JP4629442B2 - Process for producing β-L-homofuconojirimycin by microorganism - Google Patents

Description

  The present invention relates to a method for producing β-L-homofuconojirimycin using a microorganism capable of producing β-L-homofuconojirimycin.

  Glycosidases are involved in many biological functions as metabolic enzymes in the biosynthesis and degradation of glycoconjugates. Fucosidase, a kind of glycosidase, is thought to be involved in cancer cell metastasis and virus infection. Therefore, fucosidase inhibitors have the potential to be used as therapeutic agents for diseases caused by cancer and viruses.

β-L-homofuconojirimycin is a pseudo sugar having fucosidase inhibitory activity and is synthesized as a fucosidase inhibitor by a chemical synthesis method. (E.g., Patent Document 1, Non-Patent Document 1) Further, in the non-chemical synthesis method, Angylocalyx pynaertii leguminous vegetable (e.g., see Non-Patent Documents 2 and 3) of and Liliaceae Scilla sibirica (e.g., non It can be obtained by extraction from Patent Document 4).
US Pat. No. 5,100,007 “Tetrahedron Letters” (UK), 1989, 30, p. 4439-4442 “European Journal of Biochemistry” (UK), 2001, 268, p. 35-41 “Journal of Natural Products” (USA), 2002, 65, p. 198-202 “Journal of Natural Products” (USA), 2002, 65, p. 1875-1881

  Although the above-described chemical synthesis method or the method for producing β-L-homofuconojirimycin using plant materials itself achieves its purpose, it is not always satisfactory from the viewpoint of industrial use. Not what you get.

  In other words, the chemical synthesis method has a problem in terms of complexity of operation or cost as a method for producing on an industrial scale. On the other hand, in the method of preparing from a plant, it takes time until the plant is cultivated and harvested, and the cultivation of the plant depends on the weather, and there is a problem that the acquisition of the material lacks stability.

  Therefore, there is still a need to develop and create a microorganism that can stably and efficiently produce β-L-homofuconojirimycin. An object of the present invention is to provide a method for producing β-L-homofuconojirimycin using a microorganism capable of producing β-L-homofuconojirimycin.

The inventors of the present invention have intensively studied to achieve the above object. As a result, among various filamentous fungi isolated from various sources by the present inventors, Penicillium sp. AB5577 ( Penicillium sp. AB5577) strain (FERM P-19515) Found for the first time to produce. The present invention was completed by successfully producing β-L-homofuconojirimycin from the culture of this filamentous fungus.

That is, the present invention is characterized by culturing a microorganism belonging to the genus Penicillium in a medium and collecting β-L-homofuconojirimycin and / or a salt thereof from the culture. This is a method for producing homofuconojirimycin. Here, salts of β-L-homofuconojirimycin are inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc., or acetic acid, propionic acid, glycolic acid, lactic acid, malonic acid, succinic acid. , Salts with organic carboxylic acids such as fumaric acid, malic acid, tartaric acid and citric acid.

  The method for producing β-L-homofuconojirimycin by the microorganism of the present invention realizes stable production and supply of β-L-homofuconojirimycin, and efficiently produces β-L-homofuconojirimycin. It becomes possible to manufacture at low cost.

The microorganism producing β-L-homofuconojirimycin used in the method of the present invention may be any microorganism as long as it has the ability to produce β-L-homofuconojirimycin or a salt thereof. A specific example is AB5577 strain isolated from soil collected by the present inventors from Minamiashigara City, Kanagawa Prefecture. This strain belongs to the genus Penicillium , and has been deposited as an α-homonojirimycin-producing bacterium with the accession number of FERM P-19515 at the National Institute of Advanced Industrial Science and Technology, Patent Deposit Center. It is an example of a β-L-homofuconojirimycin producing bacterium that is an α-L-fucosidase inhibitory active substance most effectively used in the method.

  In general, the bacteriological properties of fungi are very variable and are not constant. Fungi are natural or commonly used ultraviolet irradiation, X-ray irradiation, mutagenesis agents (eg, N-methyl-N-nitro-N-nitrosoguanidine or ethylmethanesulfonate) or artificial recombination using genetic recombination. It is a well-known fact that mutation is performed by means of mutation. All strains belonging to the genus Penicillium and capable of producing β-L-homofuconojirimycin, including such natural mutants and artificial mutants, can be used in the present invention.

  The medium used in the present invention may be any medium as long as it contains a carbon source, a nitrogen source, inorganic salts, growth factors, and nutrients required by the microorganism used. As the carbon source, those capable of assimilating β-L-homofuconojirimycin producing bacteria such as glucose, maltose, glycerin, starch, dextrin, starch syrup, molasses, animal and vegetable oils can be used. As the nitrogen source, soybean powder, wheat germ, fish meal, corn steep liquor, yeast extract, sodium nitrate, ammonium nitrate, ammonium sulfate and the like can be used. If necessary, trace metal salts such as cobalt, iron, nickel and manganese can be added. In addition, inorganic and organic substances that assist the growth of bacteria and promote the production of β-L-homofuconojirimycin can be appropriately added.

  The strain is cultured under aerobic conditions by shaking culture, stirring culture, or the like. Usually, the culture temperature is in the range of 15 to 37 ° C, preferably 24 to 30 ° C. The pH during the culture is preferably in the range of 3.0 to 8.0. The number of days of culture is 3 to 14 days. However, when the amount of β-L-homofuconojirimycin accumulated in the culture solution reaches the maximum, the culture is stopped and a fermentation product is collected from the culture solution. Isolation may be performed according to the method. The culture conditions such as medium composition, medium liquidity, culture temperature, agitation speed, and aeration rate are adjusted or selected for convenience so that preferable results are obtained according to the type of strain used and external conditions. When there is foaming in liquid culture, antifoaming agents such as silicone oil, vegetable oil and surfactant are conveniently used.

  Collect β-L-homofuconojirimycin from the culture solution by centrifugation, filtration through a filter, etc., followed by purification using a cation / anion exchange resin, decolorization, chromatography, and crystallization. You can.

  The present invention will be described in more detail with reference to examples, but the present invention is not limited to these specific examples.

  In the following examples,% is (weight / volume)% unless otherwise specified.

  The α-L-fucosidase inhibitory activity is measured according to a method already reported (European Journal of Organic Chemistry 2000, p. 2089-2093). Specifically, the measurement was performed by the following operation method.

  17 μM of a mixture of p-nitrophenyl α-L-fucopyranoside (340 μg), α-L-fucosidase (produced by bovine kidney-derived Sigma, 1.3 ng), BSA (bovine serum albumin) (38 μg), and test solution 5 μL After reacting in citrate buffer (pH 6.0, 45 μL) at 27 ° C. for 60 minutes, 50 mM glycine buffer (pH 10.1, 90 μL) was added to stop the reaction, and the absorbance at 400 nm was measured. The absorbance is As. Moreover, the absorbance when no test solution was added was simultaneously measured as Aw, and the absorbance when α-L-fucosidase was not added as Ab. From the absorbance, α-L-fucosidase inhibitory activity was determined by the following formula.

¹ H and ¹³ C-NMR were measured using a nuclear magnetic resonance apparatus (model JNM-LA300) manufactured by JEOL Ltd. in a solution of heavy water (D ₂ O) unless otherwise specified.

Flask culture: soluble starch 1.5%, glucose 0.5%, malt extract 0.05%, yeast extract 0.02%, polypeptone 0.1%, potassium nitrate 0.1%, calcium carbonate Place 100 ml of 0.1%, pH 6.0 medium in a 500 ml Erlenmeyer flask with baffle and inoculate a penicillium sp. AB5577 ( Penicillium sp. AB5577) strain from a slant and rotate at 27 ° C. for 8 days. Aerobic culture was performed on a rotary shaker at 180 rpm.

1 L of the culture solution collected from the 10 culture flasks was separated into cells and supernatant by centrifugation. The culture supernatant was passed through a column packed with 400 ml of strongly acidic cation exchange resin Duolite (registered trademark) C-20 (H ⁺ type), and then 1.2 L of deionized water was passed through this column. Washed and eluted with 2.0 L of 2.0% aqueous ammonia. The eluate containing the α-L-fucosidase inhibitory active ingredient was concentrated under reduced pressure to 100 ml, and passed through a column packed with 80 ml of strongly basic anion exchange resin Duolite (registered trademark) A-113 (OH ⁻ type). Thereafter, 250 mL of deionized exchange water was passed through the column for washing. The passing liquid and washing liquid obtained by the above operation were concentrated to dryness under reduced pressure, dissolved in a small amount of distilled water, and 70 ml of weakly acidic cation exchange resin Amberlite (registered trademark) IRC-50 (NH ₄ ⁺ type) was added. The solution was applied to a packed column and eluted with distilled water to fractionate the eluate. α-L-fucosidase inhibitory activity fractions were collected, concentrated and freeze-dried to obtain 25 mg of α-L-fucosidase inhibitory active substance. NMR analysis of the thus obtained α-L-fucosidase inhibitory active substance revealed that its ¹ H-NMR chemical shift value and ¹³ C-NMR chemical shift value were β-L-homofuconojirimycin. Consistent with literature values.
¹ HNMR data:
δ (ppm): 0.96 (3H, d, J = 6.8 Hz), 2.34-2.38 (1H, m), 2.70 (1H, dd, J = 7.0, 7. 0 Hz), 3.40-3.42 (2H, m), 3.56-3.66 (3H, m)
¹³ C NMR data:
δ (ppm): 77.7, 75.0, 70.5, 63.2, 63.0, 55.8, 18.8

Soluble starch 1.0%, glucose 5.0%, yeast extract 1.0%, potassium nitrate 0.2%, calcium carbonate Sterilized by placing 2.0 L of 0.1%, pH 6.0 medium in a 4 L jar fermenter, inoculated with Penicillium sp. AB5577 ( Penicillium sp. AB5577) pre-cultured, stirred at a culture temperature of 27 ° C. Culture was performed for 7 days at a rotation speed of 450 rpm and an aeration rate of 2 L / min.

The culture solution (2.0 L) was separated into cells and supernatant by centrifugation. The culture supernatant was passed through a column packed with 600 ml of strongly acidic cation exchange resin Duolite (registered trademark) C-20 (H ⁺ type), and then 1.8 L of deionized water was passed through this column. Washed and eluted with 3.0 L of 2.0% aqueous ammonia. The eluate containing the α-L-fucosidase inhibitory active ingredient was concentrated under reduced pressure to 500 ml, and passed through a column packed with 75 ml of strongly basic anion exchange resin Duolite (registered trademark) A-113 (OH ⁻ type). Thereafter, 250 mL of deionized exchange water was passed through this column for washing.

The passing liquid and washing liquid obtained above were concentrated to dryness under reduced pressure, dissolved in a small amount of distilled water, and filled with 300 ml of weakly acidic cation exchange resin Amberlite (registered trademark) IRC-50 (NH ₄ ⁺ type). The eluate was fractionated by elution with distilled water. α-L-fucosidase inhibitory activity fractions were collected, concentrated and lyophilized to obtain 92 mg of α-L-fucosidase inhibitory substance. The α-L-fucosidase inhibitory active substance thus obtained was subjected to NMR analysis. As a result, the ¹ H-NMR chemical shift value and ¹³ C-NMR chemical shift value of β-L-homofuconojirimycin Consistent with literature values.

Claims (2)

Filamentous fungi belonging to the genus Penicillium having β-L-homofuconojirimycin-producing ability or mutants thereof are cultured in a medium, and β-L-homofuconojirimycin and / or salts thereof are collected from the culture. A process for producing β-L-homofuconojirimycin, The filamentous fungus capable of producing β-L -homofuconojirimycin is Penicillium sp. AB5577 (FERM P-19515) (Penicillium sp. AB5577 FERM P-19515 ) or a mutant thereof, Item 2. A process for producing β-L-homofuconojirimycin according to Item 1 .