JP2012005396A - Method of industrially manufacturing (r)-1,1,1-trifluoro-2-propanol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of industrially manufacturing (R)-1,1,1-trifluoro-2-propanol.SOLUTION: The (R)-1,1,1-trifluoro-2-propanol can be efficiently manufactured at high optical purity by having at least one of microorganism selected from a group including Pichia farinosa act on 1,1,1-trifluoro acetone. As the microorganism found in nature is made to act in the method of manufacture, problems in case of using genetically modified organisms, etc. can be avoided, and industrial implementation of the method is easy.

Description

  The present invention relates to an industrial process for producing (R) -1,1,1-trifluoro-2-propanol.

  (R) -1,1,1-trifluoro-2-propanol is an important compound as an intermediate for various medicines and agricultural chemicals. In addition to conventional methods using chemical catalysts, biology in which microbial enzymes are allowed to act on 1,1,1-trifluoroacetone to reduce it to (R) -1,1,1-trifluoro-2-propanol. Alternative methods have been investigated. For example, in Patent Document 1, a microorganism in which a specific alcohol dehydrogenase gene is incorporated into 1,1,1-trifluoroacetone is allowed to act to reduce enantioselectively and have an enantiomeric excess of 99% ee or more. The method for producing (R) -1,1,1-trifluoro-2-propanol is disclosed in Patent Document 2 as a microorganism or a transformant that functionally expresses an enzyme such as alcohol dehydrogenase or carbonyl reductase. Non-patent document 1 discloses a method for producing (R) -1,1,1-trifluoro-2-propanol, which includes a step of allowing the treated product to act on 1,1,1-trifluoroacetone. By reducing 1,1,1-trifluoroacetone using a microorganism incorporating a specific alcohol dehydrogenase gene, (R) -1,1,1-trifluoro Manufacturing method for obtaining the Oro-2-propanol is disclosed.

  The production method of (R) -1,1,1-trifluoro-2-propanol is only a method using a gene recombinant, but as a related technique, (S) -1,1,1-trifluoro is used. In the production method of 2-propanol, a production method using a non-gene recombinant is also examined. In Patent Document 3 and Non-Patent Document 2, commercially available dry bread is used for 1,1,1-trifluoroacetone. (S) -1,1,1-trifluoro-2-propanol is obtained by allowing yeast to act.

  As a technique related to the present invention, the present applicant selects a microorganism that naturally obtains the target product with high optical purity and produces (S) -1,1,1-trifluoro-2-propanol. An application has been filed (Japanese Patent Application No. 2010-030365).

International Publication No. 2007/054411 International Publication No. 2007/142210 International Publication No. 2007/006650

TC Rosen et al., Chimica Oggi Suppl, 43-45, 2004 M. Buccierielli et al., Synthesis, 11: 897-899, 1983

  In the methods of Patent Document 1, Patent Document 2, and Non-Patent Document 1, (R) -1,1,1-trifluoro-2-propanol having high optical purity is obtained by using a genetically modified product. However, genetically modified organisms may have unexpected traits due to random introduction of genes, and there is a risk that products derived from them will be mixed into products as impurities, and if they are diffused into nature, Since there are concerns about the effects on animals and plants, it is necessary to prove safety and to provide special equipment for preventing the spread of the microorganisms, and the implementation is not always easy.

  On the other hand, a method that does not use a gene recombinant has been studied in the opposite three-dimensional (S) -1,1,1-trifluoro-2-propanol shown in Patent Document 3 and Non-Patent Document 2. The reaction requires problems such as an excessive amount of microorganisms relative to the substrate, treatment of cells by heat treatment, and low optical purity and reaction efficiency compared to genetically modified organisms. From the viewpoint of productivity, it was difficult to adopt.

  An object of the present invention is to provide a method capable of producing (R) -1,1,1-trifluoro-2-propanol economically and simply on an industrial scale.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have made 1,1,1-trifluoroacetone act on specific microorganisms to produce industrially adoptable (R) The inventors have found a method capable of producing -1,1,1-trifluoro-2-propanol with high optical purity and good yield, and have completed the present invention.

  That is, the present invention provides the inventions described in the following [Invention 1] to [Invention 7].

[Invention 1]
1,1,1-trifluoroacetone represented by the formula [1]

(R) -1,1,1-trifluoro-2 represented by the formula [2], which is characterized by contacting a cell obtained by culturing Pichia farinosa as a microorganism. -Propanol

Manufacturing method.

[Invention 2]
The method according to invention 1, wherein the microorganism is a deposit number shown below.

[Invention 3]
A cell suspension of microorganisms was prepared so as to be 10 ⁷ to 10 ¹¹ cfu / ml, and 1,1,1-trifluoroacetone was added to the suspension, and the concentration of the acetone was 0.05 to 3. The method according to the invention 1 or 2, wherein the addition is performed so as to be 3% (w / v).

[Invention 4]
The method according to any one of inventions 1 to 3, wherein the reaction is carried out in a range of 20 to 30 ° C.

[Invention 5]
The method according to any one of Inventions 1 to 4, wherein the pH during the reaction is in the range of 6.0 to 9.0.

[Invention 6]
When contacting the cells obtained by culturing microorganisms, the coenzyme NAD (P) H used is regenerated with the microorganism's own dehydrogenase, and no new coenzyme NAD (P) H is added from the outside. A method according to any one of inventions 1 to 5, wherein

[Invention 7]
The method according to any one of inventions 1 to 6, wherein glucose is used as a substrate for dehydrogenase in the regeneration of coenzyme NAD (P) H.

As described above, a production method for obtaining (R) -1,1,1-trifluoro-2-propanol by reducing a microorganism by acting on 1,1,1-trifluoroacetone is conventionally known. However, all of them used genetic recombinants. Therefore, the present inventors inherently have a specific microorganism (“" having the ability to reduce 1,1,1-trifluoroacetone to (R) -1,1,1-trifluoro-2-propanol with high optical purity. The concentration of 1,1,1-trifluoroacetone in a suspension of the microorganism selected from microorganisms found in nature and adjusted to a density of 10 ⁶ to 10 ¹² cfu / ml Is added so as to be 0.01 to 5% (w / v) and reacted at a reaction temperature of 5 to 40 ° C. and a pH of 4.0 to 10.0, whereby (R) -1,1 having high optical purity is obtained. , 1-trifluoro-2-propanol was newly found to be simple and can be produced on an industrial scale. Further, in the present invention, since the coenzyme NAD (P) H used for the reduction reaction can be regenerated by the microorganism's own dehydrogenase, it is not necessary to newly add the coenzyme NAD (P) H from the outside. (Of course, the reaction can also be carried out by adding the coenzyme NAD (P) H).

  The term “microorganisms found in nature” as used herein refers to microbial cells that have not been subjected to genetic manipulation such as transformation, and refers to wild strains that are stored in various microbial depository institutions. In addition, cultured microorganisms can be used as they are for the suspension of microorganisms, as well as bacterial bodies crushed with ultrasonic waves or glass beads, bacterial bodies conjugated with gels such as acrylamide, organic substances such as acetone and glutaraldehyde. Cells treated with a compound, cells supported on inorganic simple substances such as alumina, silica, zeolite and diatomaceous earth can also be used.

  In the present invention, the concentration of 1,1,1-trifluoroacetone means the concentration (w / v) of the acetone in the suspension of the microorganism (concentration of the reduced product is considered). Not (excluded)) does not define the total amount of acetone added throughout the reaction.

  As in the present invention, microorganisms that naturally give a target with high optical purity are selected from microorganisms found from the natural world, and 1,1,1, By reacting 1-trifluoroacetone in a specific concentration range, (R) -1,1,1-trifluoro-2-propanol with high optical purity (˜100% ee) is produced on an industrial scale. The knowledge that can be obtained has never been known.

  (R) -1,1,1-trifluoro-2-propanol, which is important as a pharmaceutical and agrochemical intermediate, can be produced industrially simply and efficiently.

  The microorganism used in the present invention can efficiently reduce the carbonyl group of 1,1,1-trifluoroacetone to a hydroxyl group in a stereoselective manner. (R) -1,1,1-trifluoro-2 was difficult to devise industrially by devising a method of adding so as to maintain a low concentration suitable for microbial enzyme reaction in the body suspension. -Propanol can be provided with high optical purity and high productivity.

  The present invention is described in detail below. 1,1,1-trifluoroacetone used in the present invention is a known compound, and may be appropriately prepared by those skilled in the art based on the prior art, or commercially available ones may be used.

  In the present invention, as for 1,1,1-trifluoroacetone, the compound itself can of course be used as well as an adduct with water or an alcohol having 1 to 4 carbon atoms. The microorganisms that can obtain (R) -1,1,1-trifluoro-2-propanol by adopting the above reaction conditions are Pichia farinosa NBRC 10231, Pichia farinosa (Pichia farinosa). ) NBRC 1163, Pichia farinosa NBRC 0459, at least one strain selected from the group consisting of Pichia farinosa NBRC 0462, preferably Pichia farinosa NBRC 10231, Examples include Pichia farinosa NBRC 0459 and Pichia farinosa NBRC 0462, more preferably Pichia farinosa NBRC 10231 and Pichia farinosa NBRC 0462.

These microorganisms have been deposited with various institutions with the following deposit numbers. These microorganisms are generally commercially available and can be easily obtained by those skilled in the art.

  For the culture of the microorganism, a medium (solid medium or liquid medium) containing nutrient components usually used for culturing the microorganism can be used, but a reduction reaction of water-soluble 1,1,1-trifluoroacetone is performed. In some cases, a liquid medium is preferred. The medium is a carbon source such as glucose, sucrose, maltose, lactose, fructose, sucrose, trehalose, mannose, mannitol, dextrose and other sugars, methanol, ethanol, propanol, butanol, pentanol, hexanol, glycerol and other alcohols, Organic acids such as acid, glutamic acid and malic acid are used, and ammonium salts, peptone, polypeptone, casamino acid, urea, yeast extract, malt extract, corn steep liquor and the like are used as nitrogen sources. Furthermore, nutrient sources such as other inorganic salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate can be added as appropriate.

  Of these carbon source, nitrogen source, and inorganic salt, it is preferable to add an amount sufficient for the microorganism to grow and not inhibit growth, and usually 5 to 80 g, preferably 10 to 1 L of the medium. Add ~ 40g. The same applies to the nitrogen source, and it is preferable to add an amount that allows the microorganism to grow sufficiently and does not inhibit the growth. Usually, 5 to 60 g, preferably 10 to 50 g, and inorganic salt as a nutrient source for 1 L of the medium. It is necessary to add elements necessary for the growth of microorganisms, but since growth is inhibited at a high concentration, 0.001 to 20 g, preferably 0.005 to 10 g, is usually added to 1 L of the medium. In addition, these can be used combining several types according to microorganisms.

  The pH in the medium needs to be adjusted within a range suitable for the growth of microorganisms, and is usually 4.0 to 10.0, preferably 6.0 to 9.0. The temperature range in the culture needs to be adjusted in a range suitable for the growth of microorganisms, and is usually 10 to 50 ° C, preferably 20 to 40 ° C. During the culture, it is necessary to ventilate the medium with air, preferably 0.3 to 4 vvm (“vvm” means the aeration amount with respect to the medium volume per minute. Volume / volume / minute), more preferably 0. Perform at 5-2 vvm. For microorganisms that require a large amount of oxygen, air with an increased oxygen concentration may be ventilated using an oxygen generator or the like. For instruments that are difficult to set an arbitrary ventilation rate such as test tubes and flasks, the volume of the medium is set to 5 to 40%, preferably 10 to 30% with respect to the volume of the instrument. What is necessary is just to attach vent plugs, such as. In order to facilitate the cultivation, it is preferable to stir the medium. In the case of a fermenter, the stirring ability of the apparatus is preferably 10 to 100%, more preferably 20 to 90%. On the other hand, in the case of a small-scale instrument such as a test tube or a flask, it may be performed using a shaker, preferably 50 to 300 rpm, more preferably 100 to 250 rpm. The culture time may be a time required for the growth of microorganisms to converge, and is 6 to 72 hours, preferably 12 to 48 hours.

  In order to allow the microorganism to act on 1,1,1-trifluoroacetone, which is a substrate, usually, a suspension in which the microorganism is cultured can be used for the reaction as it is. If the components produced during the culture adversely affect the reduction reaction, the cells are isolated from the culture once by an operation such as centrifugation, and the suspension is again made using the obtained cells (wet cells). May be prepared and used in the reaction. Moreover, the above-mentioned microbial cell disruption and microbial cell processing can also be performed.

In order to advance the reaction efficiently, it is necessary to increase the density of the cells in these suspensions. However, if the density is too high, the reaction is inhibited by the production of autolytic enzymes, accumulation of terminal metabolites, etc. In some cases, usually 10 ⁶ to 10 ¹² cfu / ml (“cfu” means the number of colonies formed on the agar medium; colony forming units), preferably 10 ⁷ to 10 ¹¹ cfu / ml, more preferably Is carried out at 10 ⁸ to 10 ¹⁰ cfu / ml.

  In the addition of 1,1,1-trifluoroacetone to these suspensions, it is necessary to maintain the concentration of acetone so that the reduction reaction proceeds smoothly and does not adversely affect the survival of microorganisms. At concentrations higher than 5% (w / v), microorganisms may be killed or the optical purity may be reduced, so concentrations below this value, that is, usually 0.01 to 5% (w / v), preferably 0.05-3% (w / v). The basis of the volume for calculating the acetone concentration may be considered, for example, by using the amount of the culture solution dispensed in the test tube in Example 1 and the total amount of the suspension of the microorganism after the culture in Example 4. Moreover, about the addition method of 1,1,1-trifluoroacetone to this suspension, it is preferable to add sequentially so that a preferable range may be maintained, monitoring a reductive reaction. In addition, the total amount of 1,1,1-trifluoroacetone added has a concentration at which the reaction converges due to the accumulation of products. Therefore, it is necessary to adjust a suitable range according to the microorganisms. 0.1-30% (w / v) is preferable with respect to a liquid, and 0.2-20% (w / v) is more preferable.

  The reaction temperature must be maintained in a range suitable for the enzymatic reaction of the selected microorganism, and is usually 5 to 40 ° C, preferably 20 to 30 ° C. Moreover, it is necessary to maintain the pH during the reaction in a range suitable for the enzymatic reaction of the selected microorganism, and it is usually 4.0 to 10.0, preferably 6.0 to 9.0.

  When the suspension is in a stationary state, microorganisms settle and the reaction efficiency decreases, so the reaction is performed while stirring the suspension. Further, it is necessary to ventilate in order to supply oxygen necessary for respiration of microorganisms, but 1,1,1-trifluoroacetone and (R) -1,1,1- Since trifluoro-2-propanol is scattered as a gas outside the system, it is preferably 0.01 to 0.3 vvm, more preferably 0.02 to 0.1 vvm. The reaction time is determined depending on how the target product is produced, and is usually 6 to 312 hours.

  In the present invention, the coenzyme NAD (P) H (hydrogen donor) used for the reduction reaction is regenerated from the coenzyme NAD (P) using the dehydrogenase possessed by the microorganism. It is preferable to carry out the reduction reaction separately in the presence of a substrate serving as an energy source for enzyme regeneration. For example, saccharides and alcohols described as the carbon source described in the section of culturing microorganisms can be used. Although glucose is used in the present invention, glucose is a substrate that can be used by many microorganisms, and it can be easily measured for concentration using a commercially available analyzer, so it is used as an energy source for coenzyme regeneration. . Glucose may be added directly to the suspension of bacterial cells, or may be mixed in advance with the substrate 1,1,1-trifluoroacetone and added to the suspension. Coenzyme NAD (P) H can be reduced by adding a commercially available one, but it is very expensive and is not economical. By regenerating by the microorganism itself without newly adding the coenzyme NAD (P) H from the outside as in the present invention, the number of reductions per cell increases, and the target product can be obtained economically and with high productivity. Can be manufactured.

  The glucose added here must be added in an amount that does not inhibit the reaction, and the concentration in the suspension is maintained at 0.05 to 10% (w / v), preferably 0.1 to 5%. Add to. For example, as shown in Example 4 to be described later, using a concentration control device with a sugar concentration sensor or the like to perform the reaction while always maintaining the glucose concentration in the reaction system at 0.5%, the reaction is smooth. This is one of the particularly preferred embodiments in the present invention.

  The method of the present invention aims at an industrial production method when converting 1,1,1-trifluoroacetone to (R) -1,1,1-trifluoro-2-propanol. By adopting the conditions, it is possible to manufacture in large quantities.

  In the present invention, R-type alcohol, that is, (R) -1,1,1-trifluoro-2-propanol, in a preferred embodiment, an optical purity that can be practically employed is 75% ee or more, particularly preferably. Can be obtained at 98% ee or higher.

  In order to recover the produced (R) -1,1,1-trifluoro-2-propanol from the reaction completion liquid, a general isolation method in organic synthesis can be employed. After completion of the reaction, a crude product can be obtained by performing ordinary post-treatment operations such as extraction with an organic solvent. In particular, the reaction-finished liquid or the filtered washing liquid after removing the bacterial cells as necessary can be easily and efficiently recovered by subjecting it to distillation. The obtained crude product can be subjected to purification operations such as dehydration, activated carbon, distillation, recrystallization, column chromatography and the like, if necessary. It is also possible to perform an operation to further increase the optical purity of the obtained product.

  EXAMPLES Next, examples will be shown, but the present invention is not limited to the following examples.

[ Results of investigation of reactivity to 1,1,1-trifluoroacetone (primary screening) ]
Prepare a liquid medium consisting of 1000 ml of ion-exchanged water, 20 g of glucose, 10 g of polypeptone, 6 g of yeast extract, 6 g of malt extract, 6 g of potassium dihydrogen phosphate and 4 g of dipotassium hydrogen phosphate, and add 100 ml to a 500 ml Erlenmeyer flask with baffle. Dispensed and steam sterilized at 121 ° C. for 15 minutes.

Microorganisms shown in Table 3 were inoculated into this liquid medium and cultured with shaking at 28 ° C. and 160 rpm (swivel) for 48 hours to prepare each cell suspension. Dispense 5 ml of the cell suspension into a test tube (φ1.4 cm × 18 cm), add 0.5% (25 μl) of 1,1,1-trifluoroacetone, 250 μl of 2M glucose, and further add 28 ° C. The reaction was carried out by shaking at 160 rpm (reciprocating) for 48 hours. After the reaction, n-butyl acetate was added to each reaction solution and mixed, and then the n-butyl acetate layer was separated by centrifugation. The obtained n-butyl acetate layer was measured for optical purity of the product under the analysis conditions described later, and is shown in Table 3.

  Thus, when Pichia farinosa NBRC 0462 was used, it was confirmed that (R) -1,1,1-trifluoro-2-propanol was produced with a high enantiomeric excess.

[Analysis conditions]
The optical purity was analyzed by gas chromatography. BGB-174 (30 m × 0.25 mm × 0.25 mm) manufactured by BGB was used for the gas chromatography column, the carrier gas was helium, the pressure was 100 kPa, and the column temperature was 60 to 85 ° C. (1 ° C./min). The optical purity was calculated from the peak area obtained under the analysis condition of ˜110 ° C. (5 ° C./min) and the vaporization chamber / detector (FID) temperature of 230 ° C. The retention time of each enantiomer of 1,1,1-trifluoro-2-propanol was 22.1 min for the R isomer and 23.9 min for the S isomer.

[ Results of investigation of reactivity to 1,1,1-trifluoroacetone (secondary screening) ]
Prepare a liquid medium consisting of 1000 ml of ion-exchanged water, 20 g of glucose, 10 g of polypeptone, 6 g of yeast extract, 6 g of malt extract, 6 g of potassium dihydrogen phosphate and 4 g of dipotassium hydrogen phosphate, and add 100 ml to a 500 ml Erlenmeyer flask with baffle. Dispensed and steam sterilized at 121 ° C. for 15 minutes.

The liquid medium was inoculated with the microorganisms shown in Table 4, and cultured with shaking at 28 ° C. and 160 rpm (swivel) for 48 hours to prepare each cell suspension. Dispense 5 ml of the cell suspension into a test tube (φ1.4 cm × 18 cm), add 0.25% (12.5 μl) of 1,1,1-trifluoroacetone, and add 250 μl of 2M glucose, and further add 28 The reaction was carried out by shaking for 48 hours at 160 ° C. (reciprocating). After the reaction, n-butyl acetate was added to each reaction solution and mixed, and then the n-butyl acetate layer was separated by centrifugation. The obtained n-butyl acetate layer was measured for optical purity of the product under the analysis conditions described above, and is shown in Table 4.

  Thus, when Pichia farinosa NBRC 10231, Pichia farinosa NBRC 0459, and Pichia farionosa NBRC 0462 strains are used, (R) -1,1,1-trifluoro-2-propanol is produced with a high enantiomeric excess. I was able to confirm.

[ Results of investigation of the reactivity to 1,1,1-trifluoroacetone (concentration dependence) ]
Prepare a liquid medium consisting of 1000 ml of ion-exchanged water, 20 g of glucose, 10 g of polypeptone, 6 g of yeast extract, 6 g of malt extract, 6 g of potassium dihydrogen phosphate and 4 g of dipotassium hydrogen phosphate, and add 100 ml to a 500 ml Erlenmeyer flask with baffle. The solution was dispensed one by one and autoclaved at 121 ° C. for 15 minutes.

This liquid medium was inoculated with Pichia farinosa NBRC 0462 and cultured with shaking at 28 ° C. for 48 hours to prepare a 1.1 × 10 ⁹ cfu / ml suspension. Dispense 5 ml of the cell suspension into a test tube (φ1.4 cm × 18 cm), add 1,1,1-trifluoroacetone 0.1-2.0% and 20% glucose aqueous solution 250 μl uniformly. Further, the reaction was carried out by shaking at 28 ° C. for 48 hours. After the reaction, n-butyl acetate is added to each reaction solution for extraction, and the optical purity of the product is measured by gas chromatography (column: BGB-174 manufactured by BGB (30 m × 0.25 mm × 0.25 mm)). The results are shown in Table 5. The yield was measured by ¹⁹ F-NMR internal standard method.

  Thus, when the 1,1,1-trifluoroacetone concentration was low, it was confirmed that (R) -1,1,1-trifluoro-2-propanol was produced with a high enantiomeric excess.

  Next, among the microorganisms described in Example 1 and Example 2, Pichia farinosa NBRC 0462, which gives the target product with high optical purity, was used, and 1,1,1-trifluoroacetone was suspended. The reaction results obtained by adding the acetone so that the total amount with respect to the suspension is 2% (w / v) (Example 4) and 2.8% (w / v) (Example 5) are shown below.

[Production of (R) -1,1,1-trifluoro-2-propanol (2% (w / v))]
A liquid culture medium having a composition of 2000 ml of ion-exchanged water, 60 g of glucose, 30 g of peptone, 50 g of yeast extract, 4.8 g of potassium dihydrogen phosphate, and 2.5 g of dipotassium hydrogen phosphate was prepared. ) Pasted on Maruhishi Bio-Engine, MDN type 5L (S)), steam sterilization was performed at 121 ° C for 60 minutes. This liquid medium was inoculated with 80 ml of a 1.7 × 10 ⁹ cfu / ml suspension of Pichia farinosa NBRC 0462 pre-cultured with 100 ml of the same composition, and cultured for 16 hours at 28 ° C., aeration 1 vvm, and stirring 500 rpm. A suspension of 1.3 × 10 ⁹ cfu / ml (133 g / L as wet weight) was prepared. Adjustment of pH at this time was performed using ammonia water, and it adjusted to 6.5. After completion of the culture, the aeration was changed to 0.1 vvm, the stirring was changed to 50 rpm, and 1,01-trifluoroacetone 50.08 g and glucose 90 g were dissolved in 200 ml of ion-exchanged water prepared in a separate container. Using an online sugar concentration sensor (online biosensor BF-410, manufactured by Biot Co., Ltd.) for the body suspension, the computer program automatically turns the suspension into a suspension so as to maintain the glucose concentration at 0.5%. Added. After the prepared substrate was completely added, 200 g glucose was dissolved in 300 ml ion exchange and added to the cell suspension in the same manner. The reduction of the substrate by the microorganism was monitored every 24 hours, and it was confirmed that the yield was 100% after 64 hours.

Distillation was performed to recover 1,1,1-trifluoro-2-propanol generated from the reaction solution after the reaction was completed. 93.5 ml of the distillate was recovered and found to contain 44.58 g of 1,1,1-trifluoro-2-propanol by an internal standard method of ¹⁹ F-NMR. The optical purity was measured under the analysis conditions described above and confirmed to be 100% ee (R form).

[Production of (R) -1,1,1-trifluoro-2-propanol (2.8% (w / v))]
A liquid culture medium having a composition of 2000 ml of ion-exchanged water, 60 g of glucose, 30 g of peptone, 50 g of yeast extract, 4.8 g of potassium dihydrogen phosphate, and 2.5 g of dipotassium hydrogen phosphate was prepared. ) Pasted on Maruhishi Bio-Engine, MDN type 5L (S)), steam sterilization was performed at 121 ° C for 60 minutes. This liquid medium was inoculated with 80 ml of a 1.2 × 10 ⁹ cfu / ml suspension of Pichia farinosa NBRC 0462 pre-cultured with 100 ml of the same composition, and cultured for 16 hours at 28 ° C., aeration 1 vvm, and stirring at 500 rpm. A suspension of 1.3 × 10 ⁹ cfu / ml (120 g / L as wet weight) was prepared. Adjustment of pH at this time was performed using ammonia water, and it adjusted to 6.5. After completion of the culture, aeration was changed to 0.1 vvm, stirring was changed to 50 rpm, and 400 ml of 1,1,1-trifluoroacetone and 400 g of glucose were dissolved in 400 ml of ion-exchanged water prepared in a separate container. The glucose concentration was automatically added to the suspension by a computer program so as to maintain the glucose concentration at 0.5% using a sugar concentration sensor (online biosensor BF-410, manufactured by Biot Co., Ltd.). The reduction of the substrate by the microorganism was monitored every 24 hours, and after 88 hours, the reaction was completed after confirming that the sugar consumption was stagnant. The total amount of substrate added at this time was 2.8%, and the yield was 81.8%.

Distillation was performed to recover 1,1,1-trifluoro-2-propanol generated from the reaction solution after the reaction was completed. 158 ml of the distillate was recovered and found to contain 58.9 g of 1,1,1-trifluoro-2-propanol by the internal standard method of ¹⁹ F-NMR. The optical purity was measured under the analysis conditions described above, and it was confirmed that it was 75.1% ee (R form).

  The (R) -1,1,1-trifluoro-2-propanol targeted in the present invention can be used as an intermediate for medical and agricultural chemicals.

Claims (7)

1,1,1-trifluoroacetone represented by the formula [1]

(R) -1,1,1-trifluoro-2 represented by the formula [2], which is characterized by contacting a cell obtained by culturing Pichia farinosa as a microorganism. -Propanol

Manufacturing method. The method according to claim 1, wherein the microorganism is a deposit number shown below.

A cell suspension of microorganisms was prepared so as to be 10 ⁷ to 10 ¹¹ cfu / ml, and 1,1,1-trifluoroacetone was added to the suspension, and the concentration of the acetone was 0.05 to It adds so that it may become 3% (w / v), The method of Claim 1 or 2 characterized by the above-mentioned.   The method according to any one of claims 1 to 3, wherein the reaction is carried out in a range of 20 to 30 ° C.   The method according to any one of claims 1 to 4, wherein the pH during the reaction is in the range of 6.0 to 9.0.   When contacting the cells obtained by culturing microorganisms, the coenzyme NAD (P) H used is regenerated with the microorganism's own dehydrogenase, and no new coenzyme NAD (P) H is added from the outside. The method according to claim 1, characterized in that:   The method according to any one of claims 1 to 6, wherein glucose is used as a substrate for dehydrogenase in the regeneration of coenzyme NAD (P) H.