JP4854418B2 - Method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material - Google Patents

Description

  The present invention produces dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material by microorganisms, and then dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1 The present invention relates to a method for producing a dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan material, which separates the furan material from microorganisms.

  Dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan (sometimes referred to as hydroambroxan (TM)) is a highly persistent fragrance, mainly salvia sclarea Manufactured by chemical conversion from Sclareol extracted from. A process for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan from sclareol is shown in FIG. As shown in FIG. 8, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate includes decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene. Ethanol and sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan-2 (1H) -one; (Sclareolide)) are known.

  The conversion reaction of sclareol to dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate by microorganisms is described in, for example, Patent Documents 1 to 4. Patent Document 1 discloses the production of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol by Hyphozyma roseoniger ATCC20624. Patent Document 2 discloses the production of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate by Cryptococcus laurentii ATCC20920, and Patent Document 3 discloses dodecahydro by Bensingtonia cilliata ATCC20919. -3a, 6,6,9a-Tetramethylnaphtho [2,1-b] furan intermediate production is disclosed in US Pat. No. 6,057,086, which is dodecahydro-3a, 6,6,9a by Cryptococcus albidus ATCC20918 and Cryptococcus albidus ATCC20921 -The production of tetramethylnaphtho [2,1-b] furan intermediates is disclosed. Thus, by producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate using microorganisms, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [ The cost of 2,1-b] furan synthesis can be reduced.

  In the production process of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate using microorganisms, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1 -b] Efficient separation and purification of furan intermediate is required. Patent Document 4 discloses a method for separating and purifying dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate (sclareolide) using the above-mentioned microorganisms. . The method disclosed in Patent Document 4 is a method of crystallization following a solvent extraction method using ethyl acetate and filtration using a mesh.

  However, in the conventional method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan, dodecahydro-3a, 6,6,9a-tetramethylnaphtho produced by microorganisms [ There was a problem that the yield in separating and purifying the 2,1-b] furan intermediate was poor.

Japanese Patent No. 2547713 Patent No. 2802588 Patent No. 3002654 Patent No. 2063550

  Therefore, in view of the actual situation as described above, the present invention can efficiently separate a dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate produced by a microorganism. An object of the present invention is to provide a method for producing a dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material.

  In order to achieve the above-described object, the present inventors have conducted intensive studies, and as a result, in a solution containing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate and bacterial cells, It has been found that the two can be separated by adjusting the density, and the present invention has been completed.

  That is, the present invention includes the following.

  That is, dodecahydro-3a, 6,6,9a-, which comprises a step of separating microbial cells from dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material produced by microorganisms A process for producing tetramethylnaphtho [2,1-b] furan raw material, wherein dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material and microbial cells are suspended. The dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material and cells are separated by adjusting the solution density of the solution. This is a method for producing a 6,9a-tetramethylnaphtho [2,1-b] furan raw material.

  Here, the dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material is synthesized from dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan Means an intermediate in the process. Such intermediates include decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan-2 (1H) -on).

  In the method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material according to the present invention, the solution density of the solution is preferably 1.07 g / mL or more. Moreover, it is preferable that the solution density of the said solution is less than the value from which a microbial cell does not precipitate and floats by centrifugation.

  Further, according to the method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan according to the present invention, two or more kinds of dodecahydro-3a, 6,6,9a-tetra From a solution containing methylnaphtho [2,1-b] furan feedstock, one dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan feedstock is mixed with the remaining dodecahydro-3a, 6, It is also possible to separate 6,9a-tetramethylnaphtho [2,1-b] furan from microbial cells. Specifically, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene from a solution containing ethanol, sclareolide and a microorganism, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene Ethanol can be separated from sclareolide and microorganisms.

  Furthermore, according to the method for producing a dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material according to the present invention, two or more kinds of dodecahydro-3a, 6,6,9a-tetra After separating the methylnaphtho [2,1-b] furan raw material and the cells, one kind of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material is used as the remaining dodecahydro- It is also possible to separate from 3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material. Specifically, from a solution containing decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol, sclareolide and a microorganism, first, decahydro-2-hydroxy-2,5,5,8a-tetra Methylnaphthaleneethanol and sclareolide can be separated from the microorganism, and then decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol can be separated from sclareolide.

  According to the method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan according to the present invention, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1 -b] The furan raw material can be efficiently separated from the microorganism that produced the dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material. Therefore, according to the method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan according to the present invention, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2 , 1-b] furan dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material can be obtained at low cost.

Hereinafter, the present invention will be described in detail.
Dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material producing microorganism In the present invention, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan Dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate in the synthesis system of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan Any microorganism can be used as long as it produces microorganisms. That is, in the method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan according to the present invention, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2, 1-b] Microorganisms isolated from soil can be used with the ability to produce a furan intermediate as an index. The ability to produce dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate is obtained by culturing a test microorganism in a sclareol-containing medium, and dodecahydro-3a, 6 contained in the medium. , 6,9a-tetramethylnaphtho [2,1-b] furan intermediate can be detected and detected. The dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate contained in the medium is obtained using an organic solvent from the medium after removing the test microorganisms. , 6,9a-tetramethylnaphtho [2,1-b] furan intermediate can be extracted and then detected by, for example, gas chromatography (GC).

  In addition, detection of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate is not limited to GC, for example, gas-liquid chromatography (GLC), thin layer chromatography (TLC) Conventionally known analytical methods such as high pressure liquid chromatography (HPLC), infrared spectrum (IR) and nuclear magnetic resonance (NMR) can be used.

Here, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate means decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide ( Decahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan-2 (1H) -one).

  For example, in the method for producing a dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material according to the present invention, the inventors of the present application disclosed in a previous application (Japanese Patent Application No. 2006-48550). The disclosed microorganisms can be used. As a result of analysis, this microorganism is a yeast belonging to the Ascomycetes, named Ascomycete sp. KSM-JL2842, and incorporated by the National Institute of Technology and Evaluation (NITE) Deposited as FERM P-20759 on January 12, 2006, at 2-5-8 Kazusa Kamashishi, Kisarazu City, Chiba Prefecture.

  In the method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan raw material according to the present invention, ascomycetes identified by the accession number FERM P-20759 Is classified into the same genus as the yeast belonging to, preferably the same species as the yeast, more preferably the same strain as the yeast and dodecahydro-3a, 6,6,9a-tetramethylnaphtho [ Microorganisms capable of producing a 2,1-b] furan intermediate can also be used.

  Further, in the method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan according to the present invention, a conventionally known dodecahydro-3a, 6,6,9a-tetramethylnaphtho [ 2,1-b] furan intermediate producing microorganisms can also be used. For example, Hyphozyma roseoniger ATCC20624 disclosed in Patent No. 2547713 referred to as Patent Document 1, Cryptococcus laurentii ATCC20920 disclosed in Patent No. 2802588 referred to as Patent Document 2, and Patent No. 3002654 referred to as Patent Document 3 Bensingtonia cilliata ATCC20919 and Cryptococcus albidus ATCC20918 and Cryptococcus albidus ATCC20921 disclosed in Japanese Patent No. 2063550 referred to as Patent Document 4 can be used.

Production of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate by microorganisms By culturing the above-mentioned microorganisms under predetermined conditions, dodecahydro-3a, 6, 6,9a-tetramethylnaphtho [2,1-b] furan intermediate can be produced. Specifically, the above-described microorganism is cultured in a medium containing sclareol. As the medium, a medium having a preferred composition can be appropriately used according to the microorganism to be used. For example, when using the ascomycetous fungus net (hereinafter referred to as JL2842 strain) specified by the above-mentioned accession number FERM P-20759, usable media include carbon source, nitrogen source, metal minerals and vitamins, etc. Examples thereof include a solid medium and a liquid medium. Note that a surfactant or an antifoaming agent may be added to the medium for culturing the JL2842 strain depending on the culture conditions and the like.

  Examples of the carbon source added to the medium include monosaccharides, disaccharides, oligosaccharides, and polysaccharides, and two or more of these may be used in combination. Examples of carbon sources other than carbohydrates include organic acid salts such as acetate. Here, as the carbon source, each of these components may be used alone, or a plurality of components may be mixed and used as necessary.

  Examples of the nitrogen source include inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium phosphate and ammonium acetate, urea, peptone, meat extract, yeast extract and casein hydrolyzate. Examples thereof include nitrogen-containing organic substances, amino acids such as glycine, glutamic acid, alanine, and methionine. Here, as the nitrogen source, each of these components may be used alone, or a plurality of components may be mixed and used as necessary.

  Furthermore, examples of metal minerals include sodium chloride, ferrous sulfate, magnesium sulfate, manganese sulfate, zinc sulfate, and calcium carbonate. Here, as metal minerals, each of these components may be used alone, or a plurality of components may be mixed and used as necessary.

  The culture conditions for culturing the JL2842 strain are not particularly limited, and are adjusted to the optimum pH and temperature. Specifically, the optimum range of pH is 3 to 8, preferably 4 to 8, and more preferably 5 to 7. Moreover, the optimal range of temperature is 10-35 degreeC, Preferably it is 15-30 degreeC, More preferably, it is 20-30 degreeC. In addition to shaking culture, anaerobic culture, stationary culture, and culture using a fermenter, culture can be carried out by resting cell reaction and immobilized cell reaction.

  The concentration of sclareol added to the medium having such a composition is not particularly limited, but is preferably 0.1% to 50%. Sclareol may be added to the medium prior to culturing or may be added during the culturing (fed-batch culture). In addition, compositions other than sclareol, such as carbon sources, nitrogen sources, vitamins, minerals, surfactants and antifoaming agents, can be fed simultaneously.

  Microorganisms other than the JL2842 strain can also be cultured under optimal culture conditions using an optimal medium composition by appropriately adjusting the medium composition.

Separation of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate and fungus As described above, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2, Since the 1-b] furan intermediate is produced by microorganisms, it is first obtained as a solution containing the cells. Therefore, in the method for producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan according to the present invention, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2, The 1-b] furan intermediate is separated from the bacterial cells, and the dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate is efficiently recovered.

  Specifically, as described above, after culturing the microorganism in the presence of sclareol, the culture solution is centrifuged to collect the precipitate.

  The dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate and bacterial cells collected by centrifugation were prepared to a predetermined solution density (hereinafter, for separation (Referred to as a solution). The separation solution is preferably an aqueous solution, but may be an organic solution. Moreover, it does not specifically limit as a solute, For example, salts, such as sodium tartrate, sodium chloride, sodium citrate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, can be used. Moreover, sugar alcohols, such as sorbitol, can also be used as a solute.

  In particular, by suspending dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate and cells in the separation solution, dodecahydro-3a, 6,6,9a- Tetramethylnaphtho [2,1-b] furan intermediate can be floated in the solution for separation to allow the bacterial cells to settle. Therefore, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate and cells are suspended in the separation solution, and after the cells settle, the solution supernatant is recovered. Thus, the bacterial cells and dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate can be separated. To suspend only the cells after suspending the dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate and the cells, for example, use a centrifugation method be able to. As a condition for centrifugation, for example, it can be 5 minutes at 3000 rpm.

  In addition, after suspending the cells and dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate in a solution for separation, the cells are allowed to settle to settle only the cells. May be. In the case of standing still, conditions such as room temperature for 24 hours can be set as an example. Here, the separation solution can separate the dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate and the cells by setting the solution density to a predetermined value. Specifically, the solution density of the separation solution is such that dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate can be floated in the solution, and The density should be such that it settles without floating in the solution. More specifically, the dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate is added to the solution by setting the solution density of the separation solution to 1.06 g / mL or more. Can float and allow cells to settle.

  Furthermore, by setting the solution density of the separation solution to 1.07 g / mL or more, among the dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediates, especially decahydro-2- Hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol can be separated with high efficiency. In addition, when the solution density of the separation solution is 1.23 g / mL or more, both decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide can be separated with high efficiency.

  In addition, as described above, when dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate is produced by a microorganism, a plurality of dodecahydro-3a, 6, If it contains 6,9a-tetramethylnaphtho [2,1-b] furan intermediates (eg decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide) By adjusting the solution density, one kind of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate can be transformed into bacterial and other dodecahydro-3a, 6,6,9a -Can be separated from the tetramethylnaphtho [2,1-b] furan intermediate. For example, when decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide are included in the culture solution, the solution density of the separation solution is 1.07 g / mL or more and 1.15 g / mL or less By doing so, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol is mainly floated in the solution, and sclareolide can be precipitated together with the cells.

  Furthermore, in this case, sclareolide is suspended by resuspending the precipitated cells and sclareolide in a different separation solution (for example, a solution density of 1.23 g / mL) with a solution density greater than 1.15 g / mL. It can be floated mainly in the solution, and only the bacterial cells can be settled. In this way, by sequentially using a plurality of separation solutions having different solution densities, a plurality of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediates are sequentially removed from the cells. Can be separated.

  In order to sequentially separate a plurality of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediates, first, a plurality of dodecahydro-3a, 6,6,9a-tetra Methylnaphtho [2,1-b] furan intermediate is isolated from the cell and then one of several dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediates The dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate may be sequentially separated. In this case, first, for example, specifically, a solution for separation having a solution density of more than 1.15 g / mL is suspended, and decahydro-2-hydroxy-2,5,5,8a- is suspended in the solution. Both tetramethylnaphthalene ethanol and sclareolide are floated, and only the cells are allowed to settle. Next, collect the supernatant fraction excluding bacterial cells and adjust the solution density to 1.07 g / mL or more and 1.15 g / mL or less, or different separations that are 1.07 g / mL or more and 1.15 g / mL or less Resuspend in working solution. Thereby, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol mainly floats in the solution, and sclareolide mainly precipitates. In this way, by sequentially using a plurality of separation solutions having different solution densities, a plurality of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediates are separated from the cells. The body can be separated sequentially.

  On the other hand, when decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol is separated, the pH of the separation solution is preferably 3 or more. When the pH of the separation solution is less than 3, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol is added to dodecahydro-3a, 6,6,9a-tetramethyl in the separation solution. There is a possibility that the reaction changing to naphtho [2,1-b] furan proceeds. Therefore, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol is converted to dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1 by using a separation solution having a pH of 3 or higher. -b] Can be recovered without changing to francs.

Synthesis of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan As described above, dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2, The 1-b] furan intermediate is used as a raw material for the production of dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan, a high-value-added, high-perfume fragrance can do. Dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate is used to produce dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan It does not specifically limit as a method, A conventionally well-known method can be used suitably. Specifically, sclareolide is decahydro-2-hydroxy-2,5,5,8a-tetramethyl, for example by reduction with lithium aluminum hydride, sodium borohydride, or potassium borohydride / lithium chloride mixture. Convert to naphthalene ethanol. Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol is used with acidic catalysts such as p-toluenesulfonic acid, p-toluenesulfonic acid chloride, catalytic amounts of sulfuric acid and acidic ion exchangers. It is converted to dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan by dehydration cyclization in various solvents.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1] Separation of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol from a culture solution by adjusting the solution density [I]
Ascomycete sp. KSM-JL2842 (FERM P-20759) was inoculated with 1 platinum ear in 2.1% YM broth (Becton Dickinson) and cultured with shaking at 25 ° C. for 3 days. Next, inoculate 1% of the inoculum into a medium consisting of 0.2% yeast extract, 0.2% ammonium nitrate, 0.1% monopotassium phosphate, 0.05% magnesium sulfate heptahydrate and 1.0% glucose, and shake at 25 ° C. Culture was performed. On the third day of culture, sclareol and Tween 80 were added to a final concentration of 1.0% and 0.5%, respectively, and further cultured for 5 days.

  After completion of the culture, the culture solution was centrifuged at 3000 rpm for 5 minutes to obtain a dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate (decahydro-2-hydroxy-2,5 , 5,8a-tetramethylnaphthaleneethanol) and bacterial cells were precipitated. The supernatant was removed, and a mixed precipitate of the cells and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol was collected. The collected precipitate was suspended using a sodium tartrate solution or a saline solution (separation solution) adjusted to a solution density of 1.00 to 1.14 g / mL, and then centrifuged (3000 rpm for 5 minutes).

  Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol contained in the supernatant fraction after centrifugation was quantified for each separation solution having a different solution density. Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol was quantified by gas chromatography (GC) analysis. The results are shown in FIGS. From the results shown in FIGS. 1 and 2, when the solution density is 1.06 g / mL or more, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol floats in the supernatant fraction, and the cells are A phenomenon such as precipitation was observed, and it was clarified that decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and bacterial cells could be separated. Furthermore, when the solution density of the separation solution is 1.07 g / mL or more, the recovery rate of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol in the supernatant fraction should be 96% or more. Became clear.

  In this example and the following examples, the recovery rate is a ratio of the value obtained by subtracting the product amount contained in the precipitate portion after centrifugation, assuming that the product amount contained in the culture solution before centrifugation is 100%. Calculated as

[Example 2] Separation of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol from a culture solution by adjusting the solution density [II]
As in Example 1, Ascomycete sp. After culturing KSM-JL2842 (FERM P-20759), a mixed precipitate of the cells and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol was collected by centrifugation. In this example, a citric acid solution, a disodium hydrogen phosphate solution, a sodium tartrate solution, a saline solution, a dipotassium hydrogen phosphate solution, or a sorbitol solution (solution for separation) prepared at a solution density in the range of 1.00 to 1.15 g / mL. ) Was used to suspend the collected precipitate and centrifuged again (3000 rpm for 5 minutes).
Table 1 shows the density of the various solutions.

  About each said solution for separation, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol contained in the supernatant fraction after centrifugation was quantified. Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol was quantified by GC analysis in the same manner as in Example 1. The results are shown in FIGS. From the results shown in FIGS. 3 and 4, when the solution density is 1.06 g / mL or more, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol floats in the supernatant fraction, and the cells are precipitated. This phenomenon was observed, and it was revealed that decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and bacterial cells could be separated. Moreover, it was revealed that 97% or more of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol was recovered by using any separation solution.

  On the other hand, when the supernatant fraction was observed, a phenomenon in which the cells floated was not observed. In order to examine in detail, the supernatant fraction of each sample was observed with a microscope, and the cells contained in the supernatant fraction were counted using a hemocytometer. The number of cells floating in the supernatant increased depending on the solution density, but even when the solution density was 1.23 g / mL, the solution density was about 1.40 g / mL to about 1/40 of the cells contained in the culture solution. It decreased to 1/100 or less at the following times. Therefore, in the range of the solution density of the separation solution prepared in this example, separation between the JL2842 strain and dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate was hindered. Not suggested.

[Example 3] Separation of sclareolide from culture medium by adjusting solution density In this example, sclareolide was isolated from bacterial cells as dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan intermediate. We examined how to do this. First, as in Example 1, Ascomycete sp. After culturing KSM-JL3571 (FERM P-20758), the mixed precipitate of the cells and sclareolide was recovered by centrifugation. The collected precipitate was suspended using a sodium tartrate solution or a saline solution (separation solution) adjusted to a solution density of 1.00 to 1.23 g / mL, and then centrifuged (3000 rpm for 5 minutes).

  For each separation solution having a different solution density, sclareolide contained in the supernatant fraction after centrifugation was quantified. Squaleolide was quantified by gas chromatography (GC) analysis. The results are shown in FIGS. From the results shown in FIGS. 5 and 6, in the separation solution having a solution density of 1.07 g / mL or more, a phenomenon was observed in which the microbial cells settled in the precipitate portion and sclareolide floated in the supernatant fraction. It became clear that can be separated. The recovery rate of sclareolide in the supernatant fraction was maximized (about 80%) when a separation solution having a solution density of 1.23 g / mL was used.

[Example 4] Separation of sclareolide and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol by adjusting the solution density In this example, a plurality of dodecahydro-3a, 6,6,9a-tetramethylnaphtho As the [2,1-b] furan intermediate, sclareolide and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol were separated from the cells, and sclareolide and decahydro-2-hydroxy-2,5 The method of separating 5,8a-tetramethylnaphthaleneethanol from each other was studied.

  First, also in this example, ascomycete sp. After culturing KSM-JL3571 (FERM P-20758), a mixed precipitate of the cells and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide was collected by centrifugation. The suspension was suspended using a sodium tartrate solution (separation solution) adjusted to a solution density of 1.11 g / mL, and then centrifuged (3000 rpm for 5 minutes). Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide were quantified by GC analysis under the same conditions as in Examples 1-3. As a result, it was revealed that both decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide floated in the supernatant fraction, and these intermediates could be separated from the cells. The recovery rate of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol floating in the supernatant fraction was 66.4%, and the recovery rate of sclareolide was 17.9%.

  From the results of this example, it became clear that both sclareolide and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol can be separated from the cells by using the separation solution.

  Next, a graph showing the recovery rate of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol shown in FIG. 2 (Example 1) and a recovery rate of sclareolide shown in FIG. FIG. 7 is a diagram obtained by superimposing the figure. From FIG. 7, the optimum range of the solution density of the separation solution that can recover decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol at the maximum and the solution density of the separation solution that can recover the maximum of sclareolide are shown. It became clear that the optimum range was different. Therefore, using a solution for separation having a solution density with high recovery efficiency of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol and low recovery efficiency of sclareolide, decahydro-2-hydroxy-2 5,5,8a-Tetramethylnaphthalene ethanol was found to be mainly separated. In addition, after mainly recovering decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol, sclareolide was added to the separation solution by using a separation solution having a high solution density for recovery of sclareolide. It was found that it was mainly recoverable.

The state after suspending and centrifuging a mixed precipitate of bacterial cells and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol using the separation solution prepared in Example 1 is shown. It is a photograph. After suspending and centrifuging the mixed precipitate of bacterial cells and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol using the separation solution prepared in Example 1, the supernatant fraction FIG. 5 is a characteristic diagram showing the recovery rate of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol contained in the minute. The state after suspending and centrifuging a mixed precipitate of bacterial cells and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol using the separation solution prepared in Example 2 is shown. It is a photograph. After suspending and centrifuging the mixed precipitate of bacterial cells and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol using the separation solution prepared in Example 2, the supernatant fraction FIG. 5 is a characteristic diagram showing the recovery rate of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol contained in the minute. It is a photograph which shows the state after suspending and centrifuging the mixed precipitation of a microbial cell and a sclareolide using the solution for isolation | separation prepared in Example 3. FIG. It is a characteristic view which shows the recovery rate of the sclareolide contained in a supernatant fraction, after suspending and centrifuging the mixed precipitation of a microbial cell and sclareolide using the solution for isolation | separation prepared in Example 3. FIG. The profile showing the recovery rate of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol shown in FIG. 2 and the profile showing the recovery rate of sclareolide shown in FIG. FIG. It is a figure which shows the process of producing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan from sclareol.

Claims (5)

Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and / or sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan- produced by microorganisms Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and / or sclareolide (decahydro-3a, 6 ) comprising the step of separating microbial cells from 2 (1H) -one (Sclareolide)) , 6,9a-Tetramethylnaphtho [2,1-b] furan-2 (1H) -one; (Sclareolide)) , comprising decahydro-2-hydroxy-2,5,5,8a-tetra Methylnaphthalene ethanol and / or sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan-2 (1H) -one; (Sclareolide)) and microbial cells are suspended The solution density was adjusted to 1.07-1.23 g / ml to decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene It is characterized by separating microbial cells from tanol and / or sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan-2 (1H) -one; (Sclareolide)) And said manufacturing method . Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan-2 (1H) -one ; (Sclareolide)) is an intermediate in the process of synthesizing dodecahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan, which is synthesized using Sclareol as a substrate. The manufacturing method according to claim 1. Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan-2 (1H) -one ; (Sclareolide)) from the suspended solution of decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol to the remaining sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2 , 1-b] furan -2 (IH) - on; (Sclareolide)) and the production method according to claim 1 or 2, wherein the separating the microbial cells. Decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol and sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2,1-b] furan-2 (1H) -one; (Sclareolide)) and bacterial cells, decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol was added to the remaining sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [ 2,1-b] furan -2 (1H) - one; the method according to claim 1 or 2, wherein the separating from (Sclareolide)). The solution density was adjusted to 1.07-1.15 g / ml, and decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthalene ethanol was added to sclareolide (decahydro-3a, 6,6,9a-tetramethylnaphtho [2 , 1-b] furan -2 (1H) - one; the method according to claim 3 or 4, wherein the separating from (Sclareolide)).

Images