at 40 ° C, stirring the solution. The plate is then washed well with distilled water and immersed in diluted J2-pacTBOp (4 ml of Jj-pacTBOpa per 500 ml of PgO; L2-solution: 2.2 g of J2 + 4.4 g KJ dissolved in 100 ml of H2O) to stain non-digested starch . After 1 min, an optimal color is obtained.

Preparation of amylase gel

1 g of agarose and 100 ° C are dissolved in 100 ml of 0.2 M sodium glycerol phosphate / 0.01 M CAC buffer with a pH of 6.9, after cooling 5 to 50 ° C, 100 ml of Triton X-100 (2 g Triton X-100 + 8 g of ethanol). Before the spraying, 100 ml of a suspension of amylase crystals (10 mg of pig pancreatic amylase / ml of a saturated HH4-sulphate solution of Beringer, No. 15017) were added before spraying.

Example 3. In a 1 L Erlenmeyer flask with 120 ml of a user liquid consisting of 4% starch, 2.4% glucose, 0.9% hydrochloride casein lysate and 0.9% yeast extract, the pH value of which is adjusted to 7.6, to which 0.4% CaCO3 was added, and which was sterilized for SO minutes at 121 ° C, were sown with 3 ml of Pre-Sem SE 82 grown in nutrient liquid from 2% starch, 1% glucose, 0.5% hydrolyzate casein and 1% yeast extract, the pH of which by means of NaOH was increased to 7.2, to which

5 added 0.4% CaCO3 and which was sterilized for 30 minutes at 121 ° C, and incubated for 5 days at 28 ° C on a rocking chair. Get culture solution with 122000 units. ING. amylase / ml

0 For further processing, the mycelium is centrifuged at 12000 rpm and separated from the combined culture liquids, 300 ml of the culture filtrate is acidified with half-concentrated nitric acid to

5 pH 2.5 and for 10 minutes unmixed with 2.5 g of activated carbon. After separation of the carbon at 12,000 rpm, to the solution neutralized to a pH value of 6 by Yun. KOH, 300 ml of methanol are added, kept for a short time and the precipitate is removed at 12000 rpm. The residue is added dropwise to 3 liters of ethanol, the precipitate after a brief exposure is centrifuged at 12,000 rpm, twice washed with absolute ethanol and once with ether, dried in vacuo, 2.23 g of product are obtained with 7.45-10 units . ING. amylase / g, which contains more than 95% homologues of aminosugar derivatives c.

Example 4. In a 1 liter Erlenmeyer flask with 120 ml of a nutrient fluid consisting of 3.5% glucose, 2% starch, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% CaCO3 and 0, 3% K2PRO4, whose pH value before sterilization is set at 7.8 and sterilized for 30 minutes at 121 ° C, is seeded with 6 ml of a preliminary culture of strain SE 50/110 grown in a nutrient fluid consisting of 3% soy flour, 3% glycerol and 0.2% CaCO3, and incubated for 3-4 days at 24 ° C on a rocking chair. Get a culture solution containing 153000 units. ing. amyla5 zy / ml I-12200 units ing. sucrase / l.

1 l of the culture solution is acidified with nitric acid to pli 2.5, stirred for 10 min with 5 g of activated carbon and then centrifuged for 5 min at 5000 rpm. Neutralize 25 g of Amberlite IRA 410 (OH-form). The residue is evaporated to 100 ml, 100 ml of methanol are added and filtered. The filtrate is added to 2 liters of alcohol, the precipitate is filtered under suction, washed three times with acetone and ether and dried in vacuo.

Output 14 g of white powder with 5-10 units. ing. amylase / g, which predominantly contains homologues with.

Example 5. Work similarly to example 4, but with the addition of 0.5% starch and after a four-day fermentation receive a culture solution with 40000 units. ing. amylase and 184 units. ING. sucrase / ml The culture solution contains homologues, starting with.

Example 6. In a 1 liter Erlenmeyer flask with 120 ml of a nutrient fluid consisting of 3% glucose, 0.6% casein hydrolyzate, 1.6% yeast extract, 0.3% CaCO3, 0.3% CdPRO, RP value which before sterilization by KOH brought up to 7.8, seeded with a preliminary culture of sandwich SE 50/110 in example 4 and incubated for 4 days at 24 ° C on a rocking chair. Get a culture solution with 10,800 units. ing. sucrase / l, which mainly contains a derivative of amino sugars with.

5 liters of the culture filtrate at 1300 rpm are separated from the mycelium, the solution is acidified to pH 2.5 with semi-concentrated nitric acid and placed with 55 g of activated carbon (Merck) and 200 g of Clarcela for 15 minutes. After the solids are removed by suction, the solution is neutralized with concentrated ammonia to pH 7, evaporated to 1.5 liters and precipitated with a fivefold amount of ethanol. The resulting flocculated precipitate is separated by centrifugation at 1200 rpm, the yellowish residue is evaporated to 150 ml and centrifuged at low revolutions to separate minor insoluble particles. 50 ml of this solution are applied to an A-120-filled. (H + -form) column (30X300 mm; 30 ml P2O per hour). 300 ml of the eluate containing inert saccharides are collected, as well as the proportion of non-adsorbed inhibitor-active components, the ion exchanger from about 400 ml of water is poured into a beaker, and with stirring, concentrated ammonia is added to a ppm of 11.5. The mixture is stirred for another 30 minutes and the ion exchanger is separated, the residue is concentrated to 1/20 of the volume, filtered through a column (20X150 mm) filled with amberlite IRA-410 (PSO3 form), and at a speed of 30 ml / hour, 500 ml of eluate are collected. which is concentrated and then lyophilized, and 1.3 g of crude product are obtained.

For further purification, the crude product is fractionated with a Bio-Gel P-2 100--200 mesh.

(Bno-Rad, Munich). For this purpose, a column with a diameter of 50 ml and a length of 450 mm, which runs on water at a speed of 40 ml / hour, is used, and fractions of 10 ml are collected. All fractions are examined for the presence of carbohydrates by means of an anthron sample and by means of a sample of the reduction of sucrose for the presence of inhibitor-active components.

The fractions containing the inhibitor sucrase, in addition, thin-layer chromatography as in example 2 are examined for the content of the individual components. The fractions containing the amiposugar derivative are combined, concentrated and lyophilized. Obtain 35 mg of the derivative of amino sugar with a p with 0.3-105 units. ING. amylase / g and 30000 units ING. sucrase / g

Example 7. In an Erlenmeyer flask with 120 ml

a nutrient fluid consisting of 5% starch, 1% yeast extract, 0.2% K2PRO4, seeded with 2 ml of the preliminary culture in example 4. After a three-day incubation at 28 ° C, cultural

solutions with the following release of the amylase inhibitor (mainly consisting of amino-sugar derivatives c).

Inhibitor

Amylase strain, u / l

37,000

SE 50

109000 SE 50/13

53500 SE 50; BY

Example 8. In a 1 L Erleimeyera flask with 120 ml of a nutrient fluid consisting of 1.3% maltose, 3.5% glucose, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% CaCO3, 0.3% K2PRO4, seeded with 2 ml of pre-culture according to number 4. After four days)) incubation at 24 ° C on rocking chairs with different strains, solutions are obtained with the following inhibitor yields (mainly consisting of amino-sugar derivatives).

Strain Inhibitor of Sakha Inhibitor

times, u / mlamylase, edml

SE 5025580

SE 50 / 1314,814 (50

SE 50 / 11057,9755

Example 9. In a fermenter with 100 liters of nutrient fluid consisting of 3.5% glucose, 2.5% dry powder malt extract,

0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% CaCO3, 0.3% C.NPO and 0.1% antifoaming agent, seeded with 5 l of pre-culture as in example 4 and incubated for 5 days while stirring and airing at 24 ° C. Get cultured

solution with 73000 units ipg sucrase / l, which

mainly contains proctal amnposahar s / g 2.

90 l of fermentation liquid with mycelium concentrated nitric acid under II

acidified to pH 2.5 and 900 g (1%) of activated carbon (Merck) are added to adsorb the bulk of the dyes formed. The mixture is stirred for 15 minutes, centrifuge and 3000 rpm the mycelium is separated and the main amount of coal and the residue, with an addition of 3 kg of Clarcela, is filtered through an ioutch filter, working pressure. 65 l of a yellow-brown, transparent filtrate with 60,000 units is obtained. ig. sucrase / l, the filtrate is adjusted to pH 7 with concentrated ammonia and 1300 g (2%) of activated carbon (Merck) are unmixed for 30 minutes to adsorb the active substance. Filtered through suction, working iodine pressure, and the precipitate of activated carbon 3 times and discard 10 liters of distilled water. Then the coal is pressed down to dryness and three times, each time for 15 minutes, it is moved from 4 l of 50% acetone and pH 2.5 to desorb the active substance from coal. After the carbon has been removed by filtration, the acetone eluates are combined. The combined eluate is distilled to 250 ml, an equal volume (250 ml) of methanol is added and filtered through a folded filter. The filtrate (480 ml), and carefully placed dropwise, is added to 5 liters of en, etone. The residual precipitate is frlt through a coup and washed 3 times with acetone and ether. Then the vacuum valve is at 35 ° C. Output 230 g with 8500 units. itgg. sucrase / g 25 g of the crude product are dissolved in 1 l of water and in a stream for 30 minutes, they are drenched with 300 g of DowexR 50 AVX 4 P + (200-400 mesh). Filter TREE and three oases washed with 2 l. 0.001 and. HCI. The washed Dowex is then suspended in 500 ml of water, and the suspension is adjusted to a pH of 9.0 by addition of a 25% ammonia solution. Then it is stripped 2 more times with 500 ml of a 0.6% -iToro ammonia solution, the eluates are combined and evaporated to 100 ml. To discolor the solution, it is stirred for 5 minutes with 2 g of DAE-cellulose (ITLEKHERER and PTUL LU 02035, 0.6 mash / g) and then centrifuged. An equal volume (100 ml) of methanol is added to the light yellow residue and then 2 l of acetone is added dropwise with stirring. The pellets were filtered by coup putsch and tromoned with acetone and ether and dried at 35 ° C in vacuo.

Yield 4.2 g with 26,000 units. ing. sucrase / g For further fine purification, 4.0 g of the inhibitor in portions of 0.5 g is subjected to gel filtration through biogel P-2. For this purpose, 0.5 g of the drug is dissolved in 10 mt of IAO and paio t pa column with biogel P-2 (200-400 mesh, Bio-Rad) with a diameter of 5 cm and a length of 95 cm. water with a degree of fluidity of 80 ml / h. Collect 12 ml fractions. In all fractions, the total carbohydrate content was determined (by an inropic sample as ectinction at Ev2o). and the content of the inhibitor sucrase and amylase. In addition, the fraction is subjected to thin-layer chromatography (color inhibitor of enzymes in example 2).

12

The fractions in which the aminosugar derivatives with l 4-6 are found are combined, evaporated to 10 ml and precipitated by dropwise addition to 200 ml of alcohol. The precipitate is removed by centrifugation, washed with acetome and ether and taken up in vacuo; the output of 4.0 g of the total inhibitor: 0.2 g of the derivative of amino sugars with p 4-6 with 17.5-10 units. ing. amylase / g and 8500 units ING. sucrase / g The fractions containing the derivative of amino sugars with L 3 are treated in the same way, and the precipitation is carried out with 200 ml of acetone; yield of 4.0 g of crude inhibitor: 0.1 g of an amino sugar derivative with “3 with 1.4–106 units. ING. amylase / g and

21,000 units ING. sucrase / g From the fractions containing the derivative of amino sugars with L 2 (precipitation with acetone), 0.9 g of the derivative of amino sugars with L 2 with 0.3-10 units is isolated. ing. amylase / g and 68000 units ing. sucrase / g

Example 10. In three small fermenters, each of which contains 8 liters of nutrient fluid from 7.5% dry powder malt extract, 0.3% caseyi hydrolyzate, 0.7% yeast extract, 0.3%

CaCO3, 0.3% K2PR04, seeded with 5% of a preliminary culture of strain SE 50/110 (prepared according to example 4). After a five day incubation at 24 ° C, a culture solution of 73 units is obtained. ing. sucrase / ml, which

mainly contains derivatives of amino sugars with l 2.

After centrifugation (30 min., 3000 rpm) to separate the mycelium, 20.5 L of an intensive brown brown organic solution with 67,000 units is obtained. ipg sucrase / l. The pH was adjusted to 3.5 by the addition of nitric acid and 60 g of Lewapola (Ca 9221, grain size 0.35 mm, Bayer) / l 1.23 kg of Lewapola were added for bleaching. After stirring for twenty minutes, the filter is filtered through a Zeitz C 3 filter. The bleached culture solution is neutralized with ammonia (18.5 L, 67000 U.S. Sharazy / L). Then, to adsorb the active substance, 20 g of active carbon / l of 370 g are added and stirred for 30 minutes, filtered through a Seitz K 3 filter, on which a layer of Clarcel BcnoMoraTCvibnoro filtering substance was removed and the filtrate was removed (17.5 L,

3600 units ig. sucrase / l). The coal residue is washed three times with 2 liters of distilled water. For desorption of the active substance from the coal, it is carried out 3 times for 15 minutes and 1 liter of 80% acetone is washed, and the pH value of the concentrate PS1 is adjusted to 2.5. Eluates were pooled (2.4 L, 371,000 units of ng. Surase / L). To the eluate, add 20 g of Dowex (H +) (Dowex 50 WX 4, H + form, Serva, Heidelberg) 46 g of Dowax and mix for 20 minutes. The resin is then filtered off (fraction of Dazex I) and sprinkled with a small amount of 75% acetone. The filtrate and the washing liquid (3 l 215 000 units of ing. Sucrase / l) are mixed

with 60 g of Amberlite IRA 410 (OH — form, from the company Serva, Heidelberg) / l until the pH value is set to 7. Then filtered to the filtrate (2.8 l, 219000 units of ingarate sucrase / l) 72 g of Doweks H + and stirred for 20 minutes With a hanging porous nylon bag filled with amberlite IRA 410 OH, the pH is maintained at 3.0. Then the Dowex is filtered (Dowex II fraction) and the filtrate is removed (2.6 L, 27,000 units ingarats / l). Each of the Douex fractions I and II is washed 3 times with 75% acetone at pH 3.5 and then desorbed three times with 100 ml (the Dowex fraction I ) or 150 ml (fraction Dawex) with 0.6% ammonia solution. During the first desorption, in which the amount of ammonia was insufficient to neutralize the dowax resin, the pH was adjusted to 9 with concentrated NHa. Three eluates of fractions I and II The Dowex is combined and evaporated to dryness, dissolved in 50 ml of water, adjusted to pH 3-4 with hydrochloric acid and 50 ml of methanol are added. The solutions, stirred under stirring, are added dropwise to 1.5 liters of absolute acetone, the precipitated filtered on a coup and washed 3 times with acetone and 1 time with ether. Dry in vacuum. Output: Fraction I 65 g, 25,000 units. inhibitor of sucrase / g. Fractions II 12.3 g, 36,000 units. inhibitor of sucrase / g. Fractions I and 111 as inhibitory components predominantly contain the derivative of amnosaccharides s / g 2, along with a small number of the following higher homologs (). The table shows the data of the compounds obtained.

Example 11. 200 g of the preparation described in Example 1 are dissolved in 940 ml of distilled water and in 60 ml of conc. sulfuric acid and heated for 4 hours to reflux (internal temperature 98-100 ° C; oil bath temperature 140 ° C). To the cooled black-brown solution, 10 g of active carbon (Merck No. 2186) are added and stirred for 1 hour. Then the active carbon is sucked off, washed with water and the filtrate is about 250 ml of 10 n. KOH is adjusted to pH 7-8. The solution is stirred for an hour with 50 g of activated carbon. The coal is sucked off, washed with 2 L of water and the filtrate is separated. For desorption

coal is digested overnight with 2 liters of 30% alcohol. Then coal is sucked off and the alcohol solution is evaporated. The residue is 6.2 g. The crude product (6.2 g) is dissolved in 500 ml of water and gently mixed with 30 g of Amberlite IR 120 (P + -form) for an hour. The cation exchanger is filtered and washed with distilled water until the filtrate is neutral. The ion exchanger is then stirred with 15 ml of 25% ammonia in 1000 ml of water overnight, separated and removed. The filtrate is evaporated. The residue is 3.7 g. Cellulose chromatography is carried out for further purification. 4.5 g of desorbed material from the ion exchanger is applied to a 1 m long column filled with cellulose and

15

2.5 cm wide. Etaiol / water 5; 1, 3: 1 ethanol / water was used to elute the produced ammonia sugars. At a speed of 20 drops per minute, 14 ml fractions are collected. Separate fractions are checked by thin layer chromatography.

After evaporation of the 47-85 fraction, 1.6 g of a brownish derivative of amino sugars are obtained. C. The colored impurities do not have any quantitative value. A derivative of amino sugars with l 1 is obtained as a colorless gum if the cleaning is carried out with a strongly acidic ion exchanger not on a batch process, but on a column.

Example 12. 200 g of the preparation described in Example 1 are dissolved in 940 ml of distilled water and 60 ml of conc. P28O4 and heated for 1/4 hour to reflux (internal temperature 98-100 ° C; oil bath temperature 140 ° C). 10 g of activated carbon (Merck, No. 2186) is added to the cooled black-brown solution and stirred for an hour. Then activated carbon is sucked off, washed with water and the filtrate is 250 ml of 10 n. CPC is brought to RP 7-8. The solution is stirred for an hour with 50 g of activated carbon. The coal is sucked off, washed with 2 l of water and the filtrate is evaporated. For desorption, coal is digested overnight with 2 liters of 30% alcohol. Then the carbon is filtered, and the alcohol solution is evaporated. Balance 8.0 g

The residue was dissolved in 15 ml of PaO and applied to a column filled with 50 g of Amberlite IR 120 (H + -form) column (height 20 cm, diameter 2.4 cm). Served at a rate of 3 drops in 1 MNP and washed with water (12 kagül / min) until all major components are removed. The main products are then removed with 0.5% ammonia from the column by elution (12 drops / min) and the aqueous solution is evaporated to dryness. The remainder 4.1 g.

2 g of this residue is dissolved in a small amount of water and applied to a column filled with Sephadex G-15 (height 200 cm, diameter 3.0 cm). Elute with water. At a speed of 8 ml / h, fractions of 2 ml are collected. Separate fractions are examined by thin layer chromatography. Fractions 85-94 give 280 mg of ammonia sugar with n 2 with a specific activity of 50,000 units. ig. sucrase / g

Example 13. 2 g of the preparation described in example 1 in 60 ml of 20 mM sodium glonophosphate buffer with a 6.9 n RP im IMM CaCU are continuously incubated for 120 hours at 37 ° C and 1 g of a-amylase from Y is incubated zreggillus spec, (firm Serva, No. 13418), then heated to 100 ° C for 5 minutes and the insoluble parts are removed by centrifugation at 4000 rpm. After lyophilization of the solution, 1.9 g of product is obtained with 350 units. ing. sucrase / g and 2-10 units ING. amylase / g If this product is examined by single-layer chromatography or by staining inhibition of Ca 16

according to Example 2, it turns out that, as inhibitory-active compounds, mainly amino-sugar derivatives with n 1, 2 and 3 are obtained.

Example 14. If 2 g of the preparation described in example 1 in 30 ml of 20 mM acetate buffer with RP 4.8 with 1.25 mg of sweet potato p-amylase are incubated with continuous stirring for 120 hours at 37 ° C

(Böhringer 15471), then heated to 100 ° C for 5 minutes and insoluble parts removed by centrifugation at 4000 rpm, then after lyophilization of the solution, 1.5 g of product are obtained with 1800 units. ing. sucrase / g and 3.8-10 units ing. amylase / g

If this product is examined by thin layer chromatography or by staining the inhibition of sucrase according to example 2, then it turns out that as inhibitory

compounds mainly get derivatives of amino sugars with l 2 and 3.

Example 15. If a 200 ml Erlenmeyer flask with 25 ml of nutrient fluid consisting of 0.1% K2PRO4, 0.2% (NP4) 25O4,

0.05% MgSO4, 0.05% KCl, 0.01% FeSO4, 2% of the preparation described in Example 1, are inoculated with a suspension of Asp spores. Niger ATCC 11304 is incubated on a rocking chair at 28 ° C, then after 6 days the titer decreases from 210,000 to

53000 units ING. amylase / ml and after 10 days to 21 300 units. ING. amylase / ml At the same time content units ing. sucrase / ml from 7.0 to 72 units. inhibitor of sucrase / ml. 20 ml of the solution, incubated with a spore suspension for 10 days, are centrifuged at 3000 rpm for 30 minutes to separate the mycelium. From 15 ml of residue 72,000 units. ing. sucrase / l) under 30 minutes stirring with 2 g amberlite IRC 50 H + and 1 g amberlite IRA 140 OP remove salts (electrical conductivity less than 2 ms). The solution is filtered and the filtrate is passed through 5 ml / h through equilibrated with Dowex H + in 0.001 n. HCl column (diameter 1 cm смЮ cm), then washed with 200 ml of 0.001 n. PS1. For desorption, the column is washed with a 0.6% ammonia solution (10 ml / hour) and 5 ml fractions are collected. The fractions that have an inhibitory effect on sucrose are evaporated to 2 ml and

2 ml of methanol. This solution is adjusted to a pH of 3-4 and added dropwise to 100 ml of acetone to precipitate. The precipitate is filtered on suction, washed with acetone and ether and dried. Yield 26 mg with 28000 units. ing.

sucrase / g from derivatives of amino sugars with n 2 and 3. A pure derivative of amino sugars with n 2 is obtained from this product, as described in example 9, by gel filtration through a biogel P-2 through a column. Obtain 7 mg of the derivative of amino sugars with n 2 with 60,000 units. ing. sucrase / g

Example 16. 2 l of culture filtrate obtained from the fermentation solution according to example 6 by removing the mycelium

by centrifugation at 13,000 rpm, having 17

activity 13000 units. ing. sucrase / g to reduce the salt content (the electron conductivity of the culture filtrate is about 10 ms) for an hour, it is not mixed with 500 g of a mixture of 2.5 parts Amberlite GC-50 (H + form) and 1 part Amberlite IRA-410 (OH-form), filtered, evaporated to 100 ml and centrifuged at 20000 U / min for 15 minutes to remove insoluble parts. The residue is brought to 100 ml of its electrical conductivity of 3.5 ms) and applied for further purification on a column (55X400 mm) with P-cellulose (Serva N ° 45130; obtained by known methods and equilibrated in 5 mM ammonium phosphate buffer, pH 5 ,five). The indicated phosphate buffer (90 ml / hour) is used as a solvent and 18 ml fractions are collected.

The aluate fractions are checked for carbohydrate content (anthrone sample) and components inhibiting sucrase (sucrase inhibitory breakdown), then fractions that, according to the anthrone test, contain almost no carbohydrates and at the same time according to the test with sucrose, combined, evaporated to 150 ml and filtered through a column (50x300 mm) with amberlite IRA-410 (HCOj-form). To better control deionization, the eluate is collected in fractions (10 ml per fraction per 20 min) and tested for carbohydrates (by anthropic sample; almost negative), for phosphate (by means of ascorbic acid – molybdate reagent: almost negative), and for inhibition of sucrase ( by enzymatic test). The inhibitory fractions (3-30) are collected, concentrated and lyophilized, redissolved and lyophilized, and 280 mg of crude inhibitor is obtained.

For further purification, the crude inhibitor according to irimer 6 is fractionated through Biogel P-2. From the fractions containing the pure derivative of amino sugars with, after lyophilization, 30 mg of product with 0.3-10 units is obtained. ING. amylase / g and 35,000 units. ing. sucrase / g

Example 17. For the preparation of derivatives of amino sugar with p 5-7, for example, the preparation described in Example 1 is prepared.

For this, 30 g of the preparation according to Example 1 is dissolved in 250 ml of H20. The conductivity of this solution is 10 ms, pH 5.5. For desalting, 60 g of Amberlite IRC 50 H + (weakly acid cation exchanger, which only minimally binds the derivatives of amino sugars from an aqueous solution) and 20 g of Amberlite IRA 410 OH- are added to the solution and mixed for 20 minutes. The filtrate (electrical conductivity 0.5 ms, pH 3.5) by 1 n. The HC1 is adjusted to a pH of 3.0 (electrical conductivity of 0.6 ms). With a solution of 42 ml / h, this solution is passed through a column filled with Dowex 50 W +) (H +) column (diameter 2.5 cm, height 40 cm, equilibrated in

18

0.001 n. MCI) and then iromyut 2 l 0.001 n. HC1. After washing, the columns are eluted with 1.2% aqueous and 10 ml fractions are collected. Inhibitor-active fractions

the ammonia is removed under vacuum and then the solution is evaporated to 30 ml. It is precipitated by dropping in 600 ml of alcohol, the precipitate is filtered off under suction and after washing with ethanol and ether is dried in a vacuum. Output

4.4 g with 26.5-10 units ing. amylase / g

0.5 g for fine purification according to Example 9 is applied to a column with biogel P-2 and developed. Fractions that are according to thin layer chromatography (inhibition color

amylases) contain amine sugar derivatives with a p 5-7, combine, evaporate in vacuo and precipitate with alcohol in this way. Yield from 0.5 g of crude product: 0.2 g of amino sugar derivatives c / g 5-7 s 30-U units. NNG

amylase / g and 2500 units. ing. sucrase / g

Example 19 (an example of acidic desorption of the proposed compounds).

A column with a diameter of 1.5 cm is filled with 30 g

raw dowex 50WX4 (P +) 200-400 mesh in 0.001 n. HC1. Then, for about an hour, a mixed eluate (400,000 UH, sucrase / L, pH 2.5, 60% acetone) passed through the column, obtained according to Example 10, and

then washed with 500 ml of 0.001 n. PS1. Under these conditions, only minimal activity is eluted. Then desorbed 0,0125 n. PS1, and register the electrical conductivity of the eluate column. In addition, research on the content of units. ing. sucrase eluate. The 74-100 activated fractions are combined and neutralized with amberlite IRA 410 OP-, then evaporated to 5 ml, 5 ml of methanol are added and precipitated by addition of 200 ml of acetone. After washing with acetone and ether, dried in vacuo.

The output of 1 g of the derivative of amino sugar with / g 2 with 65000 units ppp sucrase / g

From active pre-fractions get derivatives of aminosugar with p 3 and p 4.

This method of acid desorption as opposed to the main desorption allows

to fractionate homologous derivatives of amino sugars.

Individuality of the derived amino sugar 1, where R is glucose, and confirmed by thin layer chromatography,

p-1 by gas chromatography of its trimethylsilyl derivative, the angle of turn.

The structure of the derived amino sugar 1, where R is glucose, and confirmed

IR spectra, NMR spectra of partially deuterated derivatives, mass spectra of acetylated pi methylated derivatives.

The structure of higher homologues confirmed

Claims (3)

degradation products. Claims 1. A method for producing amino-derivative derivatives of general formula I BUT, he is. OF where R is an oligosaccharine chain with hexose units, characterized in that the organism of the actinoplanet family in an aqueous nutrient medium containing sources of carbon, nitrogen, salt, is grown under aerobic conditions at a pH of 5.0 to 8.5 with the following release of the target product famous tricks. 2. A method according to claim 1.0 tonnes, which is perceived by the fact that, as a microorganism of the actinoplanet family, an organism is taken of the form actinoplanes. 3. A method according to claim 1, characterized in that the aqueous nutrient medium contains an antifoam agent.