Method for manufacturing 2-amino-2-deoxy-D-mannitol.

0001The present invention relates to a method for manufacturing 2-amino-2-deoxy-D-mannitol which is the useful substance, as described below.

0002More particularly, the present invention relates to a method for manufacturing 2-amino-2-deoxy-D-mannitol which comprises culturing a microorganism belonging to the genus Streptomyces and capable of producing 2-amino-2-deoxy-D-mannitol in a medium and isolationg 2 amino-2-deoxy-D-mannitol from the obtained culture broth.

00032-Amino-2-deoxy-D-mannitol in accordance with the present invention is not only per se useful as a cosmetic to be formulated (Japanese Published Unexamined Patent Application No. 212421/1984) or as a starting material for preparing X ray contrast media (Japanese Published Unexamined Patent Application No. 128346/1977), but also a useful substance having a specific activity that 2-amino-2-deoxy-D-mannitol enhances an yield of moranoline in the production of moranoline, which exhibits inhiting action of blood sugar increase after having meals and is expected as a remedy for diabetes mellitus, (Japanese Published Examined Patent Application No. 009919/1981) by microorganisms belonging to the genus Streptomyces.

(Prior Art)

00042-Amino-2-deoxy-D-mannitol is a known compound which has been hitherto prepared by reducing N-acetylamino-2-deoxy-D-­mannose with sodium borohydride, etc. to convert into N-acetylamino-2-deoxy-D-mannitol and then hydrolyzing N-acetylamino-2-deoxy D-mannitol with an alkali or an acid.

(Problems to be solved by the Invention)

0005According to the preparation method described above, however, the method involves defects that N-acetylamino-2-deoxy-D-mannose used as a starting material is expensive, resources are poor, complicated steps for synthesis and isolation are required, and the like.

0006As a result of investigations on a method for manufacturing 2-amino-2-deoxy-D-mannitol by fermentation over a wide range, the present inventors have found the fact that a microorganism belonging to the genus Streptomyces can produce 2-amino-2-deoxy-­D-mannitol in a free form in large quantities and have come to accomplish the present invention.

0007A representative example of the microorganism in accordance with the present invention is <u style="single">Streptomyces lavendulae</u> SEN-158 (hereafter referred to as "SEN-158 strain") isolated from a soil in Sapporo City, Japan by the present inventors.

0008SEN-158 strain has been deposited in the Fermentation Research Institute of the Agency of Industrial Science &amp; Technology of Japan under Accession Number of FERM P-4301. Its bacteriologial properties are described in Japanese Published Unexamined Patent Application No. 084094/1979. Further this strain has been deposited in the American Type Culture Collection, Rockville Md., under Accession Number of ATCC 31434.

0009In the present invention, any strain other than the SEN-158 strain is usable as far as it belongs to the genus Streptomyces and is capable of producing 2-amino-2-deoxy-D-mannitol. Further, artificial mutants obtained by applying to these strains variation means conventionally used such as a treatment for irradiating with ultraviolet rays or 60Co, etc., a variation treatment with a variation inducer such as nitrogen mustard, azaserine, nitric acid, nitrosoguanidine or 2-aminopurine, etc., transduction, transformation, cell fusion, etc. and naturally occurring variants are also suited for the purpose of the present invention.

0010The present invention can be practiced by using the 2-amino-2-deoxy-D-mannitol-producing strain and culturing the strain in a conventional manner for culturing Actinomycetes.

0011A medium can be liquid or solid but shake culture or submerged culture in liquid medium can ordinarily be used.

0012The medium can be any one that is suited for growth of Actinomycetes and capable of producing 2-amino-2-deoxy-­D-mannitol.

0013As the carbon sources, there can be used glucose, galactose, mannitol, sucrose, maltose, glycerine, dextrin, starch, or the like.

0014As the nitrogen sources, there can be used soybean powders, peptone, yeast extract, meat extract, corn steep liquor, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, etc.

0015In addition, supplementation of sodium chloride, potassium chloride, calcium carbonate, various phosphates or the like in a suitable amount can provide good results. Furthermore, a suitable amount of iron, magnesium, etc. may also be added.

0016If necessary, organic or inorganic compounds, vitamins, etc. can be supplemented to accelerate growth of the microorganisms used or production of 2-amino-2-deoxy-D-mannitol.

0017In case that foaming is remarkable during fermentation, a defoaming agent may be appropriately added.

0018Conditions for incubation such as a pH value of the medium, incubation temperature and the like can be appropriately varied within such a range that produces 2-amino-2-deoxy-D-mannitol. For example, in shake culture or submerged culture, it is desired to culture at pH of from 6 to 9 at an incubation temperature of from approximately 20 to 35°C, preferably 25 to 30°C.

0019A time period for incubation varies depending upon scale of culture and other conditions but it is generally sufficient for 2 to 20 days.

0020After incubation, the cells are separated and the product is isolated from the obtained culture broth and purified.

0021In order to isolate and purify the substance of the present invention from the culture broth , conventional technique used for isolating and purifying microorganism metabolites from the culture broth can be used.

0022For example, adsorption and desorption with various adsorbents (e.g., silica gel, alumina, activated charcoal, ion exchange resin, etc.), chromatography, partition chromatography, fractional crystallization, recrystallization, etc., can be used singly or in combination.

0023Hereafter the present invention is described in more detail by referring to an example and a test example below.

Example 1

0024In a 500 ml Erlenmeyer flask, 100 ml medium containing (8% of soluble starch, 1% of soybean powders, 1% of yeast extract, 0.05% of potassium chloride, 0.05% of magnesium sulfate, 0.5% of sodium chloride and 0.2% of sodium nitrate, pH 7.2) was charged and sterilized. Several platinum loops of SEN-158 strain were inoculated to the medium from the slant followed by shake culture at 27°C for 3 days to give a preculture solution. This preculture solution, 300 ml, was inoculated on 15 liters of culture solution (components are the same as in the preculture solution) charged in a 30 liter volume jar fermenter followed by culture at 27°C for 11 days.

0025As a defoaming agent, NISSAN DISFOAM CB-442 was used; an aeration rate and an agitation speed were 20 liters/min. and 300 rpm, respectively.

0026After 12.9 liters of the obtained culture broth was centrifuged at 9000 rpm for 20 minutes, the obtained supernatant was passed through a column packed with strongly acidic ion exchange resin Dowex 50W x 2 (H⁺) (1 liter). After thoroughly washing with water, elution was performed with 1 N ammonia water.

0027The eluate was concentrated under reduced pressure. The concentrate was passed through strongly basic ion exchange resin Diaion SA-11A (OH⁻) (500 ml) followed by eluting with water. The eluate was combined with the washing liquid. The mixture was concentrated under reduced pressure. The concentrate was allowed to stand at 5°C for several days. The formed crystals were collected and recrystallized from 20% hydrated ethanol to give 1 g of 2-amino-2-deoxy-D-mannitol. Physical Properties of this product are as follows. Melting point: 161-163°C Elemental analysis (C₆H₁₅NO₅) <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Calcd. (%)</entry><entry namest="col2" nameend="col2" align="char" char=".">C: 39.77</entry><entry namest="col3" nameend="col3" align="char" char=".">H: 8.34</entry><entry namest="col4" nameend="col4" align="char" char=".">N: 7.73</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Found (%)</entry><entry namest="col2" nameend="col2" align="char" char=".">C: 39.62</entry><entry namest="col3" nameend="col3" align="char" char=".">H: 8.17</entry><entry namest="col4" nameend="col4" align="char" char=".">N: 7.81</entry></row></tbody></tgroup></table></tables> Specific rotary power [α]<img file="EP0322571A1_D0001.tif" /> +4.0° (C= 1%, water) ¹³C-NMR ppm; (D₂O, internal standard; methanol 49.8 ppm) 53.84 64.05 64.31 70.73 71.29 71.85 ¹H-NMR ppm; (D₂O, internal standard; DSS) 2.98-3.12 (1H, m), 3.56-3.90 (7H, m)

0028The structure of this product was confirmed by the fact that the properties were fully identical with those of a compound synthesized by reducing N-acetylamino-2-deoxy-D-mannitol with sodium borohydride and then hydrolyzing it with hydrochloric acid.

Test Example

Effect of 2-amino-2-deoxy-D-mannitol on moranoline production

0029Variant GC-148 of newly isolated moranoline-producing strain MB-733 belonging to the genus Streptomyces was inoculated on a medium shown below. After shake culture at 27°C for 7 days, 10 ml of the culture broth was centrifuged. The supernatant was passed through a column packed with strongly acidic ion exchange resin Dowex 50W x 2 (H⁺) (1 liter). After thoroughly washing with water, elution was performed with 0.5 N ammonia water. After the eluate was concentrated to dryness under reduced pressure, the residue was dissolved in 1 ml of water. The solution was subjected to high performance liquid chromatography to quantitatively determine moranoline.

0030Conditions for high performance liquid chromatography were as follows: column, Nucleosil 5NH₂; developing solvent, acetonitrile-water = 7 : 3; detection, differential refractometer.

0031Medium A: 2% of soluble starch, 1% of soybean powders, 1% of yeast extract, 0.05% of potassium chloride, 0.05% of hydrated magnesium sulfate, 0.5% of sodium chloride, 0.2% of sodium nitrate and 0.35% of calcium carbonate; pH 7.0

0032Medium B: medium obtained by supplementing 1% of 2-amino-2-deoxy-D-mannitol to Medium A

0033The results are shown in the table below. <tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center"><u style="single">pH</u></entry><entry namest="col3" nameend="col3" align="center"><u style="single">Moranoline (µg/ml)</u></entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Medium A</entry><entry namest="col2" nameend="col2" align="char" char=".">8.6</entry><entry namest="col3" nameend="col3" align="right">981</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Medium B</entry><entry namest="col2" nameend="col2" align="char" char=".">7.6</entry><entry namest="col3" nameend="col3" align="right">4428</entry></row></tbody></tgroup></table></tables>

0034The moranoline-producing effect of 2-amino-2-deoxy-­D-mannitol in accordance with the present invention is clear from the results.